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Biochemistry and its Application
BIOCHEMISTRY AND ITS
APPLICATION

Edited by:
Papita H Gourkhede

ARCLER
P r e s s

www.arclerpress.com
Biochemistry and its Application
Papita H Gourkhede

Arcler Press
224 Shoreacres Road
Burlington, ON L7L 2H2
Canada
www.arclerpress.com
Email: [email protected]

e-book Edition 2023

ISBN: 978-1-774695-64-7 (e-book)

This book contains information obtained from highly regarded resources. Reprinted material
sources are indicated and copyright remains with the original owners. Copyright for images and
other graphics remains with the original owners as indicated. A Wide variety of references are
listed. Reasonable efforts have been made to publish reliable data. Authors or Editors or Publish-
ers are not responsible for the accuracy of the information in the published chapters or conse-
quences of their use. The publisher assumes no responsibility for any damage or grievance to the
persons or property arising out of the use of any materials, instructions, methods or thoughts in
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ABOUT THE EDITOR

Dr.(Mrs). Papita H Gourkhede (1977) is presently serving as Assistant Professor,

Department of Soil Science and Agriculture Chemistry, college of Agriculture,
Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani. She obtained her B. Sc.(Ag.)
in 2001 from College of Agriculture , Nagpur from Dr. Panjabrao Deshmukh Krishi
Vidyapeeth, Akola. Pursued M. Sc. (Ag.) in 2007 and Ph. D. in Soil Science And
Agriculture Chemistry in 2012 from Vasantrao Naik Marathwada Krishi Vidhyapeeth,
Parbhani. She started her career as Assistant professor in 2012. Her field of specialization
is soil fertility, nutrient management, micronutrients, heavy metal remediation, Remote
Sensing and Organic Farming. She has published 05 books and 35 research paper in
National and international journals of reputed. She has participated in many National,
State Seminar and symposiums. She has written 06 practical manuals for under graduate
course for the benefit of students besides this delivered several Radio talk, Lectures in
farmer traning programme. Dr. Papita has also written 112 popular articles in Agroone,
Shetibhati, RCFsheti patrika and other reputed Magazines. She has received young
Scientists award in 2016, ICAR national Award for research innovation in Dryland
Agriculture in 2018, women Scientists Award in 2019, Agrocare Award in 2017, State
level Best program officer award in 2020.
TABLE OF CONTENTS

List of Figures.........................................................................................................xi
List of Abbreviations.............................................................................................xv
Glossary.............................................................................................................. xix
Preface.......................................................................................................... ....xxiii

Chapter 1 Introduction to Biochemistry..................................................................... 1

1.1. Introduction......................................................................................... 2
1.2. The Foundations of Biochemistry......................................................... 4
1.3. An Introduction to Enzymes............................................................... 14
1.4. Carbohydrates and Glycobiology....................................................... 20
1.5. Amino Acids, Peptides, and Proteins.................................................. 26
1.6. Hormones.......................................................................................... 30
1.7. Conclusion........................................................................................ 32
References................................................................................................ 34

Chapter 2 Biosynthesis and Immunochemical techniques........................................ 35

2.1. Introduction to Biosynthesis............................................................... 36
2.2. The Biosynthesis of Cell Constituents................................................. 37
2.3. Biosynthesis of Sulfur-Containing Small Biomolecules in Plants......... 42
2.4. Biosynthesis of Volatile Plant Secondary Metabolites and Its
Interconnection with Primary Metabolism....................................... 44
2.5. Flavonoid Biosynthesis....................................................................... 48
2.6. Immunochemical Techniques............................................................ 52
2.7. Principles of Immunochemical Techniques Used in
Clinical Laboratories....................................................................... 56
2.8. Conclusion........................................................................................ 59
References................................................................................................ 61
Chapter 3 Genetic Information Transfer................................................................... 63
3.1. Introduction....................................................................................... 64
3.2. Transformation................................................................................... 66
3.3. Transduction...................................................................................... 67
3.4. Conjugation....................................................................................... 68
3.5. Genetic Information In Microbes....................................................... 69
3.6. Transcription / DNA Transcription...................................................... 70
3.7. Translation......................................................................................... 74
3.8. The Steps of Translation...................................................................... 75
3.9. Genetic Information Transfer Promotes Cooperation in Bacteria......... 77
3.10. Cellular Organization of the Transfer of Genetic Information........... 78
3.11. The Flow of Genetic Information...................................................... 81
3.12. Horizontal Gene Transfer................................................................. 82
3.13. Conclusion...................................................................................... 89
References................................................................................................ 90

