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Some Key Topics in Chemistry
and Biochemistry for
Biotechnologists

Editor
Munishwar Nath Gupta
Former Emeritus Professor
Department of Biochemical Engineering and Biotechnology
Indian Institute of Technology Delhi
New Delhi, India

p,
p,
A SCIENCE PUBLISHERS BOOK
A SCIENCE PUBLISHERS BOOK
Cover credit: Image taken from Chapter 4 of this book. Reproduced by kind courtesy of authors of
the chapter.

First edition published 2023

by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN

© 2023 Munishwar Nath Gupta

CRC Press is an imprint of Taylor & Francis Group, LLC

Reasonable efforts have been made to publish reliable data and information, but the author and
publisher cannot assume responsibility for the validity of all materials or the consequences of
their use. The authors and publishers have attempted to trace the copyright holders of all material
reproduced in this publication and apologize to copyright holders if permission to publish in this
form has not been obtained. If any copyright material has not been acknowledged please write and
let us know so we may rectify in any future reprint.
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Trademark notice: Product or corporate names may be trademarks or registered trademarks and are
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Library of Congress Cataloging‑in‑Publication Data (applied for)

ISBN: 978-1-032-26301-4 (hbk)

ISBN: 978-1-032-26303-8 (pbk)
ISBN: 978-1-003-28759-9 (ebk)
DOI: 10.1201/9781003287599

Typeset in Times New Roman

by Radiant Productions
Preface

Covid-19 brought out some fault lines in our preparation for a crisis which needed
coordinated efforts from scientists and technologists belonging to different disciplines.
Lately, there has been a debate about the relative values of a specialist versus a
generalist. The experience with Covid-19 perhaps teaches us that it is best to have
specialists who are also reasonably familiar with basic concepts of other disciplines
which have interfaces with their own special subject. It is in line with this thinking
that engineers and doctors are taught chemical sciences at an undergraduate level. As
science and technology grows, it is becoming increasingly difficult to optimise this
exposure. Not everything is covered in standard text books. The present volume is
an effort to bring together some topics which are generally not covered adequately
in the books/syllabi of undergraduate or even post graduate courses. The idea is
to encourage scientists/technologists to read more widely. That would enable these
persons to deal more effectively with such crisis by rounding off their knowledge in
some basic areas in chemical and biochemical sciences.
Chapter 1 covers flow-based systems which are increasingly becoming important
in both chemical and biological sciences. Biologists would profit from learning
how rates of biocatalytic reactions are monitored in such systems. Chapters 2 and 3
describe chemiproteomics and high throughput screening methods. Both topics are
valuable in the context of drug discovery/design.
Enzymes function in many different milieu. Among such non-conventional
media, solid-gas biphasic medium is perhaps most under appreciated. Gases like
hydrogen sulphide and nitric oxide are now known to be metabolic intermediates of
high biological significance. Hence the choice of the topic of Chapter 4 as solid-gas
biocatalysis. Biocatalysis in non-conventional media and at high temperatures needs
enzymes with high stability. More robust enzymes derived from salty environments
(marine resources being the most important one) are described in Chapters 5 and 6.
Nanosciences/nanotechnology is now relevant to many area of biotechnology
and medicine. Quantum dots, extremely small nanoparticles have some fascinating
properties. Among these is their value as fluorescent materials. Chapter 7 discusses
these nanoparticles. While most reviews focus on their applications; this chapter
highlights synthesis. This may enable one to design and tailor QDs for specific
applications. While chirality of organic molecules is generally a familiar topic among
chemists; chirality of nanomaterials is not so well known. Chapter 8 deals with this
somewhat obscure topic.
iv Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Chapter 9 covers basics of colloids and immunochemistry in the context of

Covid-19. This last chapter points out how being clear about some basic chemistry
and biochemistry would have prevented some mis-steps in dealing with Covid-19 at
various levels.
I hope this volume would be useful as a reference as well as a supplementary
reading material by chemists, biotechnologists and those in the medical profession.
I thank various authors. I thank Mr. Raju Primlani (from Science publishers-an
imprint of CRC which is publishing this book) to encourage me to put together this
volume. I also thank others from CRC who have worked in producing this volume.
7th September, 2022 Munishwar Nath Gupta
Delhi
Contents

Preface iii
1. Uses of Flow-based Systems for Enzyme Kinetics 1
Supaporn Kradtap Hartwell
2. Chemoproteomics: An Extremely Powerful Kit in Drug Discovery 26
Toolbox
Anupama Binoy, Revathy Sahadevan and Sushabhan Sadhukhan
3. Combinatorial Chemistry and High Throughput Screening Methods 44
Vaishnavi Puppala, Shivcharan Prasad and Ipsita Roy
4. Fundamentals and Applications of Solid/Gas Biocatalysis 64
Sergio Huerta‑Ochoa, Carlos Omar Castillo‑Araiza,
Itza Nallely Cordero‑Soto, Yahir Alejandro Cruz‑Martínez,
Lilia Arely Prado‑Barragán and Olga Miriam Rutiaga Quiñones
5. Biochemistry and Biomolecules of Halophiles: Recent Trends 91
and Prospects
Sumit Kumar, Bibhuti Bhusan Das, Nitin Srivastava and Sunil Kumar Khare
6. Marine Enzymes: Exploiting Bacterial Resource for 117
Blue Biotechnology
Yasmin Khambhaty
7. Quantum Dots in Biological Sciences 152
Kamla Rawat and Himadri B. Bohidar
8. Chirality in Nanomaterials: Occurrence, Methods of 177
Determination and Biochemical Significance
Wells Utembe
9. Surface Chemistry and Immunochemistry at a Crossroad 191
called COVID-19
Munishwar Nath Gupta
Index 215
Chapter 1
Uses of Flow-based Systems
for Enzyme Kinetics
Supaporn Kradtap Hartwell

Introduction
Enzymes are proteins that accelerate chemical reactions upon binding to a specific
reagent called a substrate. Enzymes have been incorporated in numerous chemical
and biochemical analyses and biotechnological processes to catalyze the target
reactions. The shape and properties of the binding sites on the enzyme, arisen from
the unique amino acids sequence, is complimentarily specific for a certain substrate
and is conventionally known as a Lock and Key fit. The induced fit model further
explains that a binding site on an enzyme is not rigid and can adjust its shape to
better accommodate a substrate molecule (Koshland 1958, Boyer 2022, Vasella
et al. 2022). Some enzymes facilitate bond breaking and transition state formation by
physically altering the substrate molecules (Garcia-Viloca et al. 2004). Even though
some enzymes may form chemical bonds with the substrate during the reaction,
these bonds are temporary and active sites will return to normal after releasing the
products. In other words, an enzyme catalyzes a chemical reaction without itself
being altered, and therefore, can resume its function after removal of products.
Figure 1 shows the reversible binding of enzyme-substrate, followed by the releasing
of products and freeing of enzyme. Thus, apart from having high selectivity, enzymes
have signal amplification ability by continuously reacting the released enzyme with
more incoming substrate molecules. This helps to increase the number of products
and promote sensitivity, which is beneficial when using enzymatic reaction as part
of a chemical detection process such as in enzyme-based immunoassay techniques
(Calabria et al. 2021). In addition, the fact that enzyme molecules can resume their

Department of Chemistry, 3800 Victory Parkway, Xavier University, Cincinnati, Ohio, USA 45207.
Email: [email protected]
2 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 1. Enzyme-substrate reaction a) Reversible binding of enzyme-substrate, and b) irreversible

releasing of products. E, S, and P represent enzyme, substrate, and product, respectively.

function makes it feasible to study enzyme kinetics in the heterogeneous format

because the immobilized enzyme molecules can be reused without having to flush
them out of the system.
Knowledge about enzyme kinetics is essential in evaluating characteristics and
possible applications of a particular enzyme and in identifying suitable substrate
for that enzyme. Kinetic parameters of an enzyme aid in understanding of the
mechanism of enzyme-catalyzed reactions which allow better manipulation and
optimization of the conditions for effective enzymatic reaction. Enzymes serve as
catalysts, which are not consumed in the reaction, and so their concentrations are
unchanged. Therefore, enzyme kinetics are usually evaluated from the rate of change
in concentration of substrate or products either in transient state (before reaching
steady state) or at steady state when concentration of enzyme-substrate product no
longer changes. The steady-state approach often cannot reveal detailed understanding
of enzyme mechanisms, whereas the pre-steady state condition, referred to when
enzyme and substrate are mixed at t = 0 until either a steady state or equilibrium is
established, provides more details about mechanism of the catalytic process (Fisher
2005). In steady state approach, the concentration of the intermediate E-S does not
change. In the equilibrium approach, the rate of formation of E-S is equal to its rate of
transformation to either back to the substrate or product. Any standard book dealing
with enzyme kinetics can be consulted to learn how both approaches/assumptions
lead to a similar equation called Michaelis-Menten equation (please see below for
a brief introduction to Michaelis-Menten model). For the system with multiple
substrates, enzyme kinetics can also predict the sequence of substrates binding as
well as the sequence of products releasing.
A simple model developed by Michaelis and Menten to explain enzyme kinetics
assumes that enzyme (E), substrate (S), and enzyme-substrate complex (ES) are in
rapid equilibrium. Reactivity of an enzyme towards a particular substrate can be
expressed with kinetic parameters namely Kcat, Vmax, and Km. The catalytic constant
Kcat (or turnover number) represents the number of substrate molecules that can be
converted to product per enzyme molecule per unit time. Michaelis constant Km and
maximum velocity Vmax indicate how easily the enzyme becomes saturated with a
particular substrate. Vmax is basically the maximum rate that the enzyme reaches at
the point of being saturated by the substrate, where Km is the substrate concentration
at which the reaction velocity is half of the maximum velocity (or V = Vmax/2).
 Uses of Flow‑based Systems for Enzyme Kinetics 3

Enzyme kinetic assays are generally carried out batch-wise by mixing enzyme at a
constant concentration with a substrate at various concentrations in several cuvettes.
The initial rates (velocity V) of the individual reactions are observed for a period of
time, e.g., measurement of products in a spectrophotometer for a set period of time
such as a few seconds or a few minutes; V = d[P]/dt. Initial rate is preferred because
it is easiest to observe the rate of reaction when the reaction is just started without
the effect of accumulated products that slow down the reaction. At low substrate
concentration ([S] << Km), the reaction is first order with respect to substrate as
rate of the reaction is directly proportional to the concentration of substrate. At high
substrate concentration ([S] >> Km) where it is assumed to be a constant parameter
in the rate law, the reaction is assumed zero order with respect to substrate as rate
of the reaction is at maximum and independent of substrate concentration (Atkins
and de Paula 2017). Figure 2 shows three common plots used for determination of
the important kinetic parameters Km and Vmax of an enzyme. Plot of V vs [S] is non-
linear. Transformation of the curve to linear graph, either by plotting 1/V vs 1/[S] as
proposed by Lineweaver and Burk, or V vs V/[S] as proposed by Eddie and Hofstee,
makes it easier to estimate Km and Vmax from slope and XY intercepts.
Apart from physical conditions such as temperature, pH, and ionic strength, the
presence of enzyme inhibitors can alter Vmax or Km of the enzyme. Inhibitors may
be in the form of small organic molecules or small peptide analog of the natural
substrate. Table 1 summarizes common types of inhibitors and their effects on
kinetic parameters of an enzyme (Kuddas 2019, Strelow et al. 2012). Nevertheless,
inhibitors can be used as tools to validate purity of an enzyme. A detailed discussion
on this is beyond the scope of this chapter but is available elsewhere (Scott and
Williams 2012).
As mentioned, enzyme kinetic assays usually involve mixing enzyme at a
constant concentration with various concentrations of substrate. When performed
manually, the process can be tedious and slow. The commonly used 96 well-plate
does not always answer the demand for high throughput analysis (Pereira et al. 2020),
for example to evaluate sources of enzyme, and drug discovery. Limitations of the
common 96 well-plate include insufficient space to accommodate the experiments
that involve many different conditions to be studied. It also requires relatively large
volumes of reagents, is not portable nor fully automated, and still requires well trained

Figure 2. Graphs representing relationship of reaction velocity and concentration of substrate. a)

Michaelis-Menten plot, b) Lineweaver-Burk (double reciprocal) plot, c) Eddie-Hofstee plot.
4 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Table 1. Common types of inhibitors and their effects on Vmax and Km.

Type of inhibitors Characteristics of inhibition Effect on Km and Vmax

Competitive • Inhibitors bind only to free enzyme. Vmax is unchanged, but Km is
inhibitors • Inhibitors compete for the active sites on higher.
the enzyme, preventing the real substrate
from binding.
• Possible ways of inhibition include The level of inhibition
steric hindrance blocking accessibility of depends on the relative
substrate to the active site of the enzyme, concentration of substrate and
overlapping of binding site, binding to the inhibitor. To achieve Vmax,
same active site or sharing some binding higher substrate concentration
pockets, or causing conformational change is required to compete and
of the active site. beat inhibitor to the enzyme.
Noncompetitive • Inhibitors bind equally well to both free Vmax is lower, but Km is
inhibitors enzyme and the enzyme-substrate complex. unchanged.
• Inhibitors bind at a separate site from Noncompetitive inhibitors
active site for substrate. are not affected by substrate
• Possible ways of inhibition include concentration.
deformation of the structure of the enzyme,
conformational change that inhibits
catalytic capability of the enzyme, or
hindering the binding and releasing of
substrate.
Uncompetitive • Inhibitors bind only to the enzyme- Both Vmax and Km are lower.
inhibitors substrate complex at a location outside the
active site.
• The resulting enzyme-substrate-inhibitor
complex is enzymatically inactive.
Allosteric inhibitors • Inhibitors bind to allosteric site other than Depend on type of inhibition,
or in addition to the active site, causing an allosteric inhibitor may
conformational change of the enzyme. display a competitive,
• Possible ways of inhibition include noncompetitive, or
affecting the formation of the enzyme- uncompetitive inhibition.
substrate complex, stabilization of the
transition state, or reducing the ability to
lower the activation energy of catalysis.

personnel to operate devices, such as a pipette, precisely. These limitations drive

the development for automatic and high throughput systems for enzyme kinetics.
The following sections present flow based techniques including flow injection and
sequential injection analysis techniques, microfluidic systems, as well as paper based
microfluidic devices for studies related to enzyme kinetics.

Flow based analysis formats

Flow injection and sequential injection systems
Introduced in the 1970s (Ruz̆ ic̆ ka and Hansen 1975), flow injection has been known
as a low cost automatic chemical analysis technique that can reduce analysis time
 Uses of Flow‑based Systems for Enzyme Kinetics 5

and, in many cases, amount of chemicals. The flow injection technique involves
introducing a plug of a chemical reagent, usually via an injection valve, into a
flowing stream of another reagent that is forced into a small tubing by pumping.
Chemical reaction takes place while the reagents are merging and flowing down-
stream. Product is detected when the reaction plug flows through the detector.
For studying enzyme kinetics, volumes of enzyme/substrate and degree of mixing
can be optimized by controlling flow rates. Detection of product or substrate
concentration, especially at non-equilibrium state, can be done rapidly and at a
more precise time for each replicate, as compared to the batch method. The process
becomes more automatic and helps to minimize inconsistency owing to manual
operations. This is especially useful in measuring initial rate that is often hard to
do precisely in batch mode due to the difficulty in conducting prompt and precise
solution transferring, mixing, and maneuvering cuvettes. The ability to measure
initial rate is attractive because concentration of the main substrate of interest is
still abundant at the beginning of enzymatic reaction. Therefore, it can suppress
interferences from other lower concentration substrates that may become pronounced
as the reaction progresses and the concentration of the main substrate is depleted.
Also, as the product concentration[s] are low, product inhibition does not complicate
the situation.
Conventional flow injection analysis (FIA) systems for enzyme kinetics have
been reviewed (Hartwell and Grudpan 2012). A typical flow injection system,
composed of peristaltic pump, a 6-port injection valve, tubing, and detector, can
easily be operated at milliliter level. Volume of sample/reagent is dictated by
the size of the injection loop connected to the ports on the valve, see Fig. 3a. In
loading position, the injected solution fills the loop of a selected size/volume and
excess amount goes to waste. In injection position, the valve is switched to let the
carrier solution push out the injected solution inside the loop and mix with it while
travelling through the detector. In enzyme kinetics, enzyme may be injected into
the flowing substrate carrier solution or vice versa, depending on cost of reagents
and experimental design. Due to the continuous flow in one direction, it is more
convenient to use enzyme solution at constant concentration as a carrier, and inject a
substrate of various concentrations. However, high consumption of enzyme solution
can increase analysis cost.
A more modern sequential injection analysis (SIA) system with a computer
controlled syringe pump can perform chemical analysis at lower volume at microliter
level. The flow control is not continuous, instead direction and movement of liquid
is programmed based on volume and flow rate selection. Another main component
of the SIA system is a selection valve for aspiration and dispensing of all the
solutions. Flow reversal of the syringe pump controlled by a computer software
which allows multiple solutions as well as air segments to be introduced as stacked
zones into a long holding coil located between the syringe pump and the selection
valve, see Fig. 3b. In enzyme kinetics, aliquots of enzyme, substrate, and co-
factor solutions are aspirated one at a time through different ports on the selection
valve into a holding coil. The sequence of aspiration should be optimized to allow
6 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 3. Diagrams and operation of a) 6-port injection valve used in FIA, and b) multi-port selection
valve used in SIA. R1, R2, R3 are reagents such as enzyme, substrate, and co-factor.

efficient reaction between enzyme and substrate while minimizing dispersion of

the injected enzyme-substrate reaction plug through the interface with the carrier
solution. The stacked zones are mixed by the flow reversal when those zones are
being sent through one of the ports on the selection valve into the detector. The multi-
port selection valve (usually 8-port valve) used in SIA system can accommodate
aspiration of multiple reagents/samples, whereas a 6-port injection valve used in
FIA system can only be used to inject reagents/samples into the system one at a
time through the injection port. Because these injection/selection valves are the
heart of the systems, understanding the operation principles of the sample/reagent
 Uses of Flow‑based Systems for Enzyme Kinetics 7

introduction is important for designing the analysis. Consider Fig. 3 for comparison
of operational principles of the FIA 6-port injection valve and the SIA 8-port selection
valve, as described above, for better understanding of flow direction manipulation
and accommodation.
A review on applications of SIA for enzyme kinetics include various examples of
research (van Staden and Raluca 2002, Silvestre et al. 2011), mainly in homogenous
phase where enzyme is in solution (non-immobilized enzyme). Incorporation of the
mixing chamber to one of the ports on the selection valve is most common in order
to promote mixing by increasing radial movement that may be insufficient in the
holding coil alone. Detection, then, can be done directly on the mixing chamber
(e.g., using fiber optics), or by sending an aliquot of product solution from the chamber
to the detector unit. Lab-on-Valve (LOV) and Lab-at-Valve (LAV) commonly used
with the SIA system are ways to incorporate the detection unit within or closely
adjacent to a multi-position selection valve, in order to enable higher throughput
analysis and lower volume consumption of reagents (from hundreds to tenths of
microliter). The low dead volume in the SIA system allows for faster analysis
because the new portion of solution can be sent to the detection at the same time
that the previous portion in the detector is pushed out. While the most common
detector used for the LOV system is fiber optics which can fit within the channel
specially made for it located as part of the selection valve, other devices such as
mixing chamber, filtration unit, and dialysis unit can also be incorporated around the
valve which are usually referred to as LAV. Schematic representations of basic FIA,
SIA, SIA-LOV, and SIA-LAV systems are depicted in Fig. 4. Please note that figures
shown are common conventional flow based systems. Implementation of other types
of valves such as a solenoid valve to perform hydrodynamic injection is also possible
(Khongpet et al. 2018).

