CHEMISTRY HL – OPTION B.

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Option B.2 – Proteins and Enzymes
Understanding 1: Proteins are polymers of 2-amino acids, joined by amide links (also known as
peptide bonds).
Proteins are polymers of amino acid monomer units:
Amino acids contain an amino (-NH2) and acid group (-COOH) bonded to the same C atom
- Can be referred to as 2-amino acids
- Have an R group which defines the type of amino acid

Properties:
- Crystalline compounds with high melting points, usually above 200oC
- Higher solubility in water compares to non-polar solvents
- Move in an electric field

Understanding 2: Amino acids are amphoteric and can exist as zwitterions, cations, and
anions.
> Application and Skills 2: Explanation of the solubilities and melting points of amino acids in
terms of zwitterions.
> Application and Skills 3: Application of the relationships between charge, pH and isoelectric
point for amino acids and proteins.
Zwitterions are amino acids in aqueous and crystalline forms which are able to exist with +ve and -ve charges within the
molecule
Can be referred to as internal salts – charges result from internal acid-base reaction with transfer of proton from acid (-COOH) to
basic (-NH2) group

Amino acids are amphoteric/amphiprotic as the contain an acidic and basic group – in aq solutions, they will accept and donate
H+ according to changes in the pH of the medium:

*it is the conjugates of the acid and base that are responsible for this property

pH of the medium affects the equilibrium, position of these reactions, it influences the charge of amino acids:
- High pH (low [H+]), reaction 1 is favoured as -NH3+ group loses its H+ and forms an anion
- Low pH (high [H+]), reaction 2 is favoured as the -COO group gains H+ and forms a cation

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Isoelectric point is the intermediate pH at which the amino acid is electrically neutral

- With no net charge at this pH, amino acids will not move in an electric field
- Molecules will have minimum repulsion and hence be least soluble

Amino acids such as Gly and Ala have uncharged R groups have the same isoelectric point of pH 6.0:
- BUT when R group contains an acidic or basic group then pKa and pKb values of these groups will also influence the charge
as pH changes

Amino acids also act as pH buffers by reacting to H+ and OH- ions – cause the pH to be resistant to change in addition of small
amounts of acid or alkali
- Buffering role of amino acids is important in helping to maintain a constant pH in cells, a crucial need for biological
solutions
- Manu protein components are extremely sensitive to changes in pH and can be made inactive by significant fluctuations

Understanding 3: Protein structures are diverse and are described at the primary, secondary,
tertiary, and quaternary levels.
> Application and Skills 4: Description of the four levels of protein structure, including the
origin and types of bonds and interactions involved.
Primary Structure
Is formed through a condensation reaction between two or more amino acid monomers – a peptide bond is created
- This forms the covalent backbone of the molecule

Secondary Structure
Consists of the folding of the polypeptide chain as a result of hydrogen bonding between peptide bonds -C=O group and -NH
group of another peptide bond – this causes the chain will cause the chain to fold
There are two main types of secondary structures, the α-helix, and the β-pleated sheet
α-helix β-pleated sheet
- Regular coiled configuration of the polypeptide chain - Composed of ‘side-by-side’ polypeptides which are in
- Resulting from hydrogen bonds forming between 2 extended form
peptide bonds 4 amino acids apart - Not as tightly coiled as in α-helix
- Twists the chain into a tightly coiled helix with 3.6 - Arranged in pleated sheets that are cross linked by
amino acids per turn inter-chain hydrogen bonds
- Flexible and elastic as the interchain hydrogen bonds - Flexible and inelastic
easily breaks and re-form as molecule is stretched - E.g. fibroin, protein in the fibres spun by spiders and
- E.g. keratins, structural proteins found in hair, skin.. silkworms

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Tertiary Structure
Tertiary structure refers to the further twisting, folding and, coiling of the polypeptide as a result of interactions between the R
groups (side chains)
Results in a specific and compact 3D structure
- Known as the protein’s confirmation
- Most stable arrangement of the protein accounting for the possible interactions along entire length of polypeptide
- All interactions between side chains are intra-molecular forces as they occur within one polypeptide chain

This conformation is important in globular proteins (include enzymes and protein hormones):
- Are water soluble as their structural positions cause polar (hydrophilic) side chains on the outer surface whilst non-polar
(hydrophobic) side chains are on the interior away from water

Interactions are:
(a) Hydrophobic interactions – between non-polar side chains, i.e. two alkyl side groups in Val which form through London
dispersion forces between induced dipoles which produce non-polar regions in the interior of the protein
(b) Hydrogen bonding – between polar side chains, i.e. -CH2OH group in Ser and -CH2COOH group in Asp
(c) Ionic bonding – between side chains carrying a charge, i.e. between –(CH2)4NH3+ group in Lys and -CH2COO- group in Asp
(d) Disulfide bridges – between the sulfur-containing amino acid Cys, these are covalent bonds, hence strongest of these
interactions