Chapter 4 Chromatography and Biochemistry.......................................................... 91

4.1. Introduction....................................................................................... 92
4.2. Principles of Chromatography............................................................ 93
4.3. Performance Parameters Used in Chromatography............................. 97
4.4. Chromatography Equipment............................................................ 104
4.5. Modes of Chromatography............................................................... 108
4.6. High-Performance Liquid Chromatography (HPLC).......................... 113
4.7. Membrane-Based Chromatography Systems..................................... 117
4.8. Chromatography of a Sample Protein............................................... 118
4.9. Conclusion...................................................................................... 120
References.............................................................................................. 121

Chapter 5 Mass Spectrometry and Spectroscopic Techniques................................ 123

5.1. Introduction to Mass Spectrometry................................................... 124
5.2. Principles of Mass Spectrometry...................................................... 126
5.3. Uses of Mass Spectrometry in Biochemistry..................................... 131
5.4. Mass Spectrometry of Proteins/Peptides........................................... 134
5.5. Introduction to Spectroscopic Techniques........................................ 137

viii
 5.6. Fluorescence Spectroscopy.............................................................. 140
5.7. Fluorescence Correlation Spectroscopy........................................... 142
5.8. Infrared Spectroscopy...................................................................... 144
5.9. Spectroscopic Techniques Using Plane-Polarized Light.................... 147
5.10. Conclusion.................................................................................... 150
References.............................................................................................. 151

Chapter 6 Principles of Clinical Biochemistry......................................................... 153

6.1. Introduction..................................................................................... 154
6.2. Principles of Clinical Biochemical Analysis...................................... 155
6.3. Clinical Measurements and Quality Control.................................... 162
6.4. Examples of Biochemical Aids to Clinical Diagnosis........................ 171
6.5. Conclusion...................................................................................... 180
References.............................................................................................. 181

Chapter 7 Spectroscopy Techniques in Biochemistry............................................. 183

7.1. Introduction..................................................................................... 184
7.2. Properties of Electromagnetic Radiation........................................... 186
7.3. Interaction with Matter.................................................................... 187
7.4. Lasers.............................................................................................. 189
7.5. Ultraviolet and Visible Light Spectroscopy....................................... 190
7.6. Principles......................................................................................... 192
7.7. Instrumentation................................................................................ 194
7.8. Applications.................................................................................... 197
7.9. Instrumentation................................................................................ 202
7.10. Applications.................................................................................. 203
7.11. Conclusion.................................................................................... 211
References.............................................................................................. 212

Chapter 8 Biochemistry of Lipids............................................................................ 213

8.1. Introduction..................................................................................... 214
8.2. Diversity in Lipid Structure.............................................................. 217
8.3. Properties of Lipids In Solution........................................................ 219
8.4. Engineering of Membrane Lipid Composition.................................. 223
8.5. Role of Lipids in Cell Function......................................................... 227

ix
8.6. Lipid Metabolism in Plants............................................................... 237
8.7. Plant Lipid Geography..................................................................... 238
8.8. Future Directions of Lipids............................................................... 239
8.9. Conclusion...................................................................................... 241
References.............................................................................................. 242

Index...................................................................................................... 243
LIST OF FIGURES