Figure 4. Common flow based systems a) FIA using a peristaltic pump and 6-port injection valve, b) SIA
using a syringe pump and 8-port injection valve, c) SIA-LOV where the detector is integrated onto the
selection valve, d) SIA-LAV where the detector is adjacent to the selection valve.
8 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

In general the flow of solution in the FIA system is continuous, while the flow
of solution in the SIA system is stop and go due to reversed flow operation of the
syringe pump. However, both FIA and SIA systems can be designed and adapted to
manipulate various other solution flow formats (such as continuous flow, stopped
flow, segmented flow, and quenched flow) to serve particular applications. Briefly,
continuous flow systems are based on mixing of the merged sample and reagent
solutions injected into the continuous flow of the carrier solution. In order to allow
more reaction time between enzyme and substrate, and hence increased sensitivity,
stopped flow format can be used in which the reaction zone is physically stopped
for a period of time. This is done by stopping the pump, before online detection.
This is similar but is not the same as quenched flow where the reaction zone is
stopped both physically and chemically by stopping the pump and introducing a
quenching solution. In this case, a typical FIA system with a single 6-port injection
valve and one direction flow has limitation due to number of solutions that can be
injected being limited to one. An additional valve or a pump is needed to introduce
a quencher. The 8-port selection valve with capability of reverse flow enables
introduction of more solutions using a single valve. The stopped flow technique can
be used to detect intermediates, while the quenched flow technique is mostly limited
for detection of product. The two techniques can be used complementarily (Fisher
2005). In the FIA and SIA systems, band broadening and dispersion of the sample/
reagent zone that occur at the interfaces with the carrier stream may cause dilution
and low sensitivity of detection. However, the systems can introduce air segments
before and after each reaction zone to prevent mixing/minimize dispersion, which in
turn improves sensitivity and reduces consumption of reagents (Patton and Crouch
1986).

Microfluidics
Due to the great demand for high throughput analysis along with the need for low
volume consumption of chemical/biological reagents, downscaling the analysis
system has been a major focus. Study of enzyme, in particular, requires numerous
observations of the catalytic capability of the enzyme under various conditions of
enzyme-substrate reactions which is time consuming, labor intensive, and can be
expensive due to high total volume of reagents. The progress in downscaling of the
chemical analysis system has been gearing toward a small portable unit known as
lab-on-chip or microfluidic systems.
Microfluidic devices are usually composed of a network of microchannels of
the size in the range of sub-millimeters engraved on a solid material such as glass
or plastic that can accommodate the chemical reaction in the volume of microliter
or lower. Although in some applications, top-open channels can be used (Casavant
2013), the microchannels are usually closed with another solid plate that is secured
to the plate with engraved microchannels using such physical and chemical methods
as double sided tape, magnetic clamps, or plasma treatment (Tkachenko et al. 2009,
Tsao and DeVoe 2009, Khongpet et al. 2020).
 Uses of Flow‑based Systems for Enzyme Kinetics 9

Pumping systems for microfluidics

Liquid pumping systems used for introduction of liquid into microchannels are
commonly based on either pressure or electroosmotic force (Schilling 2001).
Due to the small diameter of the microchannel, liquid inside the microfluidic
system is mainly governed by a laminar flow. Without the turbulent flow that can
cause molecules to flow in random directions, laminar flow allows the benefit of
predictable movement and, therefore, physical/chemical conditions of liquid when
flowing through the microchannel. For the pressure driven pump that produces the
force from one end of the channel, velocity of liquid adjacent to the inner wall of a
microchannel is lower than velocity of liquid in the middle of the channel, creating
a parabolic laminar flow profile, see Fig. 5a, with concentration gradient due to
diffusion. For an electroosmotic pump, electrical field is applied across the length of
the open-ended channel. Most solid surfaces, including the wall of the microchannel,
have an electric charge that attracts counter ions from the solution to form an electric
double layer. These ions will move towards the opposite polarity electrode, pulling
the body of liquid along towards the same direction. Unlike the parabolic profile that
resulted from the pressure driven flow, the electroosmotic pump creates a simple
laminar flow profile with a uniform velocity of the same frontline across the width of
the channel, see Fig. 5b. Although dispersion and band broadening still remains, they
are minimized. As compared to parabolic flow profile, this plug flow profile exhibits
rather uniform diffusion. However, an important point to consider when working with
enzyme is the possibility of protein adsorption onto the wall of the microchannel.
Apart from depleting the amount of free enzyme, the portion of enzyme which is now
immobilized by adsorption may alter the charge on the solid inner wall of the channel
and result in changing the velocity of liquid flow.
The means to deliver and manipulate liquid flow, as well as the detection of the
target substance produced within the microfluidic system, have continuously been
the main research focuses. With the advance in digital technology, microfluidics
have become a preferred approach in down scaling flow based chemical/biological
analyses, including enzyme studies and those that utilize enzymatic reaction as part
of the analyses. Microfluidic designs that offer concurrent multi-assay, accommodate
incubation time, and enable long term monitoring of the reaction are desirable (Rho
et al. 2016). Common main approaches, other than continuous flow homogeneous
phase, include enzyme immobilization (heterogeneous phases) and droplets based
microfluidics (aqueous segment in oil) which are described in the next sections.

Figure 5. Laminar flow in a microchannel where there is a) a pressure driven parabolic gradient flow
profile in which the direction of flow is due to the pressure pump, and b) an electroosmotic driven plug
flow profile in which the direction of flow is due to the movement of a layer of adsorbed ions toward the
oppositely charged electrode.
10 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Common designs in microfluidic system

Designs of microchannel network usually depend on the application of a particular
chemical reaction to be carried out, many times mimicking the process needed for
batch-wise process. With the advance in technology such as 3D printers and laser
engravers, various complicated designs can be made to serve specific purposes.
Among the various novel proposals, some common microchannel designs for
reagents introduction, reaction accommodation, and mixing and are shown in
Fig. 6. Y- and T-junctions are the most widely used designs for introducing more
than one reagent into the system either at the same time or at different times. At
a glance, these two designs are not much different, both allow for the merging of
two or more solutions. However, the difference in flow angles together with liquid
properties such as viscosity and flow rate, have significant effect on liquid delivery
through the microchannel. This aspect will be discussed more in the later section on
droplets-based microfluidics. Serpentine design helps to fit a long channel within
a short length platform. Two liquids, even though they are miscible, if introduced
through two different inlets at a suitable flow rate usually can be controlled to flow
in parallel after merging within a microchannel. Only diffusive mixing occurs at
the liquid-liquid interface while flowing downstream. If more vigorous mixing is
needed, the microchannel should be designed to disturb laminar flow and promote
turbulence. An example of repetitive split and merge design helps to promote mixing
of the reagents.

Figure 6. An example of microfluidic system with some common microchannel designs (modified from
Siegesmund and Hartwell 2018, Ellerhorst and Hartwell 2019).

Flow based method with enzyme immobilization

In the conventional study of enzyme’s kinetics, both enzyme and substrate are in
solution or homogeneous phase. They have freedom of movement to interact from all
angles, and if the orientation and energy is suitable, binding occurs. Kinetics measured
in this homogeneous condition is known as intrinsic kinetics. After the analysis,
the solution mixture, containing enzymatic product, free enzyme, and unreacted
substrate, is usually discarded as these components cannot be easily separated for
 Uses of Flow‑based Systems for Enzyme Kinetics 11

reuse. If the enzyme is high cost or low in availability, immobilization of enzyme on

a solid surface, such as on a solid support (e.g., bead or monolith material packed
in a column, or the inner wall of the capillary or microchannel), may be beneficial.
Enzymatic reaction takes place when the substrate solution flows through the reactor
with immobilized enzyme. Immobilizing enzyme molecules enables removal of
products of the enzymatic reaction while keeping enzyme in place, and therefore,
it is possible to reuse the reactor or amplify signal by introducing more substrate
molecules. This in turn, lowers the consumption of costly enzymes. Asanomi
et al. (2011) reviewed and summarized principles and advantages/disadvantages of
various methods for immobilization of enzyme in a microchannel including particle
entrapment, adsorption, and chemical crosslinking. The immobilized enzyme may
also be reused to react with substrate at different concentrations in subsequent flow
analyses. Apart from reusability, immobilization of enzyme can offer other benefits
(Miyazaki et al. 2008). By keeping enzyme molecules in place, aggregation can be
prevented which reduces the possibility of autolysis of some enzymes. Oriented
immobilization of the enzyme to the solid support can help orient the binding sites
outward for easy access by the substrate, and can also help to control and stabilize
enzyme conformation. This can be done, for example, through a biotin-avidin binding,
or through introduction of a functional group on the inner wall of the microchannel
for covalent crosslinking with enzyme (Honda et al. 2005).
Nevertheless, it is important to keep in mind that in heterogeneous phase, substrate
molecules in liquid phase have some mass transfer limitation in that they have to
diffuse in order to reach the binding sites of the immobilized enzyme. If the enzyme
turnover number is high, the rate of reaction between enzyme and substrate is faster
than the diffusion rate of that substrate, then concentration gradient or partitioning
of substrate will occur in the microporous environment. In other words, observed or
apparent kinetics of the immobilized enzyme are masked by diffusional effect and
may be distorted from the intrinsic kinetics in homogeneous phase. Therefore, an
alternative kinetic model such as the Lilly-Hornby model was used, instead of the
common Michaelis-Menten model, to evaluate apparent kinetic parameters (Lilly
et al. 1966, Seong et al. 2003). The mathematical model of the mass transfer limit
kinetics or diffusional masked kinetics usually yields Km and Vmax values different
from the normal system. According to Webster (1983), decreasing enzyme activity
within the unit volume, increasing substrate concentration, and decreasing pore size
of the support material can minimize or eliminate diffusional effect to obtain inherent
kinetics that is assumed to be the same as intrinsic kinetics and can be described
using the normal Michaelis-Menten kinetics model.
Pore size and geometry as well as electrostatic charge of the support surface
are important parameters (Webster 1983). Relatively small pore size, as compared
to the size of substrate, can cause limitation in rotation of the large size substrate
molecules and reduce the opportunity for proper interaction of substrate to the
binding site of the immobilized enzyme. Rotational masked kinetic will cause
kinetics distortion from the intrinsic condition, even with the absence of diffusional
or concentration gradient effect. Figure 7 depicts intrinsic vs inherent kinetics to
12 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 7. Different kinetic conditions a) intrinsic kinetics in homogeneous phase with no concentration
gradient and no rotational nor diffusional limitation. b) an open large pore size relative to the size of
substrate (P > W) yields inherent kinetics with no concentration gradient, and no rotational nor diffusion
limitation c) an open small pore size relative to the size of substrate (P < W) yields rotational masked
inherent kinetics d) a closed end large pore size relative to the size of substrate (P > W) yields diffusional
masked inherent kinetics e) a closed end small pore size relative to the size of substrate (P < W) yields
diffusional and rotational masked inherent kinetics. P is pore diameter. W is size of substrate molecule. S
is substrate. E is enzyme. X shown on dotted arrows indicates the binding that cannot happen.

show the need of suitable geometry and pore size large enough for free rotation of
substrate molecules in order to prevent concentration gradient, and rotational and
diffusional masking.
To improve the efficiency of the packed-bed column, a single piece of monolith
material has been used in place of small beads (He et al. 2010). Monolith material
contains interconnected pore network that allows a more free flow of solution, with
lower back pressure and better mass transfer. One last point to ponder when using
immobilized enzyme reactor for kinetic study, reusing of enzyme may be affected
by memory effect that causes deactivation or interferences from the previous
run. Therefore, cleaning prior to the next analysis cycle is important to take care
of the interferences due to the presence of unreacted substrate/product, etc., from
the previous run. As the enzyme is generally used in excess, effect due to partial
deactivation is generally insignificant.

Droplets based microfluidics

The demand for high throughput analysis has driven the development of microfluidic
systems even further. Similar to using the conventional 96 well-plate with multi-
tip micropipettes to carry out multiple reactions all at once in batch-wise process,
multiple analyses in a microfluidic system has been a big goal. Droplet based
microfluidics is the system that generates aqueous micro-droplets (dispersed phase)
in an immiscible liquid (continuous phase). The droplet serves as an individual reactor
where the chemical reaction takes place. Multiple droplets can be produced within
a microchannel to accommodate and compare various reaction conditions, similar
to the capability of a micro-well plate, but in a more automatic flow based system.
Segmented flow, where slugs of liquid are separated by air segments, serve a similar
purpose. However, the droplets platform is different in that the liquids in the droplet
are encapsulated and protected from direct contact with the wall of the microchannel,
whereas the slugs of liquids in a segmented flow system are exposed to the solid
wall. Encapsulation of liquids inside the droplet and the immiscible nature of the
carrier solution (continuous phase) will also prevent the reagents inside the droplets
from dispersing and diffusing into the carrier steam. Minimizing loss of enzyme
 Uses of Flow‑based Systems for Enzyme Kinetics 13

Figure 8. Some common droplets introduction designs a) Y-junction, b) T-junction, c) cross-flow

focusing, and d) parallel flow focusing. D is width of orifice, and L is length of orifice.