- These interactions are susceptible to changes in the medium, i.e. temperature, pH, or presence of metal ions
Denaturation is the process in which a protein loses its specific tertiary structure as a result of disruptions and will render
them biologically inactive

Quaternary Structure
Quaternary structures are comprised of more than one polypeptide chains and involves forces and bonds to tertiary structures
- E.g. protein collagen which is found in skin and tendons and is most abundant protein in human body, it is a triple helix of
3 polypeptide chains which interchain hydrogen bonds to help give s stable rope-like structure resistant to stretching
- E.g. Haemoglobin responsible for carrying O in the blood which is made up of 4 poly peptide chains that fit together tightly
in the protein assembly

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Any condition that affects the enzymes shape and binding ability affects its catalytic action:

(1) Temperature
- ROR is increased by rise in To  incr. in kinetic energy  incr. in freq. of collision, only true up to certain To.
- Further incr. in To & kinetic energy changes the conformation of the protein (by disrupting bonds and forces responsible
for holding it in its 3o structure).
- Enzyme no longer able to bind to substrate at active site  catalytic activity is diminished  explains the shape of the
curve.

1. Optimum temperature: corresponds to the max rate of reaction for a particular enzyme (most enzymes in the human have
optimum value close to 37oC)
2. Denaturation: loss of 3o structure is irreversibly
3. Deactivation: lowering To prevents the enzyme from working but does not change the 3o structure. Usually reversibly.

(2) pH
- Changes in pH directly influences the state of ionization of the proteins side chains (acidic/basic groups)
- Le Chatelier’s Principle:
 low pH (high [H+])  acidic & basic groups become protonated high
 pH (low [H+])  acidic & basic groups become deprotonated
- Precise influence of pH depends on the pKa and pKb values of the acidic & basic and varies in different enzymes.
- Changes in ionisation of the side chains of amino acids affect ability of the enzyme to form a complex with substrate.
- In most cases there is a clear optimum value for pH at which the enzyme has maximum activity.
- Typically, enzymes show max activity when pH is close to pKa value of amino groups at its active site  identify likely
amino acid at its active site.
- Unlike for T, different enzymes in the same organism can have a wide variation in pH optimum values  helps in
controlling enzyme activity

low pH  high [H+]  acidic & basic side groups become protonated
high pH  low [H+]  acidic & basic side groups become deprotonated

(3) Heavy metal ions (Hg, Pd, Ag, Cu)

- Heavy metals are poisonous due to their effect on enzymes.
- When present as +ve ions, they react with sulfhydryl groups, –SH, in the side chains of the cysteine residues in protein,
forming covalent bond with S and displacing H+
- Disrupts the folding of the protein, which changes the shape of the active site and its ability to bind substrate.

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Understanding 4: A protein’s three-dimensional shape determines its role in structural

components or in metabolic processes.
> Application and Skills 1: Deduction of the structural formulas of reactants and products in
condensation reactions of amino acids, and hydrolysis reactions of peptides.
> Understanding 6: As enzyme activity depends on the conformation, it is sensitive to
changes in temperature and pH and the presence of heavy metal ions.

Fibrous Proteins Globular Proteins

- Responsible for structure, support, and movement - Drive the reactions of metabolism
- Structural components - Tools that operate at the molecular level (e.g. enzymes,
- Elongated molecules with dominant secondary carriers, receptors)
structures - Compact spherical molecules with dominant tertiary
- Insoluble in water structure
- Soluble in water

Understanding 5: Most enzymes are proteins that act as catalysts by binding specifically to a
substrate at the active site.
Enzymes are biological catalysts which control every reaction in biochemistry as they are specific for every reaction and can be
individually controlled

Enzymes are globular proteins which exist in compact spherical shapes when in aq solutions in cells:
- Well defined tertiary structure gives a specific 3D shape which is essential for enzyme activity
- Typically, relatively large molecules, containing several hundred amino acids
- Some also have quaternary structures
- Some enzymes require non-protein molecules to be bound for activity, these are known as:
 Co-factors when they are organic
 Coenzymes when they are inorganic

They form a complex with a substrate:

- The reactant in the reaction catalysed by the enzyme is known as the substrate

Action of enzyme is due to its ability to form temp binding to substrate where it is held by relatively weak forces of attraction –
forming an enzyme-substrate complex:
- Binding occurs at the active site which is typically a pocket or groove on the surface of the protein
- Formation of complex depends on a chemical fit
- Involves non-covalent hydrophobic interactions, dipole-dipole attractions, hydrogen bonds and, ionic attractions
- Binding puts a strain on substrate molecule and facilitates bond breaking and forming