Figure 1.1. Hemoglobin molecules

Figure 1.2. Enzyme
Figure 1.3. Enzyme mechanism-a model
Figure 1.4. Five Important Monosaccharides
Figure 1.5. Three Important Polysaccharides
Figure 2.1. Four major constituents of a cell
Figure 2.2. The structural formula of carbohydrates
Figure 2.3. The structural formula of lipids
Figure 2.4. Terpene biosynthesis
Figure 2.5. Types of ELISA
Figure 3.1. Transduction pathways
Figure 3.2. DNA transcription process
Figure 3.3. Genetic codes
Figure 3.4. Schematic representation of translation process
Figure 3.5. Gene transfer in bacteria
Figure 4.1. Partitioning of biomolecules in a two-phase system. Circles and squares,
respectively, symbolize two components. An aqueous buffer and a solid stationary
phase could be the two phases. In this experimental setting, the partition coefficients of
the two samples are drastically different.
Figure 4.2. A typical liquid chromatography system. Arrows indicate the flow
direction. The sample is loaded by injecting it into a valve. If a high number of samples
are needed, an autosampler can be utilized to repeatedly refill the column after each
chromatography step.
Figure 4.3. A typical liquid chromatography experiment. Ion exchange chromatography
was used to separate a group of isoenzymes. With detection at 280 nm, a 0–100 mM NaCl
gradient (dashed line) is utilized. Proteins are frequently detected at this wavelength.
Peak 1 contains free material, whereas peaks 2–4 contain proteins that are bound with
increasing affinity.
Figure 4.4. Retention in column chromatography. A typical chromatography trace
demonstrating the separation of two components (1 and 2). The component retention
volumes are shown by V1 and V2, respectively, whereas the base peak widths are
indicated by W1 and W2. V0 stands for the void volume.
Figure 4.5. Physical causes of band broadening. (a) Eddy diffusion, (b) Mobile
phase mass transfer, (c) Stagnant mobile phase mass transfer, (d) Stationary phase
mass transfer. All of these factors may work together to broaden the applied sample’s
comparably limited starting bandwidth.
Figure 4.6. The van Deemter curve. A plot of H versus flow rate, v, is shown (solid
line).
Figure 4.7. Elution from stationary phase. The mobile phase is represented by dashed
lines. As explained in the book, the composition of this may change to give various
pH, ion strength, or hydrophobicity. (a) Continuous flow elution. When sample
components have distinct inherent affinities for the stationary phase and the mobile
phase composition and flow rate remain constant, the sample components separate. (b)
Batch flow elution. Adsorbed sample components can be eluted selectively by washing
the column with a variety of mobile phases in a sequential manner. (c) Gradient elution.
Adsorbed material is separated using a gradient consisting of two or more buffers with
a continuously changing mobile phase composition. Continuously adjusting % buffer B
in the mobile phase can produce more complex gradients (e.g., hyperbolic gradients).
Figure 4.8. The link between different chromatography systems in terms of
chromatography mode and format is depicted in this matrix. A selection of regularly
used stationary phases is depicted.
Figure 4.9. Surface charge of proteins. At a variety of pH values, the net charge on the
surface of two proteins (pI values of 5.5 and 7.5, respectively) is depicted. Take note of
how this changes with pH.
Figure 4.10. Ion exchangers. The structure of (a) diethylaminoethyl (DEAE)-cellulose,
an anion exchanger and (b) carboxymethyl (CM)-cellulose, a cation exchanger.
Figure 5.1. Mass spectrometry principle
Figure 5.2. MALDI-TOF target plate for microbial identification
Figure 5.3. Ionization chamber made by Pierre Curie, c 1895-1900
Figure 5.4. Peptide fragmentation
Figure 5.5. The new Nuclear Magnetic Resonance (NMR) instrument in BSF analyzes
small molecular samples
Figure 5.6. Raman spectroscopy enables scientists to study at the molecular level the
chemical and physical changes of ceramic materials as they undergo friction and wear
degradation.
Figure 5.7. The Fourier Transform Infrared Spectroscopy (FTIR) Chamber
Figure 6.1. Hypothalamic-pituitary-adrenal axis. ACTH, adrenocorticotropic hormone;
CRH, corticotropin-releasing hormone
Figure 6.2. Alanine transaminase reaction