or substrate can better ensure accuracy and precision of the kinetic measurements.
Although many varieties of microchannel designs are possible, depending on
the particular application, all usually incorporate some common inlet designs for
generating droplets, namely the two inlets Y- and T-junctions, and the three inlets
flow focusing in either cross junction or co-flow formats, see Fig. 8. Please note that
the dispersed phase may be originating from two or more reagents (e.g., enzyme
and substrate) merged together before intersecting with the continuous phase. The
components in the dispersed phase may not completely mix when forming a droplet.
The single shading shown in Fig. 8 is for simplicity in presentation, not necessarily
representing a homogeneous mixture. For a complete reaction, various ways of
droplet operations such as fusion, fission, and merging can be done as reviewed by
Sohrabi et al. (2020).
The Y- and T-junctions, Figs. 8a and 8b, are popular due to the simplicity of the
designs and fabrication. The critical region is the junction where the dispersed phase
is pinched off due to the pressure and the stress applied when intersecting with the
continuous phase. Droplet size, shape, and frequency of production can be varied
by changing the geometry of the channel, i.e., height to width ratio of the channels
(Wehking et al. 2014, Garstecki et al. 2006). Steegmans et al. (2009a, 2009b, and
2010) explained that droplet formation and detachment at a Y-junction occurs in one
step by shear force between the two phases. In contrast, the process is two steps in
T-junction configuration, where a droplet is formed by inertial forces of the dispersed
phase before being detached by the dominant pressure of the continuous phase that
overcomes the interfacial tension of the dispersed phase. Ushikubo et al. (2014)
also found that droplet size depends mainly on viscosity and relative velocity of
the two liquid phases, while interfacial forces is insignificant. They compared the
Y- and T-junction performances on droplet production and found that the Y-junction
is suitable for the high viscous continuous phase fluids, while T-junction is more
feasible as it can be used with all types of fluids. T-junction is also reported to offer
14 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

more uniform size droplets and its performance does not depend much on conditions
such as velocity or flow rate.
In the flow-focusing configuration, unlike the Y- and T-junctions, shear force
exerted by the continuous phase on the dispersed phase is symmetrical. Therefore,
the generated droplets by the flow-focusing method are rather stable. The angle at
the junction affects the hydrodynamic force in and around the region and changes
characteristics of the droplets (Yu et al. 2019). In the cross junction design,
Fig. 8c, the continuous phase flows in and focuses on the dispersed phase equally
from both sides. The sum of vectors results in the droplet flowing forward. Another
flow-focusing design, Fig. 8d, employs a narrow tubing (e.g., capillary) as a sub-
channel to force the dispersed phase into the continuous phase flowing inside the main
channel. Initially, the flows of both phases are parallel. The pressure increases at the
narrow orifice and it overcomes surface tension of the dispersed phase, subsequently
forcing droplets to form and detach (Nooranidoost et al. 2016). Physical parameters,
e.g., droplet size and dispersion, impact directly the quality of the analysis results,
e.g., throughput, dynamic working range, and accuracy (Rosenfeld et al. 2014). It
has been demonstrated that the droplet size is directly related to orifice width and
the distance of the orifice from the inlet, while it conversely relates to the orifice
length up to a certain limit (Gupta et al. 2014). Apart from the sizes of the channels
and orifice, the sizes of the droplets can be controlled by changing the flow rate
of the continuous phase. It was found that droplet size is decreased by increasing
flow rate of the continuous phase, or by using a lower ratio of dispersed flow rate to
continuous flow rate (Nooranidoost and Kuma 2019).
Droplet-based microfluidics has become an attractive method for small volume
enzyme kinetics because of important benefits including rapidity, biocompatible
interfacial chemistry, and a dispersion-free system. Enzyme kinetics in the droplet-
based platform can be carried out, for example, by introducing enzyme and substrate
as a combined small volume into a continuous flow of the carrier fluid. Immiscibility
of the droplets in the carrier helps to minimize dispersion and helps to retain
enzymatic reagents within the droplet. Adsorption of enzyme and substrate onto
the wall of the microchannel is also minimized. The mixing of content within the
droplet (e.g., enzyme and substrate) occurs through diffusion, and can be enhanced
with chaotic advection by using a winding microchannel such as serpentine design
(e.g., downstream portion of Fig. 6) to promote stretch and fold movement of the
liquid mixture (Song et al. 2006). The immiscible carrier fluid should be chosen
so as to prevent side reactions as well as dispersion or partitioning of the enzyme-
substrate products from the droplet. Aqueous phase enzyme-substrate droplets are
usually carried through the microfluidic system with a surfactant or oil based liquid.
Surfactants in the carrier fluid can help to lower the surface tension and adsorption
at the liquid-liquid interface between the dispersed and the carrier phases (Dickinson
1991).
Ochoa et al. (2020) described a step-by-step procedure to design and fabricate
a microfluidic chip for enzyme inhibition assay based on microfluidic droplets and
using fluorescent image analysis. Some examples are included here to demonstrate
 Uses of Flow‑based Systems for Enzyme Kinetics 15

how the droplet-based microfluidics system can mimic the batch-wise enzyme
kinetics study such as those carried out in a micro-well plate. Song et al. (2006)
also suggested that such systems as “preformed cartridge,” proposed by Zheng and
Ismagilov (2005), may be applied to identify the enzyme of a desired reactivity. In
this application, droplets (plugs) of various enzymes in the preformed cartridge, flow
and merge with a stream of a common substrate at a T-junction. The system should,
therefore, be applicable to study the kinetic of a particular enzyme. For example, the
preformed cartridge can be employed to contain a substrate of various concentrations
which is then merged with a constant concentration stream of an enzyme.
On-chip dilution has also been reported for creating different concentrations
of reagents. Song and Ismagilov (2003) utilized a three-inlet and flow of buffer in
between two reagents (e.g., enzyme and substrate) to prevent direct mixing while
varying the amount of reagents by controlling their flow rates. Later, Bui et al. (2011)
proposed an on-chip dilution based on diffusive mixing. A Y-junction is utilized
to produce laminar parallel flows of substrate and buffer solutions. Concentration
gradients of substrate were created through diffusive mixing while the two solutions
are flowing in parallel downstream, similar to the phenomenon shown in Fig. 6. The
gradient along the liquid flows can be in linear, parabolic, or exponential fashion,
depending on the concentration of the substrate, flow rate, viscosity, and channel
length (Jeon et al. 2000). In Bui’s work, the substrate linear gradient was merged with
a constant concentration enzyme before entering the T-junction where droplets of
enzyme-substrate were produced when intersecting with the immiscible continuous
phase of oil solution. Due to the concentration gradient of the substrate, each droplet
contains different substrate concentrations. Utilizing a multiple serpentine design to
accommodate numerous droplets, a high throughput analysis of various enzymatic
conditions can be carried out within a small device. Other methods for inducing
concentration gradients have been reported, but diffusive mixing with laminar flow
is the simplest and most stable.
Another example, by Jebrail and Wheeler (2010), demonstrates the use of
electroosmotic flow to control the droplets. Electrical potentials are applied to
an array of electrodes to control the mixing and movement of the droplets. The
proposed microfluidics include both two plate format where liquid and droplets
are accommodated in a closed microchannel, and one plate format where droplets
are placed on the open channel. A hydrophobic insulator coated onto the electrodes
isolated the droplets from direct contact with the electrode surfaces. Electrical charge
was created on either side of the insulator when electrical potential was applied to the
electrodes. These charges make it possible to manipulate liquid droplets to enter and
leave the system, as well as to merge, mix, and split them as desired.
The applications of the droplet-based microfluidics have also been expanded
to include encapsulation of rare cells or a single cell, e.g., bacteria, and DNA. This
technological progress allows for the detection of a single enzymatic event and the
screening of cells for a particular enzyme activity, which enables study of evolution
of cells (Agresti et al. 2010), e.g., evaluation of the activity of enzyme expressed
after repetitive cell mutation. Droplet fusion techniques (Joensson and Svahn 2012)
16 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

can be very useful in adding enzyme, substrate, or other necessary reagents to the
existing droplets that contain cells. Due to the large surface area to volume ratio
of the micro-droplets, characteristics of the interface between the droplet’s surface
and the continuous phase (water/oil) may have great influence on the content inside
the droplet. Therefore, compositions of aqueous and oil phases should be selected
so as to minimize adverse phenomenon, e.g., nonspecific protein adsorption at the
interface which can interfere with the enzymatic assay (Roach et al. 2005).

Detection
Image analysis based on fluorescence is a popular detection method in a microfluidic
system while the UV-Visible region is not as widely used because of limited choices
of the materials for microfluidics fabrication that do not absorb light in the range of
UV-Visible detection wavelengths (Urban et al. 2006). By comparing fluorescence
signals from images taken at the inlet and the outlet, rate of the enzymatic reaction
can be determined. Fluorophores are usually attached to either the natural substrates
(Joensson and Svahn 2012) or the enzyme (Girault et al. 2018). The fluorophores
remain quenched until being released when substrates bind to the enzyme, as illustrated
in Fig. 9, in which fluorescence intensity indicates the extent of enzymatic activity.
In the droplet-based microfluidics, the observed fluorescence is usually the average
signal of many droplets travelling through the detector during the particular set
time duration, e.g., every 30s. However, the averaged responses cannot differentiate
activity in each single droplet (Yin and Marshall 2012). In order to study enzyme at a
single cell level, various attempts have been made for sorting of droplets (Beneyton
et al. 2016, Vallejo et al. 2019), commonly known as Fluorescence Activated Droplets
Sorting (FADS). For example, Vallejo et al. (2019) developed a microfluidic system
to discover new enzyme variants of particular properties. Droplets containing single
cell and substrate were collected for incubation, prior to being excited by a laser. The
droplets that emit fluorescence upon exposure to the laser indicate the presence of
the preferred enzyme variant. The emitted light activated the electrode, forcing the
droplet containing the desired cell to move into a certain outlet.
Ultrahigh throughput analysis can be performed by designing a multi-
microchannel to accommodate multi enzymatic reactions in parallel, and using
more than one fluorogenic substrate. Example reports include screening for the
particular cells that contain enzyme of specific characteristics of interest, e.g.,

Figure 9. Illustration of the use of fluorogenic substrate as a tool to detect enzymatic activity.
 Uses of Flow‑based Systems for Enzyme Kinetics 17

with enantiospecificity, chemospecificity, and regiospecificity (Ma et al. 2018).

Separate lasers are used to excite droplets in each microchannel to generate different
emission wavelengths and therefore, avoid crosstalk of two fluorescence signals.
This setup can also be applied to evaluate enzymatic activities toward two substrates
simultaneously.

Paper based microfluidic devices

Despite the growth of microfluidics research, the fabrication process and related
costs remain the important challenges, especially in places with limited resources
(Ding et al. 2020). Even with advancement of technology, producing a polymer-
based and glass-based microfluidic or Lab-on-Chip system is not a simple task and
still requires well-trained personnel and some high cost equipment and/or reagents.
Microfluidic paper based analytical devices (mPADs) have been gaining interest as
simpler and more economic disposable microfluidic analysis systems (Noviana et al.
2021, Nishat et al. 2021).
The low cost, low nonspecific binding, and availability of paper (mostly cellulose
or nitrocellulose) in a wide variety of thicknesses, porosities, pore sizes, and wicking
properties make mPADs an attractive microfluidic platform. In addition, liquid
absorption and capillary action allows for liquid flow on mPADs without the need for
external power or pumps. Many reports have demonstrated simple yet effective means
of creating microchannel designs with hydrophobic and hydrophilic zones through
either physical patterning (e.g., laser engraving, and etching) or chemical patterning
with hydrophobic inks (e.g., photolithography, wax printing, inkjet printing, and
stamping). Nishat et al. (2021) has reviewed, summarized, and compared the pros
and cons of some of these patterning methods, as well as the equipment needed.
Two simple patterning methods, namely laser engraver and embedded crayon wax,
are presented here as examples of physical and chemical patterning. As shown in
Fig. 10, hydrophobic area can be easily created by manually applying crayon wax,
similar to wax printing using a printer. Then, the paper was heated faced down on a

Figure 10. Waxed paper was created by applying crayon wax onto one side of a piece of filter paper. a)
Heating of the waxed paper prevents liquid from leaking on both the front and back sides of the paper, and
b) Non-heated waxed paper has the problem of liquid leaking from the hydrophilic region on the back side
of the paper due to the lack of hydrophobic wax (modified from Nzobigeza and Hartwell 2019).
18 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 11. Creating a microfluidic pattern using a laser engraver. A piece of filter paper is taped onto
aluminum foil using double sided tape. A laser engraver is used to cut the design imported from Corel
Draw, creating a hydrophilic pattern, with the aluminum foil acting as hydrophobic barrier (modified from
DeMott and Hartwell 2020).

hot plate. Heating is necessary for complete absorption of wax into the paper and to
create a hydrophobic barrier on both sides of the paper. Without heating, liquid can
leak out of the hydrophilic area on the back side which has not been directly coated
with hydrophobic material. Adding a graphite barrier around the pattern can also
better define the hydrophobic-hydrophilic boundary (Whitford et al. 2020). In order
to create a more precise or complicated design, equipment with higher precision such
as a laser engraver may be used. A common 60 W laser can cut through paper, but not
the aluminum foil backing (Mahmud et al. 2016). As shown in Fig. 11, by taping the
paper onto aluminum foil, the hydrophobic-hydrophilic pattern can be created and be
kept intact. If backing is not needed, removable double sided tape will allow for easy
detachment of the aluminum foil backing.
Despite many benefits of the mPADs, some drawbacks have been reported
(Carrell et al. 2019), especially regarding poor detection limit, short shelf-life of
the devices with pre-immobilized reagents, e.g., enzyme, and low reproducibility
of the reaction. The main way to improve detection limit has been accomplished
by integrating sensitive detection methods such as florescence, chemiluminescence,
or electrochemical detection onto the mPADs. As mentioned earlier, a fluorescence
substance is quenched until being released due to enzymatic reaction. As for
chemiluminescence, the reaction is usually initiated by the oxidizing agent in the
presence of a catalyst such as enzyme or metal ion (Li et al. 2019). The main advantage
of chemiluminescence over the fluorescence detection method is owing to its
simplicity with no requirement of excitation source. In addition, chemiluminescence
offers a wide calibration range and low background signal. Nevertheless, most
commercially available paper based biosensors utilize electrochemical detection
with electrodes screen printed onto the paper because of their convenient mass
production, cost-effectiveness, and ease of use (Caratelli et al. 2020).
Although enzymes can be immobilized on paper materials and Vmax and Km can
be found similar to those of free enzymes, researchers have reported that enzymes
tend to denature when being deposited and left to dry on paper for extended periods.
Shelf-life of biological materials can be increased by refrigeration. However, while
Ilacas and Gomez (2019) demonstrated successful storage of enzyme for 30 days
at low temperature, many works report lowered or inhibition of enzyme activities
 Uses of Flow‑based Systems for Enzyme Kinetics 19

on paper as compared to bulk solution. Mitchell et al. (2015) proposed a solvent-

free reagent deposition method by transforming reagents into a solid form known as
reagent pencil, in which the reagent to be deposited on the mPADs (e.g., enzyme) is
mixed with polymer (e.g., polyethyleneglycol), graphite powder, water or acetone,
and is pressed into the shape of a pencil core. By simply drawing onto the mPADs,
enzymes such as glucose oxidase and horseradish peroxidase can be stabilized in
this solid form at ambient conditions for more than 2 months. The method was
demonstrated to work well with reagents with molecular weight in the range of
2000–6000 g/mol (Liu et al. 2017). The main challenge is the reproducibility of the
amount of reagent being applied which depends largely on the pressure put on during
application.
As discussed by Carrell et al. (2019), liquid flow and mixing on the paper surface
is mainly due to capillary action which is highly dependent on the physical texture
of the paper and often yields non-uniform and irreproducible mixing. Therefore,
miscible liquids cannot be assumed to achieve complete mixing. The non-uniform
distributions of liquid with aggregation of solutes around the outer edges of the
test zones as solutions dry out on paper, known as coffee ring effect (Nishat et al.
2021), is another common problem affecting accuracy and precision of mPADs.
Sedighi and Krull (2018) reported that pore size proportionally affects enzyme
amplification in solution-phase where the smaller the pore size, the lower the
amplification. On the other hand, the enzyme amplification increases with the
smaller pore size in the surface-phase reaction where reagents were immobilized
on the paper surface. Caratelli et al. (2020) developed a 3 layer electrochemical
paper based microfluidic device for detection of enzymatic byproduct, in order to
evaluate efficacy of drugs for treating Alzheimer’s through an enzyme inhibition. It
was pointed out that layers of paper origami mPADs act as diffusional barriers and
cause the Km value of the enzyme to be higher than the value found in bulk solution.
This does not mean that the device cannot be used. It merely means that, no matter
which enzymatic reactions and analysis methods are used, comparison of enzyme
activities should be done using the same system (Razak et al. 2020). In order to
improve reproducibility of the chemical reaction on mPADs, arrangement/design of
the paper based device to enhance diffusive mixing of liquids (Osborn et al. 2010),
as well as the application of an external force such as an acoustic wave (Rezk et al.
2012), have been reported.

Examples of flow based enzyme kinetics

Flow based techniques have been applied to countless chemical/biochemical
analyses involving enzymatic reactions. Although most works may not directly use
flow based techniques for studying enzyme’s kinetics, the ability of these techniques
to detect products of enzymatic reactions ensures that these flow based techniques
can be used for kinetic study. Table 2 presents some selected examples of research
related to employment of flow based systems to determine enzyme activity and/or
kinetic parameters.
20 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Table 2. Examples of flow based systems for determination of activities, kinetic parameters of enzymes,
and their applications.

Flow based system, Enzyme Application remarks

References
FIA, β-galactosidase On-line monitoring of intracellular enzyme
Kracke-Helm et al. 1991 production during cultivation of recombinant
E. coli.
FIA, Phospholipase D Investigation of enzyme characteristics
Becker et al. 1997 (PLDs, EC 3.1.4.4) (Km, Vmax, and temperature dependence) with
chemiluminescence detection of enzymatic
product.
Stopped-flow FIA, Penicillinase Evaluation of penicillin sensor through
Yerian et al. 1988 characteristics of immobilized enzyme on
cellulose (stability, response to different
substrate, kinetic response, and effect of buffer)
with flow Injection optosensing system.
SIA, β-Glucosidase Determination of kinetic constants and
Cedillo-Perez et al. 2017 behavior of free (homogeneous media) and
immobilized enzyme on controlled pore glass
(heterogeneous media).
SIA, β-Glucosidase Kinetic study of enzyme activity in sodium
Pinto et al. 2012 dodecylsulfate (SDS)/ionic liquid (IL) mixed
micelles to investigate the influence of these
potential reaction media for transglycosylation
reactions.
SIA-LOV, Cyclooxygenase Automatic evaluation of cyclooxygenase 2
Pereira et al. 2021 inhibition induced by metal-based anticancer
compounds.
Microfluidics, Horseradish peroxidase Vibrating sharp-tip mixing of multiple streams
Li et al. 2021 of fluids for kinetic study, claimed to be the
highest performance mixer to date in 3D
printed micro-devices.
Microfluidics, thermophilic Comparison of kinetic parameters and
Thomsen and Nidetzky β-glycoside hydrolase activation energy of the free and immobilized
2008 enzymes. Microreactor is made of poly(methyl
methacrylate) and poly(dimethyl-siloxane)
(PMMA/PDMS). Enzyme immobilization is
via cross-linking with glutardialdehyde onto the
amino-silanized microstructured surface.
Droplets microfluidics, Glucose oxidase System is composed of PDMS microchannel
Hassan et al. 2016 with T-junction for droplets generation, and
3D printed flow cell for optical detection.
Kinetic parameters were obtained by probing
droplets at multiple points over time, with an
example application of continuous glucose
measurement.
Table 2 contd. ...
 Uses of Flow‑based Systems for Enzyme Kinetics 21

...Table 2 contd.