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CHEMISTRY HL – OPTION B.2
The rate of enzyme-catalysed reactions can be followed with graphs of substrate concentration against rate of reaction.
- Curves show a distinctive shape of saturation and used to describe the rate of enzyme reactions

(a) At low substrate concentration:

- The rate of the reaction is proportional to the substrate concentration.
- Enzyme is available to bind to the substrate
(b) As the substrate concentration is increased:
- The rate decreases and is no longer proportional to the substrate concentration.
- Some of the enzyme has its active sites occupies by substrate and is not available
(c) At high substrate concentration:
- The rate is constant and independent of substrate concentration.
- At this point, the enzyme is saturated with substrate.

Understanding 7: Chromatography separation is based on different physical and chemical

principles.
> Application and Skills 6: Explanation of the processes of paper chromatography and gel
electrophoresis in amino acid and protein separation and identification.
Many different approaches and techniques are used in protein analysis – there are 2 main aspects of this work:
- Analysis of amino acid composition of an isolated protein
- Analysis of the protein concentration of a sample

Analysis of amino acid composition of a protein

1. To break the peptide bonds between the amino acids in the protein structure through hydrolysis reactions – usually with
acids
2. Separation of the resulting amino acid mixture into its component can then be achieved in two ways

Chromatography
Chromatography is a useful technique for separating and identifying the components of a mixture
Components have different affinities for two phases, stationary phase and a mobile phase:
- Amino acids ae colourless in solution
- Usually treated with a locating reagent at the end of the process to allow them to take colour to aid identification

Paper chromatography is an example of partition chromatography in which the components are separated on the basis of
their different solubilities in the two phases and is mainly used of qualitative analysis
1. Paper contains approx. 10% water and is called the stationary phase
2. Water is absorbed by forming hydrogen bonds with the -OH group in cellulose of paper
3. Mobile phase starts as water rises up paper through capillary action
4. As it moves, it dissolves components of mixture to different extents, carrying them at different rates

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The procedure is simple to run:

1. Small sample of amino acid mixture is spotted near the bottom of the chromatographic paper, this is known as the origin
*this should be clearly marked in pencil to not interfere with the experiment
2. Paper is then suspended in chromatographic tank containing small volume of solvent – ensuring that spot is above level of
solvent – as solvent rises up the paper, it will pass over the spot
3. Amino acids in the spot will distribute themselves between the two phases and move up paper at different speeds
4. Due to differing speeds, they become spread out according to their different solubility
5. When solvent reaches top part of paper its final position is marked – this is the solvent front
6. Paper is removed from take and developed by spraying with locating reagent ninhydrin
7. Amino acids will appear purple, is distinguished as separate isolated spots up length of paper – this is the chromatogram

Position of each amino acid can be represented as an Rf value (retention factor) – calculated as below:
distance moved by amino acid
Rf=
distance moved by solvent
Hence, for amino acids:
a
Rf=
x

Specific amino acids have characteristic Rf value when measured under same conditions – can be spotted be comparing values
with values in data tables

Electrophoresis
Electrophoresis is a technique for the analysis and separation of a mixture based on the movement of charged particles in an
electric field – this is because amino acids have different charges depending on the pH

- pH = isoelectric point, amino acids will not move as they carry no net charge
- pH > isoelectric point, amino acids exist as anions and move to the anode
- pH < isoelectric point, amino acids exist as cations and move to cathode

- Rate of movement of ions depend on no. of charges on ions and molecular mass – smaller, more charged ions travel faster
- Voltage and temperature also affect the rate of movement

Gel electrophoresis, medium is a gel – typically made of polyacrylamide

1. Amino acid is placed in wells in centre of gel and voltage is applied
2. Depending on pH of buffer used, different amino acids will move at different rates in different directions towards
oppositely charged electrodes
3. When separation is complete, they can be detected with a stain (i.e. ninhydrin) or made to fluoresce under UV light
4. Then it is identified by comparison with known samples or from data tables

Electrophoresis can also be used to separate and identify intact proteins according to their different rates of migration towards
the poles

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Guidance 1: The names and structural formulas of the amino acids are given in the data
booklet in section 33.

Guidance 2: Reference should be made to alpha helix and beta pleated sheet, and to fibrous
and globular proteins with examples of each.

Guidance 3: In paper chromatography the use of Rf values and locating agents should be
covered.

Guidance 4: In enzyme kinetics Km and Vmax are not required.