xii
Figure 6.3. Amniocentesis
Figure 6.4. Venipuncture using a BD Vacutainer
Figure 6.5. Newborn hearing screening
Figure 6.6. Patterns of topographic distribution of myocardial infarction
Figure 6.7. A kidney nephron and its structure
Figure 6.8. A graphic representation of a chronically affected kidney
Figure 7.1. UV vis spectroscopy
Figure 7.2. Electromagnetic spectrum
Figure 7.3. Laser beams
Figure 7.4. Spectro photometer
Figure 7.5. A colorimetric assay
Figure 7.6. Fluorescence Spectroscopy
Figure 7.7. Fluorescence chromatographer- working
Figure 8.1. Image showing 4 common lipids
Figure 8.2. Scheme of the general structures of membrane phospholipids and glycolipids
Figure 8.3. Lipid bilayer and micelle
Figure 8.4. The cell membrane
Figure 8.5. Protein targeting the thylakoid diagram
Figure 8.6. Diffusion across the plasma membrane
Figure 8.7. The comparison of the process of cytokinesis in plant and animal cells
Figure 8.8. Lipid metabolism

xiii
LIST OF ABBREVIATIONS

AATs Alcohol Acyltransferases

ACF Auto Correlation Function
ACTH Adrenocorticotropic Hormone
AFP Alpha -Fetoprotein
ALT Alanine Transaminase
AP Alkaline Phosphatase
APLAC Asia-Pacific Laboratory Accreditation Co-Operation
ARF Acute Renal Failure
AST Aspartate Aminotransferase
ATP Adenosine Triphosphate
ATP Adenosine triphosphate
BRET Bioluminescence Resonance Energy Transfer
BSI British Standards Institution
CA Cinnamic Acid
CFP Cyan Fluorescent Protein
CHI Community Health Index
CK Creatine Kinase
CKD Chronic Kidney Disease
CL Cardiolipin
CPA Certified Public Accountant
CSF Cerebrospinal Fluid
DHB Defense Health Board
DMAPP Dimethylallyl Diphosphate
DNA Deoxyribonucleic Acid
ECD Electron Capture Dissociation
EDTA Ethylenediamine Tetraacetic Acid
EF Enterococcus faecalis
ELISA Enzyme-Linked Immunosorbent Assay
EP Enzyme-Product
ER Endoplasmic Reticulum
ES Enzyme-Substrate
ETD Electron Transfer Dissociation
FAB Fulfillment Assurance And Billing
FAD Flavin Adenine Dinucleotide
FCS Fluorescence Correlation Spectroscopy
FLS Flavanol Synthase
FPLC Fast Protein Liquid Chromatography
FRET Fluorescence Resonance Energy Transfer
GC Gas Chromatography
GFP Green Fluorescent Protein
GFR Glomerular Filtration Rate
GGT G-Glutamyl Transferase
GLC Gas-liquid chromatography
GST Glutathione Transferase
HETP Height Equivalent to A Theoretical Plate
HGT Horizontal Gene Transfer
HPLC High-Performance Liquid Chromatography
Hz Hertz
ILAC International Accreditation Co-operation
IMS Information Management System
IPP Isopentenyl Diphosphate
ISC Intersystem Crossing
ISEs Ion-Selective Electrodes
K Kelvin
LC Liquid Chromatography
LD Lactate Dehydrogenase
LD Linear Dichroism
MALDI Matrix Assisted Laser Desorption/Ionization
MCADD Medium-Chain Acyl-CoA Dehydrogenase Deficiency
MEP Methylerythritol Phosphate
MGEs Mobile Genetic Elements
MIRS Management Information & Retrieval System
MRI Magnetic Resonance Imaging
mRNA Messenger RNA