Flow based system, Enzyme Application remarks

References
Droplets microfluidics, P. aeruginosa aryl Fabrication of PDMS microfluidic system
Ochoa et al. 2020 sulfatase using soft lithography method with fluorescent
image analysis detection. Screening of potential
inhibitor compounds by incorporating a
potential inhibitor together with the enzyme
and substrate inside the droplets generated at
T-junction.
Functional paper-based Glucose oxidase and Development of a computational model,
microfluidics Horseradish peroxidase depicting flow behavior in two different
Kim 2016 connected wicking materials, paper and
hydrogel. The reaction kinetics of free enzymes
and those entrapped in the polyacrylamide gel
were compared.
Electrochemical paper β-galactosidase Microwire electrodes were embedded within
based microfluidics the analysis channel between two paper layers.
Adkins et al. 2016 Measured enzyme kinetics were compared to
those found in the literature.

Future trends
Microfluidics have progressed toward nanofluidic systems (van den Berg et al. 2010,
Chen et al, 2021). This lower volume manipulation would require a precise reagent
delivery system, and a sensitive detection method. Reduction of cost by using
cheaper materials that enable the possibility of mass production of the microfluidic/
nanofluidic systems are also of interest. For portable and field analyses, reducing the
number of fresh reagents would be of a great benefit. Therefore, long-term and stable
storage of the device with pre-immobilized reagents is necessary, and reusability
would be a plus. In many cases, the design of a device that can detect multiple
analytes is also desirable. Finally, bio-compatible devices that would not cause after-
use negative impact on health and environment would be welcome.

References
Adkins, J.A., E. Noviana and C.S. Henry. 2016. Development of a quasi-steady flow electrochemical
paper-based analytical device. Anal. Chem. 88: 10639−10647.
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Chapter 2
Chemoproteomics
An Extremely Powerful Kit
in Drug Discovery Toolbox
Anupama Binoy,1,# Revathy Sahadevan1,# and
Sushabhan Sadhukhan1,2,*

Introduction
The human genome project (HGP) has revolutionized the scientific world by
providing a blueprint of genes encoding the entire human proteome and one cannot
overstate its importance for modern pharmacology. This allowed us to understand the
information present on approximately 20,000 protein-coding genes (Legrain et al.
2011). However, there is a huge knowledge gap on various functional and redundant
proteins in the human proteome, their cellular localization, and distribution, their
abundance as well as their interaction with small molecules. Additionally, the existence
of splice variants and the post-translational modifications (PTMs) make the human
proteome more complicated than it seems. In 2009, the human proteome project
was launched to prepare a human proteome map with a gene-centric approach (A
Gene-Centric Human Proteome Project: HUPO--the Human Proteome Organization,
2010). The intention was to generate publicly accessible information resources for
further exploration by the scientific community. The word ‘proteome’ was coined
quite early by Mark Wilkins in 1994 (Wilkins and Gooley 1997) describing it as the

1
Department of Chemistry, Physical & Chemical Biology Laboratory, Indian Institute of Technology
Palakkad, Kerala 678 623, India.
2
Department of Biological Sciences & Engineering, Indian Institute of Technology Palakkad, Kerala 678
623, India.
Emails: [email protected]; [email protected]
* Corresponding authors: [email protected]
# Authors contributed equally.
 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 27

study of a whole set of proteins present in a whole organism, or a cell or a tissue,

and also encompassing the modifications that happen on the proteins due to several
signaling and environmental factors. Over the years, proteomics has played vital
roles in drug discovery processes, particularly in understanding the mechanism of
action of a given molecule of interest such as drugs or inhibitors. It has enabled
identification of biomarkers specific to the disease which is useful in early detection,
and prognosis as well as in monitoring the progression of several diseases (Aslam
et al. 2017).
However, along with the launch of the human proteome project, the limelight
has been shifted towards the emergence of chemoproteomics and probe-based tools
for identifying the global interacting proteome set in an organism. This also brings in
a tremendous opportunity for the scientific world for the discovery of new and more
target-specific approaches to drug discovery. Chemoproteomics presents a robust
platform for the discovery of new first-in-class medicine. It is a rapidly growing
field in chemical biology with an arsenal of techniques to understand protein-
small molecule interactions. It has paved the way for a proteome-wide evaluation
of identifying the targets by characterizing interactions between small molecules
and their protein targets (Spradlin et al. 2021). This technology includes a growing
set of techniques like activity-based protein profiling (ABPP) (Wang et al. 2018)
and targeted protein degradation using proteolysis-targeting chimeras (PROTACs)
(Belcher et al. 2021, Qi et al. 2021).
Chemoproteomics provides a strong foundation for studying the interaction
between small molecules and proteins, often with the help of some chemical probes
that are synthesized based on the structure of the molecules of interest. These
probes are also known as bioorthogonal probes as they function without interfering
with the native physiology of the living system when used in the context of in
vivo conditions. They are bolstered with a functional group to label or pulldown
substrate proteins it interacts with which is most of the time the proteins of interest.
They enable probing through selective covalent bond formations with the proteins
(often in the active site). As some of the classic examples, chemoproteomics has
been successfully applied to elucidate the binding partners of Class I and II HDACs
in the native cellular state (Salisbury and Cravatt 2007), identify in situ molecular
targets of polyphenols (Wang et al. 2016), study various protein PTMs (Erkan et al.
2020), etc. Some of these are discussed in detail in the respective sections below.
Identifying the protein targets through chemoproteomics also has its limitations as
sometimes the structural modification of the target leads to perturbation in molecular
interactions and biological functions (Piazza et al. 2020). Here, in this chapter, we
will focus on chemical reactions for the development of chemoproteomics tools and
the applications of the same in the field of biotechnology; in particular the drug
discovery and identification of disease-associated biomarkers.

Structural elements in the probes used in chemoproteomics

Chemoproteomics use small molecule probes to profile proteins/enzymes based
on their activity within the native cellular environment. These probes are often
28 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

referred to as activity-based probes (ABP) (Berger et al. 2004) and are designed
to be bioorthogonal in nature, i.e., they should act without interfering with the
native physiology of live cells (Chan et al. 2021). It is generally comprised of three
components, (i) a warhead or reactive group, (ii) a reporter tag, and (iii) a linker
connecting the first two, i.e., the warhead and the reporter tag (Fig. 1). A reactive
group (also known as warhead) is designed to enable the covalent interaction between
the small molecule and its target protein. It can be the entire molecule of interest
itself or a part of it with the desired functional group which can selectively interact
with amino acids in the active site of enzymes. Several reactive functional groups
such as sulphonate ester (Adam et al. 2001, Adam et al. 2002a), epoxide (Greenbaum
et al. 2000, Chen et al. 2003, Marques et al. 2017), fluorophosphonate (Liu et al.
1999, Field et al. 2020, Faucher et al. 2020), peptide (acyloxy)methyl ketone (Sexton
et al. 2006, van de Plassche et al. 2020), etc., have been widely used to design and
synthesize reactive warheads. A reporter tag is used to visualize, pulldown, or enrich
the target protein for identification and further validation studies. Fluorescent or
radiolabeled tags are generally used for visualization, and biotin tags are used for the
purification and mass spectrometric identification of target proteins. A linker group is
introduced to connect the reporter tag with the reactive warhead. They are designed
to control the steric hindrances and specificity of the attached groups (Meng et al.
2017). Linkers can be long alkyl chains, or poly ethylene glycol (PEG), or short
peptides. To study the small molecule-protein non-covalent interactions, photo-cross
linkers such as diazirine, benzophenone groups, etc., have been introduced to the
linker region of the probe (Guo and Li 2017). Upon photo-irradiation, these photo-
crosslinkers yield reactive carbenes which are capable of capturing non-covalently
interacting proteins or adjacent proteins that reside near the active site of the target
protein. This in turn helps in the identification of the mode of action of the small
molecules by elucidating if the small molecule is working through any off-targets.
There exists another type of probe which has been designed by introducing a terminal
alkyne or azide functional group to the molecule of interest. These kinds of probes
exert the least steric hindrance in terms of their interaction with bio-macromolecules.
The reporter tags can be introduced in the downstream process via alkyne-azide
Click reactions with an alkyne/azide derivative of fluorescent counterpart or biotin
(Meldal et al. 2008).

Figure 1. Structural components of bioorthogonal probe routinely used in chemoproteomics.

 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 29

Importance of chemoproteomics in drug discovery

The discovery of new in-class active drugs for various existing and emerging diseases
has always been a challenging task due to the lack of sufficient knowledge on the
biological functions of protein(s) involved. The expression and function of proteins
inside the body are dependent on multiple factors including but not limited to post-
transcriptional processes, PTMs, cellular localization, signaling factors, protein
turnover, environmental cues, etc. The major problem encountered by the scientific
community is in determining the specific molecular and cellular functions of many
proteins encoded by the genome of an organism and that has posed a severe limitation
in the discovery of new medicines. Advancements in biochemical, molecular, and
cellular techniques have enabled the functional understanding of certain proteins
through in vitro experiments. However, the proteins will not function quite the same
in isolation as they do under physiological conditions. Further, in the era of genetic
methods dominion over pharmacology, genetic disruption of given protein resulted
in its altered expression for the rest of the organism’s life, and that held a major
limitation in its application in pharmaceutical sectors. The development of high-
throughput screening technologies has provided a platform to study the interaction
of proteins with millions of small molecules. Despite this, these advancements
were unable to bring the expected momentum in the discovery of new first-in-class
medicines. The screening of drugs is time-consuming, expensive, and encompasses
a lot of risk factors. The probability of a drug molecule entering the Phase 1 clinical
trials after a lot of strenuous preclinical testing is only around 10% (Dowden and
Munro 2019). The major reason for this failure lies in the limited drug efficacy,
on and off-targets (specific and non-specific targets), heterogeneity among various
diseases, and its impact on individuals per se. In addition, identifying the undruggable
proteome has remained the bottleneck in modern drug discovery as there is a lack of
knowledge about well-defined binding pockets for a large fraction of the proteome
and their interactions with small molecules (Dang et al. 2017). The aforementioned
issues could be resolved largely, if not fully, by exploiting chemoproteomics wherein
various probe molecules (of the ligand of interest) can be used in conjunction
with advanced mass spectrometry-based proteomics. The active site-directed
chemoproteomics strategy leverages the covalent attack mechanism used by most
of the active sites of enzymes to react with its substrate. The chemoproteomics tools
are synthesized by tagging a suicidal substrate which serves both as an inhibitor for
the respective enzyme as well as ABPs (Activity-based Probes). In addition to site-
directed proteome profiling, multiple enzyme classes can be targeted concurrently.
Chemoproteomics enables the identification of protein target and mechanism of
action of a small molecule simultaneously, unlike the typical way where the efficacy
of small molecules is tested first and their targets and mode of actions are studied
later.
30 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Chemoproteomics to identify molecular targets

of small molecules
A complex cellular system often makes it difficult to decipher the mechanism
of action of small molecules, their interaction with protein targets, and overall
pharmacological effects. This is actually the most critical step in the drug discovery
process, and chemoproteomics encompasses versatile tools for the identification
of molecular targets that are the key players behind the activity of a given small
molecule. The added advantage is that chemoproteomics does this in a very initial
stage of the drug development, which certainly helps in making the drug discovery
process more cost-effective. Identification of the therapeutic targets uses chemical
probes whose mechanism can be well explained through affinity and activity-based
chemoproteomics, and also with the recently developed label-free technologies
(Drewes and Knapp 2018).
The mechanism of action of a small molecule depends upon their interaction
(both covalent or non-covalent) with intracellular protein targets and this lays the
basis for the discovery of new drug products with effective pharmacological activity.
Therefore, target identification becomes a critical step in the development of new
therapeutics (Schenone et al. 2013, Comess et al. 2018). However, numerous studies
have time and again stated that most drugs interact with multiple proteins which
makes the identification of primary targets substantially more difficult. In the last two
decades, chemoproteomics has unraveled methods that can comprehensively reveal
multiple targets of small molecules. Several other methods also exist such as gene
expression signatures to connect the compound with its targets, chemical genomics
approaches, and yeast two-hybrid methods (Caligiuri et al. 2006, Hillenmeyer et al.
2008, Lamb et al. 2006, Zon and Peterson 2005). However, these strategies showed
multiple interferences, off-targets, and had narrow applicability.
Chemoproteomics recruits interdisciplinary approaches including chemical
synthesis, cellular and protein biology for comprehensively fishing out multiple
targets of small molecules and their enrichment, followed by mass spectrometric
analysis to identify the proteins of interest (Beroza et al. 2002). Drug discovery
process mostly uses chemoproteomics in two different ways: (i) compound centric
chemical proteomics (CCCP) which originates from the classical drug affinity
chromatography (where the molecule is immobilized on a matrix) followed by
proteomics analysis, and (ii) activity-based protein profiling where the parent
molecule of interest is modified into an activity-based probe with special attention
to its ability to enrich the protein targets by binding (Rix and Superti-Furga 2009).
In both methods, it is very important to retain the biological activity of the small
molecules of interest throughout the process. Here in this chapter, we will shed
light on routinely used chemoproteomics strategies, particularly in drug discovery
processes with some classical examples.

1. Immobilization‑based probe‑driven chemoproteomics:

This approach is based on the immobilization of the molecule of interest on inert
and biocompatible support resins such as agarose or other macroscopic beads.
 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 31

The enrichment of the target proteins is done through affinity chromatography.

Here, the compound is immobilized on solid support through a linker arm, and the
matrix is used to fish out the proteins interacting with the molecule of interest from
the cellular or tissue extract (Fig. 3). The recent advances in mass spectrometric
techniques in terms of sensitivity and high throughput nature have further enhanced
the power of immobilization-based probes for the identification of unrecognized
potential targets of small molecules (Godl et al. 2003). Immobilized affinity probes
were mostly utilized in identifying the specific cellular targets for kinase inhibitors.
Immobilization of an analog of SB203580 (Godl et al. 2003), a mitogen-activated
protein kinase p38 inhibitor on chromatographic beads led to the identification of
several unknown high-affinity targets such as cyclin G-associated kinase (GAK) and
casein kinase 1 (CK1) as well as RICK (Rip-like interacting caspase-like apoptosis-
regulatory protein kinase/Rip2/CARDIAK) apart from its known targets. SU6668, an
indolinone compound, which was initially known to selectively inhibit the receptor
tyrosine kinase involved in tumor angiogenesis was subjected to immobilization-
based chemoproteomics for the affinity capture of other potential cellular targets.
In that study, the researcher could identify previously unknown targets of SU6668
such as Aurora kinases and TANK-binding kinase 1 (Godl et al. 2005). Graves
et al. (2002), conducted a study to explain the mechanistic role of quinolines such
as 4-aminoquinoline chloroquine and the quinolinemethanol mefloquine in the
treatment of malaria. They screened several quinoline drugs against the purine
binding proteome based on their structural similarity by exploiting displacement
affinity chromatography using target capturing through gamma-phosphate-linked
ATP-Sepharose. Aldehyde dehydrogenase 1 (ALDH1) and quinone reductase
2 (QR2) were the only two human proteins specific to human red blood cell purine
binding proteome which were identified as potential targets of these quinoline
drugs (Graves et al. 2002). In another study, Schreiber’s group constructed a
FK-506 affinity matrix using its amino derivative to identify its target protein,
FK-506-binding protein (FKBP) with a cis-trans peptidyl-prolyl isomerase
activity (Harding et al. 1989). It was also observed that FK-506 (a macrolide) and
cyclosporine A (a cyclic undecapeptide), despite both serving as immunosuppressants
by inhibiting T-cell activation, had very different molecular targets. Cyclosporine A
targeted cyclosporin A-binding protein, and cyclophilin whereas FK-506 targeted
FK-506-binding protein (FKBP) (Siekierka et al. 1989a, Siekierka et al. 1989b).
Despite its significant contribution to the drug discovery process, it is important to
note that this method is applicable to cellular/tissue extracts and not directly to the
native cellular environment and that itself represents a major flaw of this strategy.
Another major limitation of immobilization-based chemical proteomics lies in the
steric hindrance which might influence the interaction between the true protein
targets and the interacting small molecule.

2. Activity‑based probe chemoproteomics:

To overcome the steric interference caused by immobilization-based
chemoproteomics, activity-based probes were conceptualized. Activity-based protein
32 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

profiling (ABPP) is based on the covalent interaction between small molecules (such
as inhibitors, ligands, drugs, etc.) and the reactive amino acid side chains present in
the active site of the enzymes; unlike other techniques which mainly focus on non-
covalent interactions. ABPP is mostly utilized under physiological conditions and it
potentially reflects true drug-target interactions taking place inside the native cellular
environment. In principle, the activity-based probes should not differ significantly
from the parent molecule in terms of its biological activities. As discussed in above,
there are two important components in an activity-based probe: (i) the reactive
group (in the compound of interest) for binding or covalently modifying the active
site of the target proteins and (ii) a reporter tag for the detection and enrichment
of the target proteins. Often, a linker is placed in between the molecule of interest
and the reporter group to avoid any possible steric hindrance. The probes are
incubated with the live cells and allowed to physiologically interact with the cellular
components before they are lysed, followed by an affinity enrichment of the target
protein using the reporter group. Biotin is the most frequently used reporter owing
to its strong affinity towards avidin, where enrichment is performed using either
avidin or streptavidin (an avidin analog with a more convenient binding constant
with biotin) beads. While, in the case of fluorescent reporters, efficient and rapid
detection, as well as visualization of proteins, can be done through an in-gel
fluorescence experiment (Fig. 3). ABPP probes have been utilized to study different
enzyme families like kinases (Yee et al. 2005, Liu et al. 2005), phosphatases (Kumar
et al. 2004), oxidoreductases (Adam et al. 2001, Adam et al. 2002b, 2002a),
glycosidases (Hekmat et al. 2005, Vocadlo and Bertozzi 2004), proteases (Liu et al.
1999, Kidd et al. 2001), etc. This strategy involved the covalent labeling of the active
site of enzymes in an activity-based manner which helped to distinguish between
functional enzymes from their inactive zymogens (Kidd et al. 2001, Liu et al. 1999,
Adam et al. 2001).
ABPP has also been successfully applied in identifying protein targets of various
natural products/drug molecules. For example, Yee et al. (2005) attached fluorescent
labels (BODIPY and rhodamine) to Wortmannin enabling the identification of its
binding partners from HEK 293T cell lysates. This led to the identification of PI-3
kinases and other associated proteins as the major targets of Wortmannin (Yee et al.
2005). With a similar strategy, Liu and co-workers identified other kinases such as
Polo-like kinase 1 and 3 (Plk1and Plk3) as the cellular targets of Wortmannin (Liu
et al. 2007). Klaic et al. (2012) developed an affinity probe of biotinylated celestrol
to elucidate its potential targets as annexin II, β-tubulin, and eukaryotic elongation
factor 1A (Klaic et al. 2012). Greenbaum et al. (2000), developed ABPP probes based
on epoxysuccinyl peptide with different reporter groups such as the radionuclide
125I, biotin, and the BODIPY (fluorophore). They were able to validate cathepsin
as the major target of the E-64, an inhibitor of cysteine protease (Greenbaum
et al. 2000). Cravatt’s group successfully profiled the global serine hydrolases in
the complex proteome by synthesizing a biotinylated fluorophosphonate (FP-biotin)
which showed affinity toward several members of the serine hydrolase family
(Liu et al. 1999). They also synthesized a variant of FP-biotin by replacing the
 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 33

hydrophobic alkyl chain linker with a more hydrophilic polyethylene glycol (PEG)
moiety to obtain FP-PEG-biotin. Both the probes showed a similar ability to fish out
multiple members of serine hydrolases like serine peptidases, lipases, and esterases
simultaneously (Kidd et al. 2001).

3. Click‑able probes:
The application of biotinylated probes poses problems due to their large sizes which
can interfere with the way they interact with the biomacromolecules as compared to
the parent analogs of the probes. Advancements in ABPP technology alleviated steric
hindrances associated with a bulky reporter like biotin or fluorescent tags by adapting
a two-step strategy that took advantage of bioorthogonal reactions. Bioorthogonal
reactions refer to the chemical reactions that can happen within the living system
without interfering its native biological processes (Sletten and Bertozzi 2009, Prescher
et al. 2004, Prescher and Bertozzi 2005). This method involves the functionalization
of the molecule of interest with a small functional group (such as a terminal alkyne
or azide) that does not interfere with the native physiological activities of a living
system, which ultimately can yield the profiling of enzyme activities in vitro as well
as in vivo systems (Fig. 3). As depicted in Fig. 2, the identification of target proteins
can be achieved by the downstream addition of reporter tags through bioorthogonal
reactions between the complements such as alkyne with an azide moiety (Vasilyeva
et al. 2016, Zhou and Fahrni 2004), alkene with tetrazine (Wu and Devaraj 2018), or
Staudinger ligation (Sletten and Bertozzi 2011, Bednarek et al. 2020).
Due to the minimal steric hindrance and bio-orthogonal nature of these
probes, they pose the least interferences to the pharmacological activity of the drug

Figure 2. Bioorthogonal reactions used in chemoproteomics. (A) Cu(I)-catalyzed alkyne-azide Click

reaction, (B) alkene-tetrazine reaction, and (C) Staudinger ligation.
34 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 3. Illustrated representation of various chemoproteomics-based tools and their mode of action for
identification and validation of protein targets.

molecules. Click chemistry is described as a group of chemical reactions with high

selectivity and high yield along with its ability to be conducted under simple reaction
conditions. Due to these unique features of Click chemistry, it has emerged as a
highly recommended tool in biomedical applications, especially in the field of drug
discovery (Hein et al. 2008). Several research groups have utilized Click chemistry-
based probes for the identification of potential protein targets of various natural
products (Gersch et al. 2012, Yang and Liu 2015, Ziegler et al. 2013, Wright and
Sieber 2016). For example, Bottcher and Sieber (2010) developed a showdomycin
probe by modifying its 5’-OH group with an alkyne moiety. The probe resulted in
the detection of a series of essential enzymes which belongs to the oxidoreductase
and transferase families in different bacterial systems as a target of showdomycin.
The identification of the molecular targets MurA1 and MurA2, which are essential
enzymes for bacterial cell-wall biosynthesis, was of particular interest in their study
(Böttcher and Sieber 2010). Qin et al. (2020) synthesized itaconate–alkyne (ITalk)
as a bioorthogonal probe for itaconate to quantify the site-specific interaction of
itaconate in a cellular extract of inflammatory macrophages (Qin et al. 2020).
Further to understand the functional role of itaconate in host-pathogen interaction,
they synthesized a series of itaconate-based bioorthogonal probes which enabled
quantitative and site-specific profiling of itaconate proteins in Salmonella. Through
this study, they were able to demonstrate that itaconate acts as a covalent inhibitor for
isocitrate lyase, a key enzyme involved in the glyoxylate cycle (Zhang et al. 2021).
Similarly, the development of bioorthogonal azide or alkyne probes for studying the
protein PTMs such as palmitoylation, and myristoylation has been well exploited
in the last decade by several groups (Hannoush and Arenas-Ramirez 2009, Charron
et al. 2009, Hang et al. 2007, Kostiuk et al. 2008, Martin and Cravatt 2009). An FDA-
approved anti-obesity drug, tetrahydrolipstatin (Orlistat) with a potential antitumor
ability was modified into a chemical probe by conjugating it with an alkyne moiety.
This alkyne-bearing analog of Orlistat was able to identify eight new targets apart
from the previously known fatty acid synthase enzyme (Yang et al. 2010).
 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 35

4. Photoaffinity labeling
It is relatively easier to identify the proteins which covalently interact with the small
molecules as the resulting complex is stable enough for downstream processing
and mass-spectrometric analysis. On the contrary, there exists a significant number
of biologically active molecules which interact non-covalently (via hydrophobic-
hydrophobic, van der Walls interaction, ionic interaction, or hydrogen bonding, etc.)
with the proteins and this results in very labile protein-small molecule complexes
which are prone to degradation as soon as cells are lysed. In this kind of situation,
photoaffinity labeling comes to the rescue. It is a unique and unbiased strategy that
utilizes photoactivation of the probe that is bearing a photo-crosslinker. These groups
remain inactive under normal chemical and biological conditions but get activated
when they are irradiated at a specific wavelength to yield highly reactive transient
species (e.g., carbenes) that crosslink the probe to its binding partners (Fig. 3). This is
followed by a Click reaction which enables the downstream enrichment of captured
proteins. Benzophenones, aryl azides, and alkyl or aryl diazirines are commonly used
photo crosslinkers in this context (Mishra et al. 2020). Hulce et al. (2013) utilized
photoaffinity probes for the global identification of cholesterol-binding proteins
(Hulce et al. 2013). They synthesized a set of structurally similar probes made of
sterol moiety connected to the photoactivatable diazirine group. HeLa cells were
incubated with this probe and further photoactivated through exposure to 365 nm
UV light to covalently cross-link the probe to its binding proteins. Later, Click reaction
with the biotin azide followed by streptavidin enrichment led to the identification of
approximately 250 cholesterol-binding proteins. Suberoylanilide hydroxamic acid
(SAHA) was the first histone deacetylase (HDAC) inhibitor to get the FDA approval.
Salisbury and Cravatt (2007) developed SAHA-BPyne as a probe for suberoylanilide
hydroxamic acid (SAHA), the first FDA-approved HDAC inhibitor to identify
the proteins that come together (non-covalently) to form the active complex for
histone deacetylases (HDACs). SAHA was modified using benzophenone moiety
(a photo-crosslinker) to covalently capture the HDAC-associated proteins like
MTA2, CoREST, and methyl CpG binding protein 3 (MBD3) (Salisbury and Cravatt
2007).

Applications of chemoproteomics for small molecule library

screening, biomarker discovery and in vivo imaging of enzymes
Chemoproteomics has also been explored to screen libraries of small molecule
inhibitors in a high-throughput manner. Previously, we have discussed different
probe-based identification of protein targets of small molecules where the probes are
designed to target a specific class of enzymes via installing known functional groups
and/or binding groups into their structures. These directed probes can covalently
modify the enzyme active sites and target a large set of closely related enzymes with
multiple isoforms (Cravatt et al. 2008, Nomura et al. 2010). It is widely used in drug
screening to identify the selectivity of a particular target (Fig. 4A). On the other
hand, in non-directed ABPP method, the action of an inhibitor can be monitored
 36 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

Figure 4. Schematic representation of application of ABPP in drug screening. (A) Direct labeling of
proteins with probes to identify the targets, (B) Competitive labeling and (C) Screening of library of
inhibitors for their specificity.

ofviaproteins with probes

chemoproteomics to identify
and identify theproteins
the target targets,(Fig.
(B)4B), or a combinatorial
library of probes can also be synthesized and screened against many enzymes to
identify specific
of library protein labeling
of inhibitors events
for their happening in cell (Fig. 4C) (Saghatelian and
specificity.
Cravatt 2005). This method has been proven to be effective as an important tool in
the discovery of both reversible and irreversible inhibitors.

1. Screening of small molecule library for drug discovery

The advantages of using chemoproteomics (using activity-based probes) in drug
discovery is two-fold. One that it can evaluate multiple enzymes at once in their
very native environment and second, it needs minimum structural information about
the target proteins to do so. On a similar note, inhibitors can also be developed
 Chemoproteomics: An Extremely Powerful Kit in Drug Discovery Toolbox 37

for the enzymes (orphan enzymes) that lack any information about their activities
or substrates. Selective inhibitors can be readily identified from the non-specific
inhibitors and this is particularly of immense importance for the ones that belong to
a large family of enzymes having multiple isoforms such as kinases or hydrolases.
Greenbaum et al. (2002) used this approach to identify the targets of cysteine
proteases using a library of irreversible inhibitors containing primary tripeptide
skeleton linked with an epoxide electrophile. The inhibitory action was visualized
with radiolabeled active site-directed probe, 125I-DCG-04 treatment for proteases
followed by SDS-PAGE and phosphorimaging. This method allowed them to
identify multiple targets of each inhibitor in a single gel-based assay without any
optimization of substrate and kinetic conditions (Greenbaum et al. 2002). A similar
strategy was used by Leung et al. (2003) to screen the serine hydrolase inhibitors
from a library of compounds containing electrophilic ketone group. The protein-
inhibitor complex was visualized through competitive assay with fluorophosphonate
rhodamine (FP-Rhodamine). They found that fatty acid amide hydrolase (FAAH),
an endocannabinoid-degrading enzyme and triacylglycerol hydrolase are the most
important targets of the serine hydrolase inhibitors (Leung et al. 2003). In another
study, Bonavia et al. (2011) found that carbamoyl-phosphate synthetase 2, and
aspartate transcarbamylase and dihydroorotase (CAD) complex are the targets of
isoxazole- pyrazole and their proline derivatives that are effective against respiratory
syncytial virus (Bonavia et al. 2011).

2. Identification of biomarkers for human diseases

The non-directed ABPP is also effective in identifying the biomarkers of various
human diseases (Jones and Neubert 2017). Biomarkers are measurable and
quantifiable molecular signatures of biological status that can serve as indicators
of diseases or targets of drugs in clinical diagnosis and therapy. Biomarkers can be
genes, proteins, or even small molecule metabolites. Among many tools to identify
the protein biomarkers, chemoproteomics stands out with the aid of strategically
designed molecular probes. These probes must be selective and sensitive to detect
a particular protein from a complex biological system (Zhang et al. 2017). For
example, a fluorescent fluorophosphonate chemical probe (FP-TAMRA) was used to
identify the enzymes plasmin and kallikrein as biomarkers of calcium, potassium, and
sodium homeostasis (Navarrete et al. 2013). In another study by Oikonomopoulou
and co-workers, a specific serine hydrolase, kallikrein-related peptidase 6 (KLK6)
was identified as a biomarker using a biotinylated probe containing a phosphonate
ester warhead in the ascites fluid from ovarian cancer patients (Oikonomopoulou
et al. 2008).

3. In vivo imaging of enzymes

In addition to PTMs and catalytic activities, the temporal expression and subcellular
distribution of proteins also play an important role in the function of that proteins.
Thus, visualizing the proteins in their native environment, i.e., their localization
inside the cell or a whole organism is of utmost significance and can reveal important
38 Some Key Topics in Chemistry and Biochemistry for Biotechnologists

information about their function. Among the many applications of chemoproteomics,

the most fascinating one is using these bioorthogonal probes for live imaging of
molecules in living systems such as live cells or whole organisms. For these
applications, the probe molecule requires certain properties such as it should be
biocompatible, cell-permeable, should not involve any toxic metal catalysts and
most importantly the labeling resulting from the protein-probe interaction should
be rapid (Lukinavicius et al. 2014). In this context, Pan et al. (2017) developed a
series of alkyne-diaziridine-containing probes, targeting bromo domain-containing
protein (BRD4) that recognizes acetylated lysine residues located on histones. BRD4
is an important enzyme involved in many cellular processes including mitosis,
angiogenesis, inflammation, etc. These probes were clicked in vivo with fluorophores
which allowed the visualization of BRD4 located throughout the nucleus of HepG2
cells (Pan et al. 2017). In another study to visualize cytoskeleton proteins tubulin
and actin, fluorogenic probes were developed namely SiR-tubulin and SiR-actin.
These probes were able to bind with the microtubules more efficiently as compared
to bovine serum albumin. The increased fluorescence intensities imparted by these
probes when bound to microtubules further justified the finding. These probes were
also efficient for live cell imaging (Lukinavičius et al. 2014). Sherratt et al. (2017)
replaced methionine of the culture media with homopropargylglycine, harboring
an alkyne functionality, which led them to perform bioorthogonal reactions with
fluorescent azide to visualize live bacteria. This allowed rapid screening and
identification of living pathogenic organisms (Sherratt et al. 2017).

Future of chemoproteomics and Conclusions

Preceding discussion clearly shows that chemoproteomics-based tools were mainly
designed to target enzymes that have defined catalytic sites. The widely targeted
enzyme superfamily belongs to kinases which hold special places in almost all
signaling pathways. But if we carefully look into the molecular and biochemical
pathways associated with diseases progression, the first set of protein family affected
in pathogenic conditions are membrane proteins like receptor proteins, ion channels,
etc. (Sigismund et al. 2018, Niemeyer et al. 2001). Therefore, we think that there
should be a paradigm shift for the application of chemoproteomics from enzymes
to other types of proteins such as ion channels, receptor, and transport proteins,
chaperons, etc., which play an instrumental role in the pathogenesis of several
dreadful diseases like cancers, viral infections like COVID 19, HIV, etc. Additionally,
it is very important to consider all the physiological conditions prevailing in a whole-
cell while trying to understand the function of a disease-associated protein target
or its interaction with any small molecule drug. To date, ABPs or chemoproteomic
tools have been synthesized mostly focusing on a single protein PTM at a time but
in the native physiological cellular environment, a particular function is modulated
or affected through heterologous modification and expression systems. Therefore, it
is very important to consider multiple protein modifications at the same time while
synthesizing a chemoproteomic probe to get a comprehensive understanding on the
PTM crosstalk. Currently, available chemoproteomic approaches seem to be limited
Another Random Scribd Document
with Unrelated Content
Vivie. Nee.

Mevr. Warren. Nee, dat weet je niet. Ik wel. Ze noemde d’r eigen een
weduwvrouw en ze had ’n winkel van gebakken visch ergens bij de Munt
en daar onderhield ze d’r eigen en d’r vier dochters van. Twee van ons ware
zusters, dat ware Lies en ik, en we zage d’r allebei goed uit, met knappe
figure. Ik vermoed, dat onze vader ’n goedgevoede man was. Moeder
beweerde, dat ’t een heer was, maar dà weet ik niet. De andere twee ware
maar halfzusters: kleine, leelijke, magere, slovende, eerlijke onderkruipsels.
Lies en ik zouen ze half vermoord hebben, als moeder òns niet half
vermoord had, om onze handen van ze af te houden. Zij waren de
fatsoenlijken van ons. Nou, weet je wat ze kregen voor d’r fatsoen? De eene
werkte in ’n loodwitfabriek,—12 uur daags voor 9 shilling in de week,
totdat ze stierf an loodwitvergiftiging. Ze dacht, dat ze alleen maar d’r
handen wat verlamd zou krijgen, maar ze ging er van dood. De ander werd
ons altijd voorgehouden als ’n voorbeeld, omdat ze met ’n werkman van de
rijkswerf trouwde en d’r kamer en d’r drie kinderen netjes en zuiver hield
van 18 shilling in de week, totdat hij aan de drank raakte. Dat was de
moeite waard om fatsoenlijk voor te zijn, niet?

Vivie (nu pensief-aandachtig). Dachten u en uw zuster dat?