xvi
MS Mass Spectrometry
MVA Mevalonic Acid
NAD Nicotinamide Adenine Dinucleotide
NAMAS National Measurement Accreditation Service
NIRS Near Infrared Spectroscopy
Nm Nanometer
NMR Nuclear Magnetic Resonance
OD Optical Thickness
PA Phosphatidic Acid
PC Phosphatidylcholine
PCA Principal Components Analysis
PEP Phosphoenolpyruvate
PG Phosphatidylglycerol
PGS Phosphatidyl Glycerophosphate Synthase
PI Phosphatidylinositol
PKU Phenylketonuria
PS Phosphatidylserine
RF Retardation Factor
RIA Radioimmunoassay
RM Restriction-Modification
RNA Ribonucleic Acid
SA Sinapinic Acid
SUF Sulfur Utilization Factor
TOF Time-Of-Flight
tRNA Transfer RNA
TSH Thyroid-Stimulating Hormone
UK NEQAS United Kingdom National External Quality Assessment Service
UTP Uridine Triphosphate
UV Ultra Violet
VOCs Volatile Organic Compounds
WEQAS Wales External Quality Assurance Scheme
YFP Yellow Fluorescent Protein

xvii
GLOSSARY

A
Absorption Spectrum- a spectrum of electromagnetic radiation transmitted through a
substance, showing dark lines or bands due to absorption at specific wavelengths.
Amniocentesis - Amniocentesis is a medical procedure used primarily in prenatal
diagnosis of chromosomal abnormalities and fetal infections as well as for sex
determination. In this procedure, a small amount of amniotic fluid, which contains fetal
tissues, is sampled from the amniotic sac surrounding a developing fetus
Amphiphilic Nature- A chemical compound possessing both hydrophilic (water-
loving, polar) and lipophilic (fat-loving) properties.
Anabolism- the synthesis of complex molecules in living organisms from simpler ones
together with the storage of energy.
Assay- An assay is an investigative procedure in laboratory medicine, mining,
pharmacology, environmental biology and molecular biology for qualitatively assessing
or quantitatively measuring the presence, amount, or functional activity of a target
entity.

B
Biomolecule- an organic molecule that includes carbohydrates, protein, lipids, and
nucleic acids.
Biopolymers- polymers that are produced by or derived from living organisms,
Biosynthesis- the production of complex molecules within living organisms or cells.

C
Cataract – A clouding or loss of transparency of the lens in the eye as a result of tissue
breakdown and protein clumping.
Chirality - In chemistry, a molecule or ion is called chiral if it cannot be superposed on
its mirror image by any combination of rotations, translations, and some conformational
changes. This geometric property is called chirality.
Chromatographic - In chemical analysis, chromatography is a laboratory technique
for the separation of a mixture into its components. The mixture is dissolved in a fluid
solvent called the mobile phase, which carries it through a system on which a material
called the stationary phase is fixed.
Colorimetry- Is a scientific technique that is used to determine the concentration of
colored compounds in solutions by the application of the Beer–Lambert law, which
states that the concentration of a solute is proportional to the absorbance.
Condensation - Condensation is the change of the state of matter from the gas phase
into the liquid phase, and is the reverse of vaporization.
Cytoplasm - Cytoplasm is a thick solution that fills each cell and is enclosed by the cell
membrane. It is mainly composed of water, salts, and proteins. In eukaryotic cells, the
cytoplasm includes all of the material inside the cell and outside of the nucleus.

E
Endoproteinases - Endopeptidase or endoproteinase are proteolytic peptidases that
break peptide bonds of nonterminal amino acids (i.e., within the molecule), in contrast
to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids.
Enzymes- are proteins that help speed up metabolism, or the chemical reactions in our
bodies.
Exons- The sequence of DNA present in mature messenger RNA, some of which
encodes the amino acids of a protein.