Mevr. Warren. Lies niet, dat kan ik je vertellen, diè was wijzer. We gingen
allebei na ’n kerkelijke school,—dat hoorde zoo bij de damesachtige
manieren die we ons gaven om meer te zijn dan de kinderen, die niks wisten
en nergens heengingen,—en daar bleven we, totdat Lies eens op ’n nacht
wegliep en nooit terug kwam. Ik weet, dat de schooljuffrouw dacht, dat ik
wel gauw d’r voorbeeld zou volgen,—want de dominé waarschuwde me
aldoor, dat Lies zou eindigen met van Waterloo-brug af te springen.—Arme
hals,—dat was àl wat hij er van wist! Maar ik had meer angst voor de
loodwitfabriek dan voor de rivier, en dat zou jij ook gehad hebben in mijn
plaats.—De dominé wist ’n betrekking voor me te krijgen, as bijhulp in de
keuken van ’n afschaffersrestauratie, waar ze uitzonden met alles wat je
hebben wou.—Toen ben ik kellnerin geworden en daarna ging ik an ’t
buffet van ’t Waterloostation,—veertien uur per dag drank bedienen en
glazen omwasschen voor 4 shillings in de week en de kost. Dat werd toen
beschouwd as ’n groote vooruitgang voor me.—Nou, op ’n kouwe,
ellendige nacht, toen ik zòò moe was, dat ik nauwelijks wakker kon blijven,
wie denk je dat er binnen kwam voor ’n halve schotsche? Lizzie;—in ’n
lange, bonte mantel, elegant en lekker, met ’n hoop goudstukke in d’r beurs!

Vivie (grimmig). Tante Lizzie.

Mevr. Warren. Ja, en ’n beste tante òok om te hebben. Ze woont nou in

Winchester, vlak bij de kathedraal, een van de meest geziene dames dáar.—
Ze begeleidt jonge meisjes naar ’t bal van de gouverneur,—asjeblieft hoor!
Geen rivier voor Lies, dank je wel.—Jij doet me wat an Lies denken: ze
was ’n eerste zakevrouw,—spaarde geld op van de beginne af,—liet nooit te
veel zien wat ze was,—raakte nooit ’r hoofd kwijt, of liet ’n gelegenheid
voorbijgaan.—Toen ze zag, dat ik knap op zou groeien, zei ze tegen me,
zoo over de toonbank heen: “Wat doe jij hier, malle meid,—je gezondheid
en je uiterlijk verwoesten voor ’n andermans profijt?” Lies was toen an ’t
opsparen, om ’n huis voor d’r eigen te nemen, in Brussel, en ze dacht, dat
we samen gauwer zouen sparen, dan ieder voor zich. Daarom leende ze me
wat geld en hielp me aan de gang; en ik spaarde geregeld an en betaalde d’r
eerst af en begon toen ’n zaak met haàr als deelgenoot. Waarom zou ik ’t
nièt gedaan hebben? ’t Huis in Brussel was er een van de eerste rang,—’n
heel wat beter plaats voor ’n vrouw, dan de fabriek, waar Annemie
vergiftigd werd. Geen één van onze meisjes werd ooit behandeld zoo als ik
behandeld werd in de bijkeuken van die afschaffersboel, of as an ’t buffet,
—of as thuis: Had je gewild dat ik daar was gebleven en ’n afgewerkte
ouwe sloof was geworden vòòr m’n 40ste jaar?

Vivie (nu geweldig geïnteresseerd). Nee, maar waarom koos u diè zaak.
Met spaarzaamheid en goed beheer kun je elke zaak er boven op werken.

Mevr. Warren. Ja, geld opsparen. Maar in welke zaak kàn ’n vrouw geld
opspare? Zou jij kenne sparen van 4 shillings in de week en je d’r van
kleeje ook? Jij niet. Natuurlijk, as je ’n dood gewoon mensch ben en niks
anders kan verdienen, of as je idee heb in muziek of ’t tooneel, of
krantegeschrijf,—dan is ’t iets anders. Maar zoomin Lies as ik hadden eenig
benul van die dingen; alles wat wij hadden was ons uiterlijk en onze slag
om de mannen in te pakken. Denk je, dat wij zulke gekken waren, om
andere menschen zaken te laten doen met ònze mooie oogen, door òns te
gebruiken as winkelmeisjes, of buffetjuffrouwen, of kellnerinnen, as wij
zèlf er zaken mee konden doen en al ’t profijt in ònze zak steken, inplaats
van hongerloonen? Wìj niet, hoor.

Vivie. U was zeker volkomen gerechtvaardigd uit ’n zakenoogpunt.

Mevr. Warren. Ja, en uit ieder ànder oogpunt ook. Waartoe wordt ieder
fatsoenlijk meisje anders grootgebracht als om ’n rijke man in te palmen en
het voordeel van z’n geld te hebben door hem te trouwen? Asof ’n
huwelijksceremonie eenig verschil maakt in het goeie of het slechte van de
zaak! O, de huichelarij van de wereld maakt me misselijk! Lies en ik
hadden te werke en te spare en te berekene net zoo goed als andere
menschen; anders zouen we nou even arm zijn als iedere nikswaardige
dronken doorbrengster van ’n vrouw, die denkt dat d’r goeie tijd altijd zal
duren (met groote energie). Ik veracht dat soort menschen;—ze hebben
geen karakter. En as d’r iets is, dat ik haat in ’n vrouw, dan is ’t gebrek an
karakter.

Vivie. Kom nou, moeder, wees ’ns eerlijk! Is ’t niet voor ’n deel wat je
noemt karakter in ’n vrouw, dat haar die afschuw moet geven van dèze
manier van geld verdienen.

Mevr. Warren. O natuurlijk. Iedereen vindt ’t onaangenaam om te moeten

werken en geld te verdienen, maar d’r is geen keus. Waarachtig, ik weet
wel, dat ik dikwijls genoeg meêlijen heb gehad met ’n arm meisje, dat moe
was en landerig, als ze probeeren moest ’n man te amuseeren, om wie ze
niks niemendal gaf—zoo’n halfdronken idioot, die denkt, dat ie zich
aangenaam maakt wanneer hij ’n vrouw plaagt en lastig valt en half doet
walgen, op ’n manier, die met geen gèld is goed te maken. Maar ze heeft die
onaangename dingen nou eenmaal te verdrage, en ’t zoete zoowel as ’t zure
te slikke, net even goed als ’n ziekenhuis-zuster of ieder ander. De hemel
mag wete, dat ’t geen werk is, dat eenige vrouw voor d’r plezier zou doen,
al zou je, as je de vromen hoort praten, denken dat ’t ’n bed van rozen was.
Vivie. U beschouwt het toch als de moeite waard. Het betaalt.

Mevr. Warren. Natuurlijk is ’t de moeite waard voor ’n arm meisje, dat d’r
eigen niet weggooit en er goed uitziet en zich verstandig en netjes gedraagt.
’t Is oneindig beter dan eenige andere betrekking, die ze hebben kan. Ik heb
altijd gevonden dat dat zoo niet moest zijn. ’t Kàn niet rechtvaardig zijn,
Vivie, dat ’n vrouw geen betere kansen zou hebben. Ik blijf er bij: dat is
verkeerd. Maar goed of verkeerd, ’t is eenmaal zoo, en ’n meisje moet ’t
neme zooas ’t is. Maar natuurlijk is ’t niet de moeite waard voor ’n dame.
Als jij die kant uitging, zou je dwaas zijn; maar ìk zou dwaas geweest zijn,
as ik ’t nièt had gedaan.

Vivie (meer en meer werkelijk ontroerd). Moeder, veronderstel eens, dat we

allebei zoo arm waren als ù was in die ellendige dagen van vroeger, bent u
zeker, dat u me dan niet raden zou om ’t stationsbuffet te probeeren, of om
’n werkman te trouwen, of om zelfs in ’n fabriek te gaan?

Mevr. Warren (verontwaardigd). Natuurlijk niet. Voor wat voor soort

moeder zie je me an! Hoe zou jij je zelfrespect kenne beware bij zòò ’n
hongerlijë en gesloof. En wat is ’n vrouw waard, wat is ’t lèven waard,
zonder zelfrespect? Waarom ben ik onafhankelijk en in staat om m’n
dochter ’n piekfijne opvoeding te geven, terwijl andere vrouwen, die net
dezelfde kansen hadden, nou in de goot legge? Omdat ik m’n eigen altijd
heb wete te respecteere en te beheersche. Waarom kijke ze op tegen Lies in
’n vrome stad? Om dezelfde reden. En waar zouden we nou an toe zijn, als
we ons gestoord hadden an de malligheid van die dominé? An vloeren
schrobben voor anderhalve shilling per dag en niks in ’t vooruitzicht as ’t
armhuis. Laat jij je niet van de wijs brengen door menschen die de wereld
niet kennen. De eenige manier voor ’n vrouw om fatsoenlijk voor d’r eigen
te zorgen is om goed te zijn voor ’n man, die ’t betalen kan om goed voor
haar te wezen. Als ze van zìjn stand is, laat ze dan zorgen, dat-ie haar
trouwt, maar is ze dat niet, dan kan ze dat niet verwachten.—Hoe zou ze? ’t
Zou niet voor d’r eigen geluk weze. Vraag-t-er iedere dame in de
Londensche wereld na, die dochters heeft,—en ze zal je hetzelfde zegge,—
behalve dat ik ’t je ronduit zeg, en zij verdraaid.—Daarin zit ’em ’t eenige
verschil.
Vivie (geboeid,—staart haar aan). Beste moeder,—u bent ’n merkwaardige
vrouw, u bent sterker dan heel Engeland. En voelt u nu werkelijk en
waarachtig niet ’n sikkepitje twijfel ... of ... of schaamte?

Mevr. Warren. Wel natuurlijk, lieverd,—’t hoort er zoo bij om je te

schame,—dat wordt eenmaal verwacht van ’n vrouw. Vrouwen moeten zich
altijd houe of ze ’n heeleboel voele wat ze niet doen. Lies was dikwijls
kwaad op me, as ik zoo botweg de waarheid er over zei. Zij zei altijd, dat,
omdat iedere vrouw genoeg kon leere van wat ze voor d’r ooge in de wereld
ziet gebeure, ’t nergens toe dient om er over te prate. Maar Lies was ook op
en top ’n dame. Ze had er ’t echte instinct van; terwijl je an mijn altijd m’n
lage kom-af kon merken: Ik plach zoo in m’n schik te zijn, wanneer je me
je portretten zond en ik zag, dat je opgroeide als Lies. Je hebt nèt haar
damesachtige, positieve manier van doen.—Maar ik kan ’t niet uitstaan, om
’t ééne te zeggen, als iedereen weet, dat ik ’t andere meen. Waar dient dat
gehuichel toe? Als de menschen de wereld op diè manier voor de vrouwen
inrichten, geeft ’t niks om net te doen, of ze anders is ingericht. Ronduit
gezegd heb ik me nooit in ’t minst geschaamd. Ik beweer, dat ik ’t recht heb
om trotsch te zijn, dat we alles zoo goed bedistelden, en er nooit iets op ons
te zeggen was, en dat de meisjes zoo goed behandeld werden. ’n Paar ervan
kwamen best terecht: één trouwde er met ’n gezant. Maar natuurlijk, daar
mag ik nou niet meer van spreken, wat zouen ze wel van me denken! (Zij
geeuwt). O Heere, ik geloof, dat ik tenslotte toch slaperig ben geworden.
(Zij rekt zichzelf luid uit, echt opgelucht door haar uitbarsting, en kalmpjes
bereid voor haar nachtrust).

Vivie. Ik geloof, dat ik ’t zal zijn, die nu niet zal kunnen slapen. (Zij gaat
naar ’t buffetje en steekt de kaars aan. Dan doet ze de lamp uit, waardoor
de kamer veel donkerder wordt). We zullen wat frissche lucht in laten vòòr
we sluiten. (Zij opent de buitendeur en ziet dat ’t heldere maan is). Kijk
eens! (Zij trekt de gordijnen van ’t raam open. Men ziet het landschap
badend in de stralen van ’n nazomermaan, die boven Blackdown rijst).

Mevr. Warren (met ’n vluchtigen blik naar buiten). Ja liefje, maar pas op,
dat je geen kou vat van de nachtlucht.
Vivie (minachtend). Gekheid!

Mevr. Warren (knorrig). Welzeker, alles wat ik zeg is gekheid volgens jou.

Vivie (keert zich haastig naar haar toe). Nee, dat is volstrekt niet waar,
moeder. U hebt ’t vannacht totaal van me gewonnen, hoewel ìk gewild had,
dat ’t andersom zou zijn geweest. Laten we nu goeie vrienden zijn.

Mevr. Warren (wat droevig haar hoofd schuddend). Dus ’t is andersom

geweest. Maar tòch zal ik wel de kleinste motten wezen. Ik trok met Lies
altijd an ’t kortste eindje, en ik denk, dat ’t nou wel ’tzelfde met jou zal
worden.

Vivie. Dat hindert niet... Kom, goeie nacht goeie, oude moeder. (Zij omarmt
haar moeder).

Mevr. Warren (teeder). Ik heb je goed grootgebracht, heb ik niet, lieverd?

Vivie. Ja, dat hebt u.

Mevr. Warren. En je zult goed zijn voor je arme, ouwe moeder, niet waar?

Vivie. Zeker moederlief (kust haar). Goeie nacht.

Mevr. Warren (zalvend). M’n zegen over m’n eigen lieveling!—’n

moeders zegen! (Zij omhelst haar dochter beschermend, terwijl zij opziet
naar boven, alsof zij ’n zegening over Vivie af wil smeeken).
 DERDE BEDRIJF.

In den tuin van de pastorie, den volgenden morgen. De zon schijnt, de

vogels zingen lustig. De tuinmuur heeft ’n houten hek, breed genoeg om ’n
rijtuig door te laten. Naast het hek hangt ’n bel aan ’n gekronkelden spiraal
die in verbinding staat met ’n trekker er buiten. Het rijpad loopt tot aan het
midden van den tuin en buigt dan naar links, waar het eindigt in ’n kleine
begrinte ronde plaats tegenover den overdekten ingang naar de pastorie.
Aan den anderen kant van het hek ziet men den stoffigen straatweg, parallel
aan den muur en aan de verste zij afgezet door ’n grasrand en ’n ònomheind
dennenbosch. Op het grasveld, tusschen het huis en den rijweg, staat ’n
gesnoeide taxusboom, waaronder in de schaduw een tuinbank. Aan den
tegenovergestelden kant is de tuin ingesloten door ’n palmhaag; op het
grasveld staat een zonnewijzer en daarnaast een ijzeren stoel. Een smal
paadje voert, achter den zonnewijzer, door de palmhaag heen.

Frank zit op den stoel bij den zonnewijzer, waarop hij de ochtendbladen
gelegd heeft en leest de Standard, (conservatief Londensch blad). Zijn vader
komt ’t huis uit, rillerig en met roode oogen;—en ontmoet Franks blik wat
onzeker.

Frank (kijkt op z’n horloge). Half twaalf, ’n Mooi uur voor ’n dominé om
te komen ontbijten.

Dominé Sam. Spot niet Frank, spot niet. Ik ben ’n beetje .... è ... (hij rilt).

Frank. Katterig?

Dominé. Nee jongmensch;—ongesteld van ochtend. Waar is je moeder?

Frank. Schrik niet, die is hier niet. Naar stad gegaan met Bessie met den
trein van 11.13. Heeft verscheiden boodschappen voor je achtergelaten.
Voel je je in staat om ze nu aan te hooren, of wil ik wachten tot je ontbeten
hebt?

Dominé. Ik hèb ontbeten, sinjeur. ’t Verwondert me, dat je moeder naar de

stad is gegaan, terwijl we logé’s hebben. Die zullen ’t heel vreemd vinden.

Frank. Daar zal ze mogelijk wel aan gedacht hebben. In ieder geval, als
Crofts hier nog blijft en jij iederen nacht tot vier uur toe met hem op blijft
zitten, om herinneringen uit je vurige jeugd op te halen, dan is ’t natuurlijk
m’n moeders plicht om naar stad te gaan en ’n vat whisky en ’n paar
honderd flesschen spuitwater te bestellen.

Dominé. Ik heb niet opgemerkt, dat Jhr. George bizonder veel heeft
gedronken.

Frank. Daar was je niet toe in staat, ouwe heer.

Dominé. Wil je te kennen geven, dat ik...?

Frank (kalm). Nog nooit heb ik ’n weleerwaarden dominé minder sober

gezien. De anekdoten uit je vroegere leven, die je verteld hebt, waren zòò
schandelijk, dat ik zeker geloof, dat Praed den nacht niet onder je dak zou
hebben doorgebracht, als ’t niet was geweest, dat hij en moeder zoo goed
met elkaar waren opgeschoten.

Dominé. Gekheid, jongmensch. Ik ben Jhr. George z’n gastheer. Ik moet

toch over iets met hem praten, en hij heeft maar één onderwerp. Waar is
mijnheer Praed nu?

Frank. Die rijdt met m’n moeder en Bessie naar ’t station.

Dominé. Is Crofts al op?

Frank. O, al lang. Die is zoo frisch als ’n hoen; hij is veel beter geoefend
dan jij:—heeft de training waarschijnlijk tot nu toe bijgehouden. Hij is
ergens gaan rooken. (Frank neemt z’n krant weer op. De dominé wendt zich
ontstemd naar het hek toe en komt dan besluiteloos terug).

Dominé. E.... Frank.

Frank. Ja.

Dominé. Geloof je, dat de Warren’s verwachten hier geïnviteerd te worden,

na gisteravond?

Frank. Ze zijn al geïnviteerd. Crofts vertelde ons aan ’t ontbijt, dat je hem
gezegd hadt om mevrouw Warren en Vivie vandaag hier te brengen en hun
te verzoeken dit huis als het hunne te beschouwen. ’t Was na diè
mededeeling, dat moeder bedacht, dat ze naar stad moest met den trein van
11.13.

Dominé (met wanhopige heftigheid). Ik heb die invitatie niet gedaan. Ik heb
noòit aan zoo iets gedacht.

Frank (medelijdend). Hoe kun je weten, ouwe heer, wàt je gisterennacht

gezegd en gedacht hebt? Allo! Hier is Praed terug.

Praed (komt binnen door ’t hek). Goeie morgen.

Dominé. Goeien morgen. Ik moet me verontschuldigen, dat ik u niet aan ’t

ontbijt heb gezien. Ik heb ’n lichte aanval van... van...

Frank. Dominé’s keelpijn, Praed. Gelukkig niet chronisch.

Praed (van onderwerp veranderend). Wel, ik moet zeggen, uw huis is

allerliefst gelegen, werkelijk allerliefst.

Dominé. Ja, dat is ’t ook. Als u wilt, zal Frank ’n eindje met u gaan
wandelen. Ik hoop dat u me zult excuseeren: ik moet de gelegenheid
waarnemen om m’n preek te schrijven, terwijl mevrouw Gardner weg is en
m’n gasten zich amuseeren. U neemt me niet kwalijk, niet waar?

Praed. Zeker niet, u moet voor mij niet de minste complimenten maken.
Dominé. Dank u. Ik zal... è... è... (stamelend gaat hij naar den ingang en
verdwijnt in huis).

Praed (gaat op ’t gras zitten en pakt z’n enkels beet). Wonderlijk moet dat
zijn om iedere week ’n preek te schrijven.

Frank. Heel wonderlijk als hij ’t deèd. Hij koopt ze. Hij is nou spuitwater
gaan drinken.

Praed. Beste jongen, ik wou dat je meer respect toonde tegenover je vader.
Je weet zelf hoe aardig je kunt zijn als je wìlt.

Frank. M’n goeie Praeddie, je vergeet dat ik met den ouden heer moet
lèven. Als twee menschen samen wonen—’t doet er niet toe of ze vader en
zoon, man en vrouw of broeder en zuster zijn—dan kunnen ze onmogelijk
de beleefde voor-den-gek-houderij volhouden, die zoo makkelijk valt voor
’n minuut of tien op ’n middagvisite. De oude heer nou, die aan veel
bewonderenswaardige huiselijke hoedanigheden paart de besluiteloosheid
van ’n schaap met de opgeblazenheid en de ongemakkelijkheid van ’n
jakhals...

Praed. Nee, asjeblieft, beste Frank. Bedenk toch, dat hij je vader is.

Frank. Daar geef ik hem alle eer van.—Maar stel je voor, dat hij Crofts
gezegd heeft om de Warrens hier te brengen!! Hij moet totaal weg zijn
geweest. Je weet beste Praeddie, dat m’n moeder haar dadelijk weg zou
kijken. Vivie moet hier niet komen, vòòrdat zij naar de stad terug is.

Praed. Maar je moeder weèt toch niets van mevrouw Warren, wel?

Frank. Ik weet ’t niet. Haar gaan naar de stad doet me denken van wel.
Niet, dat ’t m’n moeder in ’t algemeèn zou kunnen schelen. Zij heeft ’t
dikwijls kranig opgenomen voor ’n massa vrouwen, die in moeilijkheden
waren geraakt. Maar dat waren allemaal behoòrlijke vrouwen. Daarin zit
’em het verschil. Mevrouw Warren heeft zeker haar eigenaardige
verdiensten, maar ze is zoo allemachtig lawaaiig,—en m’n moeder zou haar
eenvoudig niet kunnen dulden. Daarom... Allo! (Deze uitroep wordt
veroorzaakt door de wederverschijning van den dominé, die haastig en
ontsteld z’n huis uitkomt).

Dominé. Frank, mevrouw Warren en haar dochter komen de hei over met
Crofts. Ik zag ze van uit m’n studeerkamer. Wat moèt ik zeggen van je
moeder?

Frank (energiek opspringend). Plak je hoed op je hoofd en ga hen

tegemoet en zeg hoe allerplezierigst je ’t vindt om ze te zien;—en dat Frank
in den tuin is, en dat moeder en Bessie zijn weggeroepen bij ’n ziek
familielid, en dat ’t hun zoo speet dat ze niet konden blijven, en dat je
hoopt, dat mevrouw Warren goed geslapen heeft en... en... zeg hun àl ’t
mogelijke behalve de waarheid en laat de rest aan onzen lieven Heer over.

Dominé. Maar hoe moeten we hen later kwijt raken?

Frank. Daar is nou geen tijd voor om over te denken. Hier! (Hij vliegt ’t
huis in en keert onmiddellijk terug met ’n vilten dominé’s hoed, die hij z’n
vader op ’t hoofd duwt). Maak nu, dat je weg komt. Praed en ik zullen hier
wachten, om het zaakje ’n ongezocht aanzien te geven. (De dominé,
beduusd maar gehoorzaam, snelt weg door ’t hek. Praed staat op en stoft
zichzelf af).

Frank. We moeten de oude dame op de een of andere manier naar de stad

terugzenden, Praed. Toe, zeg ’ns eerlijk, Praeddie, hoû jij er van om ze
samen te zien: Vivie en de ouwe dame?

Praed. Och, waarom niet?

Frank (z’n tanden op elkaar). Krijg jij er geen kippenvel van? Die kwaje
oude duvel, in staat tot àlles wat gemeen is, en Vivie.... brr!

Praed. Sst, asjeblieft. Daar komen ze. (De dominé en Crofts komen samen
het rijpad op, gevolgd door Mevrouw Warren en Vivie, die heel innig met
elkaar loopen.)
Frank. Kijk, ze heeft waarachtig haar arm om het middel van de oude
vrouw. ’t Is haar rechterarm; zij moet er mee begonnen zijn. God in den
hemel, ze is sentimenteel geworden! Ai! jai! Krijg je nou geen kippenvel?
(De dominé opent het hek en mevrouw Warren en Vivie gaan hem voorbij en
blijven in het midden van den tuin naar ’t huis staan kijken. Frank, in ’n
extase van veinzerij, wendt zich vroolijk naar mevrouw Warren toe en roept
uit): Alleraangenaamst om u te zien, mevrouw Warren,—deze rustige, oude
pastorie-tuin flatteert u bizonder.

Mevr. Warren. Wel, heb je ooit! Hoor je dat George? Hij zegt dat ik er zoo
goed uitzie in ’n rustigen, ouden pastorie-tuin.

Dominé (houdt het hek nog open voor Crofts, die er met ’n bizonder
landerig air doorheen slentert). U ziet er overal goed uit, mevrouw Warren.

Frank. Bravo, ouwe heer. Luister ’ns,—laten we nou ’n gezelligen tijd er

van maken vóór lunch. Eerst gaan we de kerk bekijken. Die behoort
iedereen te zien. ’t Is ’n typische oude, dertiende-eeuwsche kerk, weet je.
De oude heer voelt er erg voor, omdat hij indertijd ’n restauratiefonds op
touw heeft gezet,—en ze zes jaar geleden totaal verbouwd is geworden.
Praed zal er jullie de merkwaardigheden van aantoonen.

Dominé (allerminzaamst glimlachend tegen het gezelschap). ’t Zal me

bizonder aangenaam zijn, als Jhr. George en mevrouw Warren er werkelijk
lust toe voelen.

Mevr. Warren. Wel ja, laten we maar gaan en ’t afdoen: ’t Zal George
goed doen; de kerk zal van hèm niet veel last hebben, wed ik.

Crofts (terugkeerend naar ’t hek). Ik heb er niets tegen.

Dominé. Neen, die weg niet. We zullen door ’t veld gaan, als u ’t goed
vindt. Dèzen kant uit.

Crofts. O, mij goed. (Hij gaat met den dominé. Praed volgt met mevrouw
Warren. Vivie beweegt zich niet, maar kijkt hen na met de lijnen van
vastberadenheid scherp geteekend op haar gezicht).
Frank. Kom je niet?

Vivie. Nee. Ik wil je ’n waarschuwing geven, Frank. Je stak daarnet den

draak met m’n moeder, toen je dat zei over den pastorie-tuin. Dat is
voortaan taboe. Behandel m’n moeder asjeblieft met hetzelfde respect
waarmee je je eigen moeder behandelt.

Frank. M’n beste Vivie, dat zou ze niet apprecieëren. Ze is heel anders dan
m’n moeder: dezelfde behandeling zou voor die twee niet deugen. Maar wat
ter wereld is er met je gebeurd? Gisterenavond waren we ’t samen volmaakt
eens over je moeder en haar kliek. En van morgen stel je je sentimenteel
aan met je arm om haar middel heen.

Vivie (rood wordend). Me aanstellen!

Frank. Dièn indruk maakte ’t op me. Den eèrsten keer dat ik je iets zag
doen van twijfelachtig allooi.

Vivie (zichzelf bedwingend). Ja, Frank; er heèft ’n verandering met me

plaats gehad, maar ik geloof niet ’n verandering ten kwade. Gisteren was ik
’n ingebeeld nest.

Frank. En vandaag?

Vivie (haar gezicht vertrekt pijnlijk, dan ziet ze hem vast aan). Vandaag ken
ik m’n moeder beter dan jij.

Frank. De hemel beware je daarvoor.

Vivie. Wat bedoel je daarmee?

Frank. Viv,—er bestaat ’n vrijmetselarij tusschen door-en-door-onzedelijke

menschen, waar jij niets van afweet. Jij hebt te veel karakter. Maar dàt is de
band tusschen je moeder en mij; en dat is ook de reden, dat ik haar beter
ken dan jij ooit zult doen.
Vivie. Je vergist je, je weet niets van haar af. Als je de omstandigheden
kende, waarmee m’n moeder te worstelen heeft gehad....

Frank (ad rem haar zin voor haar afmakend). Dan zou ik weten waàrom ze
is geworden, wàt ze is,—is ’t zoo niet? Wat zou er dat toe doen?
Omstandigheden of geen omstandigheden Viv,—je zult nooit met je moeder
kunnen opschieten.

Vivie (heel boos). Waarom niet?

Frank. Omdat ze ’n slecht wijf is, Viv. Als je ooit weer in mijn bijzijn je
arm om haar middel slaat, dan schiet ik me op de plaats zelf voor m’n kop,
—als ’n protest tegen ’n vertooning, die me in opstand brengt.

Vivie. Moet ik dus kiezen tusschen jou en m’n moeder?

Frank. Dat zou de oude dame ’n veel te slechte kans geven. Nee, Viv, je
verwaande jongentje zal je in geen geval aan je lot overlaten. Maar daarom
moet hij ook zorgen, dat je geen vergissingen begaat.—’t Geeft allemaal
niks, Viv, je moeder ìs eenmaal onmogelijk. Ze mag in haar soort niet
kwaad zijn,—maar ’t soort zelf ìs slecht, door en door slecht.

Vivie (heftig). Frank! (Hij blijft kalm. Zij wendt zich af en gaat zitten op de
bank onder den taxus, worstelend om haar zelfbeheersching te herkrijgen.
Dan zegt ze). Moet ze door iedereen verlaten worden, omdat ze eenmaal is
wat jij “’n slecht soort” noemt? Heeft ze geen recht om te leven?

Frank. Daar hoef je niet bang voor te wezen, Viv; zij zal nooit verlaten
zijn. (Hij gaat naast haar zitten op de bank).

Vivie. Maar ik moet haàr zeker verlaten.

Frank (sust haar op babyachtige manier en vleit haar met z’n stem). Moèt
niet met haar leven. Familiegroepje van moeder en dochter zou geen succes
zijn. Zou òns groepje bederven.

Vivie (onder de bekoring komend). Welk groepje?

Frank. De babies in ’t bosch; Vivie en haar kleine Frank. (Hij glijdt z’n
arm om haar middel en nestelt zich tegen haar aan als ’n moe kind). Laten
we mekaar toedekken met dorre blaâren.

Vivie (hem zachtjes wiegend als ’n moeder). Vast in slaap, hand in hand,
onder de boomen.

Frank. Het wijze kleine meisje en naar dwaze kleine jongentje.

Vivie. Het lieve kleine jongentje, met het zielige kleine meisje.

Frank. Heel, hèèl rustig en bevrijd van de idiotigheid van den vader van ’t
jongentje en de rarigheid van de moe....

Vivie (het woord smorend tegen haar borst aan). St. st. stst! ’t Kleine
meisje wil alles vergeten van haar moeder. (Zij zwijgen eenige oogenblikken
elkaar wiegend. Dan komt Vivie plotseling tot bezinning en roept uit): Wat
’n paar gekken zijn we! Kom, zit overeind. Goeie hemel, je haar! (Zij strijkt
’t glad). Ik zou wel eens willen weten of alle groote menschen zoo
kinderachtig doen, als er niemand bij is. Ik heb ’t nooit gedaan als kind.

Frank. Ik ook niet. Jij bent m’n eerste speelkameraad. (Hij vat haar hand
en wil die kussen, maar houdt eerst even op om rond te kijken. Heel
onverwacht verschijnt Crofts door de palmhaag). O verdomd!

Vivie. Waarom, verdomd?

Frank (fluisterend). Sst! Daar komt die fielt van ’n Crofts aan. (Hij gaat
verder van haar af zitten met ’n heel onschuldig gezicht).

Vivie. Wees niet lomp tegen hem, Frank. Ik wil erg m’n best doen om
beleefd tegen hem te zijn. Dat zal m’n moeder plezier doen. (Frank trekt ’n
leelijk gezicht).

Crofts. Mag ik ’n paar woorden met u spreken, juffrouw Warren?

Vivie. Zeker.
Crofts (tot Frank). Je excuseert me wel, Gardner.—Ze wachten op je in de
kerk, als je er niets tegen hebt.

Frank (staat op). Ik wil je graag van dienst zijn, Crofts, behalve met naar
de kerk te gaan. Als je iets noodig hebt, Vivie, bel dan aan ’t hek, dan
verschijnt een van de dienstboden. (Hij gaat ’t huis in met kalme
beminnelijkheid).

Crofts. (Kijkt hem met ’n sluwe uitdrukking nà, terwijl ie verdwijnt, en

spreekt dan tot Vivie op ’n toon alsof hij op vertrouwelijken voet met haar
is). ’n Aardige jongen, juffrouw Vivie. Jammer, dat hij geen geld heeft, hè?

Vivie. Vindt u?

Crofts. Wel, wat moet hij uitvoeren? Heeft geen betrekking en geen
fortuin.—Waar dient hij toe?

Vivie. Ik zie volkomen goed z’n zwakke punten, Jhr. George.

Crofts (’n beetje van z’n stuk gebracht, omdat hij zoo volmaakt doorzien
wordt). O zòò meen ik ’t niet, Maar zoolang we eenmaal op deze wereld
zijn, moeten we er ook rekening mee houden,—en geld is geld. (Vivie
antwoordt niet). ’n Mooie dag, vindt u niet?

Vivie (met ternauwernood bedwongen minachting voor z’n poging tot

conversatie). Heel mooi.

Crofts (met brutale jovialiteit, alsof hij haar flinkheid bewondert). Wel,
daarover wou ik anders niet praten (met voorgewende openhartigheid).
Luister ’ns, juffrouw Vivie. Ik ben me volkomen bewust, dat ik geen man
voor dames ben.

Vivie. Heusch niet?

Crofts. Nee; en om u de waarheid te zeggen, dat wil ik ook niet zijn. Maar
als ik wat zeg, dan meen ik het;—als ik iets voel, dan voel ik ’t ècht,—en
wat ik graag wil hebben, daar wil ik ook goed voor betalen. Dàt soort van
man ben ik.

Vivie. Dat doet u alle eer aan.

Crofts. O, ik wil m’n eigen lof niet zingen. De hemel weet, dat ik m’n
fouten heb;—geen man ziet die beter dan ik. Ik weet, dat ik niet volmaakt
ben: die zelfkennis is een van de voordeelen van ’n man van middelbaren
leeftijd;—want ik bèn niet jong meer en daar geef ik me niet voor uit ook.
Mìjn moraal is heel eenvoudig en ik geloof goed: Eergevoel tusschen man
en man, trouw tusschen man en vrouw en geen malle praatjes over den een
of anderen godsdienst, maar ’n eerlijk geloof, dat de wereld geleidelijk
vooruitgaat.

Vivie (snijdend ironisch). “Een macht, niet wijzelf, die rechtvaardigheid

wil.”

Crofts (haar au sérieux nemend). O zeker, wijzelf natuurlijk niet. U

begrijpt wat ik bedoel. (Hij gaat naast haar zitten, op ’n wijze alsof hij ’n
verwante ziel had gevonden). En nu, wat practische zaken betreft. U zult
misschien meenen, dat ik m’n geld heb weggegooid, maar dat is zoo niet. Ik
ben vandaag rijker, dan toen ik indertijd m’n fortuin in handen kreeg. Ik heb
van m’n wereldkennis geprofiteerd, om m’n geld te steken in zaken, die
andere menschen over ’t hoofd hebben gezien; en wàt ik ook wezen mag,
op ’t punt van geld ben ik ’n betrouwbaar man.

Vivie. ’t Is heel vriendelijk van u, me dat allemaal te vertellen.

Crofts. Kom nou, juffrouw Vivie,—u hoeft u niet te houden, alsof u niet
weet, waar ik heen wil. Ik verlang om me te vestigen met ’n “Lady Crofts”.
—Ik vermoed, dat u me wel erg botaf vindt?

Vivie. Volstrekt niet. Ik ben er u heel dankbaar voor, dat u zoo kort en
zakelijk bent. Ik stel uw aanbod zeer op prijs: het geld, de positie, Lady
Crofts en zoo al meer. Maar, met uw welnemen, zal ik toch maar “nee”
zeggen. Liever niet. (Zij staat en drentelt naar den zonnewijzer, om wat uit
z’n onmiddellijke nabijheid te zijn).
Crofts (in ’t minst niet ontmoedigd, gebruik makend van de meerdere
plaats op de bank om er zich gemakkelijk op uit te strekken, vat het op alsof
’n paar voorloopige weigeringen ’n onvermijdelijk deel uitmaakten van den
gewonen gang van ’n huwelijksaanzoek). Ik heb geen haast. Ik wou u dit
alleen maar laten weten, voor ’t geval dat de jonge Gardner mocht
probeeren u te vangen. Denk er eens over na.