F
Fat - In nutrition, biology, and chemistry, fat usually means any ester of fatty acids, or
a mixture of such compounds, most commonly those that occur in living beings or in
food.
Fertilization- is the fusion of gametes to give rise to a new individual organism or
offspring and initiate its development.
Fluorophores- A fluorophore (or fluorochrome, similarly to a chromophore) is a
fluorescent chemical compound that can re-emit light upon light excitation.

G
Glycolipids - Glycolipids are lipids with a carbohydrate attached by a glycosidic
(covalent) bond. Their role is to maintain the stability of the cell membrane and to
facilitate cellular recognition, which is crucial to the immune response and in the
connections that allow cells to connect to one another to form tissues.

H
Hematological - Hematology is the branch of medicine concerned with the study of the
cause, prognosis, treatment, and prevention of diseases related to blood.
Hormone - A hormone is any member of a class of signaling molecules in multicellular
organisms, which are transported by intricate biological processes to distant organs to
regulate physiology and behavior. Hormones are required for the correct development
of animals, plants and fungi.

I
Inert Gas - a gas that does not undergo chemical reactions under a set of given
conditions.
Introns- are non-coding sections of an RNA transcript, or the DNA encoding it, that are
spliced out before the RNA molecule is translated into a protein.

M
Macromolecules- are very large molecules important to biophysical processes, such
as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms.
xx
Membranes - Body membranes are thin sheets of tissue that cover the body, line body
cavities, and cover organs within the cavities in hollow organs. They can be categorized
into epithelial and connective tissue membrane.
Metabolic Pathway- a linked series of chemical reactions occurring within a cell.
Microheterogeneity - Variation in the chemical structure of a substance (as the amino
acid sequence of a protein) that does not produce a major change in its properties.
Monochromator - A monochromator is an optical device that transmits a mechanically
selectable narrow band of wavelengths of light or other radiation chosen from a wider
range of wavelengths available at the input. The name is from the Greek roots mono-,
“single”, and chroma, “color”, and the Latin suffix -ator, denoting an agent.
Mutation- is a change in the DNA sequence of an organism. Mutations can result from
errors in DNA replication during cell division, exposure to mutagens or a viral infection.

N
Necrosis - Necrosis is the death of body tissue. It occurs when too little blood flows to
the tissue. This can be from injury, radiation, or chemicals.
Neurotransmitter - A neurotransmitter is a signaling molecule secreted by a neuron to
affect another cell across a synapse. The cell receiving the signal, any main body part or
target cell, may be another neuron, but could also be a gland or muscle cell.
Noncovalent- a type of chemical bond that is found mostly between macromolecules.
Nucleotide- are organic molecules consisting of a nucleoside and a phosphate.

O
Offspring- offspring are the young creation of living organisms, produced either by a
single organism or, in the case of sexual reproduction, two organisms.
Oligosaccharides - An oligosaccharide is a saccharide polymer containing a small
number of monosaccharides. Oligosaccharides can have many functions including cell
recognition and cell binding. For example, glycolipids have an important role in the
immune response.
Organelles - Organelles are specialized structures that perform various jobs inside
cells. The term literally means “little organs.” In the same way organs, such as the
heart, liver, stomach, and kidneys, serve specific functions to keep an organism alive,
organelles serve specific functions to keep a cell alive.

P
Paracrine - Of or relating to a hormone or to a secretion released by (endocrine) cells
into the adjacent cells or surrounding tissue rather than into the bloodstream.
Phenotype- refers to an individual’s observable traits, such as height, eye color and
blood type.
Phospholipids - Phospholipids are major membrane lipids that consist of lipid
bilayers. This basic cellular structure acts as a barrier to protect the cell against various
environmental insults and more importantly, enables multiple cellular processes to
occur in subcellular compartments.

xxi
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