Vivie (scherp). Mijn neen blijft neen. Ik kom er niet op terug. (Zij ziet hem
aan van uit de hoogte. Hij grijnst; buigt zich voorover met z’n elbogen op
z’n knieën om met z’n stok naar ’n ongelukkig insect in ’t gras te prikken.
Hij kijkt haar sluw aan. Ongeduldig wendt zij zich af).

Crofts. Ik ben ’n goed beetje ouder dan u,—vijf en twintig jaar, ’n kwart
eeuw. Ik heb ’t eeuwige leven niet, en ik zal zorgen, dat u goed achterblijft,
als ik er niet meer zijn zal.

Vivie. Zelfs tegen diè verleiding ben ik bestand, Jhr. George. Gelooft u niet,
dat u beter zoudt doen met m’n antwoord te accepteeren? Er is niet de
minste kans, dat ik veranderen zal.

Crofts (staat op, na ’n laatsten slag naar ’n madeliefje en begint heen en

weer te loopen.) Wel, ’t hindert niet. Ik zou u dingen kunnen vertellen, die u
gauw genoeg van gedachten zouden doen veranderen,—maar dat wil ik
niet, omdat ik u liever wil zien te winnen door eerlijke liefde. Ik ben ’n
goeie vriend voor uw moeder geweest, vraag haar dat maar eens. Zij zou
nooit ’t geld verdiend hebben, dat uw opvoeding betaald heeft, als ìk haar
niet geraden en geholpen had. Om niet te spreken van het geld, dat ik haar
heb voorgeschoten. Er zijn niet veel menschen, die haar gesteund zouden
hebben, zooals ìk ’t heb gedaan. Ik heb van ’t begin tot ’t laatst toe niet
minder dan 40,000 pond in haar zaak gestoken.

Vivie (hem aanstarend). Wilt u zeggen, dat u m’n moeders deelgenoot

geweest bent?

Crofts. Ja. Bedenk dus nou ’ns wat ’n last en explicaties ’t besparen zou,
als wij ’t heele geval onder ons hielden, om zoo te zeggen. Vraag uw
moeder maar eens of zij ’t prettig zou vinden om uitlegging van d’r zaken te
geven aan ’n totaal vreemde.

Vivie. Daar zie ik de bezwaren niet van in, nu de zaken toch aan kant zijn
gedaan en het geld belegd is.

Crofts (blijft plotseling verbaasd staan). Aan kant gedaan? Een

onderneming aan kant doen, die 35 percent uitkeert in de slechtste jaren?!
Niet waarschijnlijk, hoor. Wie heeft u dàt verteld?

Vivie (plotseling verbleekend). Bedoelt u dat ze nog...? (Zij houdt op eens

stil en legt haar hand op den zonnewijzer om zichzelf te ondersteunen. Dan
loopt ze haastig naar den ijzeren stoel en gaat zitten). Over welke
onderneming spreekt u?

Crofts. Wel, de kwestie is, dat ze nou niet precies als ’n zaak van den
eersten rang beschouwd wordt in mijn kringen, de voorname kringen, weet
u;—die ònze kringen zullen worden, als u anders over m’n aanzoek gaat
denken. Niet, dat er iets niet in den haak mee is, dat moet u niet denken. U
begrijpt door het feit, dat uw moeder er in is, dat ze volkomen fair en eerlijk
is. Ik heb haar jaren lang gekend en ik weet van haar, dat ze liever haar
hand zou afslaan, dan met iets te doen te hebben, wat niet heelemaal
behoorlijk is. Als u wilt zal ik er u alles van vertellen. Ik weet niet of u wel
eens ondervonden hebt, als u op reis was, hoe moeilijk ’t is, om ’n werkelijk
goèd ingericht familiehotel te vinden.

Vivie (wendt haar gezicht af met walging.) Ja,—ga door.

Crofts. Nu, dat is alles. Uw moeder heeft ’n zeldzame gave om die dingen
te besturen. We hebben er twee in Brussel, één in Berlijn, één in Weenen en
twee in Buda-Pest.—Natuurlijk zijn er nog anderen behalve wij in de zaak,
maar wìj hebben er het meeste kapitaal in,—en uw moeder is onmisbaar als
directrice. U hebt zeker wel gemerkt, dat zij veel reist.—Maar u begrijpt,—
over zulke dingen kun je in gezelschap niet spreken. Noem ’t woord “hotel”
maar eens en iedereen zegt, dat je ’n publiek huis houdt. U zoudt toch niet
willen, dat ze dàt van uw moeder zouden zeggen, wel? Daarom houden we
’t zoo stil. Wat ik zeggen wil, u zult ’t ook wel voòr u houden, niet? Nu ’t al
zòòlang ’n geheim is geweest, is ’t beter dat ’t dat ook blijft.

Vivie. En dit is dus de onderneming, waar u wilt, dat ik deel in zal nemen!

Crofts. Welnee. Mijn vrouw zal niets met zaken te maken hebben. U zult
er niet meer mee te maken hebben dan u altijd gedaan hebt.

Vivie. Altijd gedaan heb! Wat bedoelt u?

Crofts. Alleen maar dat u er altijd van geleefd hebt. Ze heeft betaald voor
uw opvoeding en voor de japon die u aan uw lijf hebt. Trek uw neus maar
niet op voor zaken, juffrouw Vivie. Wat zou er van uw mooie scholen
worden zonder geld?

Vivie (staat op, half buiten zichzelf). Pas op. Ik weet wàt voor zaak ’t is.

Crofts (opschrikkend, met ’n onderdrukte vloek). Wie heeft u dat verteld?

Vivie. Uw compagnon—m’n moeder.

Crofts (zwart van woede). Die ouwe.... (Vivie ziet hem haastig aan. Hij
slikt ’t woord in en staat stilletjes voor zich te razen en te vloeken. Dan
bedenkt hij zich, dat hij sympathiek moet zijn en hij neemt z’n toevlucht tot
’n edele verontwaardiging). Zij behoorde u meer te hebben ontzien. Ik zou
’t u nooit verteld hebben.

Vivie. Ik denk, dat u waarschijnlijk gewacht zoudt hebben tot we getrouwd

waren. Het zou ’n makkelijk wapen voor u geweest zijn om me mee klein te
krijgen.

Crofts (heel oprecht). Ik had ’t nooit willen doen. Op m’n woord niet.
(Vivie ziet hem verwonderd aan. Haar gevoel voor de ironie van zijn protest
kalmeert haar en geeft haar kracht. Zij antwoordt met minachtende
zelfbeheersching.)
Vivie. ’t Doet er niet toe. Ik veronderstel dat u begrijpt, dat wanneer wij
vandaag van hier weggaan, onze kennismaking tot ’n eind komt.

Crofts. Waarom? Omdat ik u moeder geholpen heb?

Vivie. Mijn moeder was ’n arme vrouw die geen keus had om anders te
handelen dan ze gedaan heeft. Maar ù was rijk en u deed hetzelfde terwille
van 35 percent. U bent mijns inziens ’n gewone, echte schurk. Dàt is m’n
opinie van u.

Crofts (staart haar even aan,—volstrekt niet gekwetst, en veel meer op z’n
gemak, nu ze op dezen ongegeneerden voet met elkaar zijn, dan toen ze
eerst wat vormelijk waren). Ha, ha, ha, ha! ga je gang, juffie, geef er me van
langs; ’t hindert me niet en ’t amuseert me. Wat weerga, waarom zou ik m’n
geld niet op die manier beleggen? Ik neem de interest van ’n kapitaal net als
alle andere menschen. Ik hoop niet, dat je vindt, dat ik er m’n eigen handen
mee vuil maak. Zeg ’ns zelf: je zoudt toch niet weigeren om kennis te
maken met m’n moeders neef, den hertog van Belgravia, omdat sommige
van de renten, die hij ontvangt, op ’n wat wonderlijke wijze verdiend
worden? Je zoudt vermoedelijk den aartsbisschop van Canterbury niet
negeeren omdat de leden van de kerkelijke commissies enkele
kroeghouders en zondaren onder hun huurders hebben? Herinner je je de
Croftsbeurs in Newnham? Nu, die is gesticht door m’n broer, het
parlementslid. Hij krijgt z’n 22 percent van ’n fabriek met 600 meisjes,
waarvan er niet één genoeg verdient om van te leven. Hoe stel je je voor,
dat die rondscharrelen? Vraag ’t je moeder maar eens. En verwacht je dan,
dat ik bedanken zou voor 35 percent, terwijl andere menschen in hun zak
steken wàt ze maar kunnen, als verstandige lui? Zòò gek ben ik niet. Als je
je kennissen wilt kiezen en uitzoeken volgens zedelijke principes, dan kun
je ’t land wel uittrekken, tenzij je jezelf buiten de heele fatsoenlijke
maatschappij wilt houden.

Vivie (met gewetenswroeging). U kunt er verder nog op wijzen, dat ik zelfs

nooit gevraagd heb, waar ’t geld, dat ik uitgaf, vandaan kwam. Ik geloof,
dat ik net even slecht ben als u.
Crofts (geheel gerustgesteld). Natuurlijk,—en dat is maar goed ook. Wat
voor kwaad doet ’t ten slotte? (met een familiare grappigheid). Nou je er
verder over nadenkt, zul je me zoo’n schurk wel niet meer vinden, wèl?

Vivie. Ik heb dezelfde voordeelen met u gedeeld, en ik ben al zoo ver

gegaan van u op vertrouwelijke wijze te vertellen wàt ik van u denk.

Crofts (met serieuze minzaamheid). Zeker, dat heb je ook.—Je zult me

zoo’n kwaje niet vinden. Ik geef me niet uit voor iemand met een prima
intellect, maar ik heb een goeie dozis eerlijk, humaan gevoel; en het oude
ras van de Croften komt uìt in ’n zekere instinctmatige haat van alles wat
min is,—waarin je zeker met me zult sympathiseeren. Geloof me, juffrouw
Vivie, de wereld is zoo kwaad niet, als sommige schreeuwers wel beweren.
Zoo lang je de maatschappij niet openlijk trotseert, zal ze je ook geen
lastige vragen doen;—en ze maakt korte metten met de ploerten, die ’t wèl
doen. Er blijven geen geheimen beter bewaard, dan dìè, die half bekend
zijn. In de kringen, waarin ik je zal introduceeren, zal geen heer of dame
zich zòò ver vergeten om de zaken van mij of je moeder te bespreken. Geen
man kan je ’n veiliger positie aanbieden.

Vivie. Ik geloof dat u heusch denkt, dat u uitstekend met me opschiet.

Crofts. Wel, ik geloof, dat ik mezelf mag vleien, dat je beter over me
denkt, dan je eerst hebt gedaan.

Vivie (kalm). Ik vind u nu nauwelijks de moeite waard, om over te denken.

(Zij staat op en gaat naar het hek toe, onderweg stilhoudend om hem te
bekijken en om bijna zachtzinnig maar met diepe overtuiging tot hem te
zeggen): Als ik denk aan de maatschappij die ù duldt, en de wet die ù
beschermt,—als ik bedenk hoe hulpeloos overgeleverd negen van de tien
meisjes zullen zijn in de handen van u en van m’n moeder:—van de vrouw
met het onnoembare bedrijf en haar kapitalist-slavenjager....

Crofts (wit van woede). Verdomd!

Vivie. U hoèft me niet meer te verdoemen. Ik voel al of ik onder de

verdoemden leef. (Zij licht den klink van ’t hek op, om het te openen en er
door te gaan. Hij volgt haar en legt z’n hand zwaar op den hoogsten
dwarsbalk om te beletten, dat ’t hek geopend wordt).

Crofts (hijgend van woede). Denk je, dat ik dit alles van je verdraag, jij
kleine duivel?

Vivie (koel). Wees bedaard. Er zal iemand komen in antwoord op de bel.

(Zonder even te aarzelen slaat zij tegen de bel met den rug van haar hand.
’t Klinkt hard, en Crofts schrikt onwilkeurig terug. Bijna onmiddellijk
verschijnt Frank in den ingang van ’t huis met z’n geweer).

Frank (met opgewekte beleefdheid). Wil jij ’t geweer hebben Viv, of zal ik
’t gebruiken?

Vivie. Heb je geluisterd, Frank?

Frank. Alleen maar naar de bel, ik verzeker ’t je,—zoodat je niet zoudt

hoeven te wachten. Ik geloof, dat ik je karakter goed heb doorzien, Crofts.

Crofts. Voor ’n kleinigheid zou ik dat geweer van je overnemen en ’t kapot

slaan op je hoofd.

Frank (voorzichtig naar hem toesluipend). Doe dat niet. Ik ga heel

onhandig met vuurwapenen om. Er zou stellig ’n noodlottig ongeluk plaats
hebben met later ’n waarschuwing van de jury voor m’n onachtzaamheid.

Vivie. Zet ’t geweer weg, Frank, ’t is volmaakt onnoodig.

Frank. Groot gelijk, Viv. Veel beter jagersmanier om hem in ’n val te

vangen. (Crofts, die de beleediging begrijpt, maakt ’n dreigende beweging).
Crofts, er zijn vijftien kogels in ’t magazijn en ik ben ’n zekere treffer van
’n afstand als deze, op ’n schijf van jouw omvang.

Crofts. O je hoeft niet bang te zijn. Ik zal je niet aanraken.

Frank. Heel grootmoedig van je onder de omstandigheden. Wel bedankt.

Crofts. Ik wil jullie dit nog zeggen vòor ik heenga. ’t Zal je misschien
interesseeren, omdat jullie zoo op elkaar gesteld bent. Sta me toe, Frank, je
voor te stellen aan je halfzuster, de oudste dochter van den eerwaarden
Samuel Gardner. Juffrouw Vivie, uw halfbroer. Goeie morgen. (Hij gaat
door ’t hek heen den weg op).

Frank (na ’n pauze van ontdaanheid, neemt z’n geweer op). Viv, je zult
voor den rechter getuigen, dat ’t een ongeluk was. (Hij mikt op de
verdwijnende figuur van Crofts. Vivie grijpt den loop en draait die rond,
tegen haar borst aan).

Vivie. Schiet nou. Nu mag je.

Frank (laat ’t eind van z’n geweer haastig vallen). Halt! Pas op! (Zij laat ’t
gaan. ’t Valt op den grond). O, wat heb je je jongentje laten schrikken! Stel
je voor, dat ’t af was gegaan.... O! (geheel ontdaan valt hij op de bank
neer).

Vivie. Ja, stel je dat voor. Begrijp je niet, dat ’t een verlichting voor me
geweest zou zijn om ’n felle lichamelijke pijn in me te voelen scheuren?

Frank (vleiend). Trek ’t je niet zoo aan, beste Viv. Bedenk maar, dat àls
m’n geweer den vent zòò heeft verschrikt, dat hij voor ’t eerst in z’n leven
de waarheid gezegd heeft, ’t ons dan in èrnst maakt tot de babies in ’t
bosch. (Hij houdt z’n armen voor haar open). Kom, laten we ons weer
toedekken met blaâren.

Vivie (met ’n kreet van afschuw). O, dat niet, dat niet! Je laat me rillen!

Frank. Waarom,—wat scheelt je?

Vivie. Adieu! (Zij gaat heen door ’t hek).

Frank (opspringend). Allo! Wacht even! Viv! Viv! (Zij draait zich om bij ’t
hek). Waar ga je naar toe? Waar kan ik je vinden?
Vivie. Op Honoria Frasers kantoor, Chancery Lane 67,—voor de rest van
m’n leven. (Zij gaat heen in de tegenovergestelde richting van Crofts).

Frank. Maar hoor dan toch ’ns... wacht even... Wat drommel! (hij rent haar
achterna).
 VIERDE BEDRIJF.

De kamers van Honoria Fraser in Chancery Lane. Een kantoor op de

hoogste verdieping met een raam van spiegelglas; geverfde muren,
electrisch licht en een vulkachel. Zaterdagmiddag. Men ziet de
schoorsteenen van Lincoln’s Inn en den hemel daarachter in ’t Westen, door
het venster. Er staat ’n dubbel schrijfbureau in het midden van de kamer
met een kistje sigaren, aschbakjes en een verplaatsbare electrische lamp, de
laatste half verborgen onder hoopen papier en boeken. Dit schrijfbureau
heeft gaten voor de knieën, rechts en links ervan staan stoelen.—Het ziet er
heel slordig uit. Het bureau van den klerk, gesloten en netjes, met ’n hoogen
stoel er voor, staat tegen den muur aan, dicht bij ’n deur, die in verbinding
staat tot de binnenkamer. In den tegenovergestelden muur is de deur, die
voert naar de algemeene gang. Zijn bovenpaneel is van matglas, waarop
met zwarte letters aan den buitenkant “Fraser en Warren”. Een groen baaien
scherm verbergt den hoek tusschen die deur en het venster.

Frank, in ’n modieus licht sport-reispak met zijn stok, handschoenen en

witte hoed in z’n handen, loopt heen en weer in ’t kantoor. Iemand probeert
de deur open te maken, met ’n sleutel.

Frank (roept). Binnen! ’t Is niet gesloten. (Vivie komt binnen met hoed op
en mantel aan. Zij blijft staan en staart hem aan).

Vivie (streng). Wat voer je hier uit?

Frank. Op je wachten. Ik hen hier al uren geweest. Is dat de manier om op

je zaken te passen? (Hij legt zijn hoed en stok op tafel, gaat met ’n sprong
boven op de klerks kruk zitten, en kijkt haar aan met al de symptomen van
’n onrustige, plagerige, lichtzinnige stemming).