Enzyme Assay - Enzyme Denaturation Mechanism and Enzyme Stabilization Strategies

Enzyme Assay

The role of enzyme assay is to

• To Identify an Enzyme - Presence or Absence - By qualitative approach.
• To check amount of enzyme activity - By quantitative approach.

Enzyme identification by catalytic action of enzyme. Enzyme activity depends on the following factors:

Temperature

pH, Nature and Strength of ions.

General Considerations for an enzyme assay

Scattering:

• Instability and Instruments
• Measurements in turbid solutions
• Contamination

Turbidity caused by weakly soluble substance, soiling, dust or air bubble.

Low scattering only possible, only if the observed component produces a signal (eg: absorption), while other component show no signal (No absorption) in the observed range.

Methods for Observing enzyme reaction

By measuring the appearance or disappearance of colored compound:

1. Photometry
2. Fluorometry
3. Turbidometry
4. Luminometry

Continuous Assay

Continuous assay is important for determination of enzyme velocity and for evaluating the enzyme activity.

Continuous assay helps in the detection of erroneous influences and artifactual disturbances and control of reaction course (Progress Curve).

Influence of pH on Enzyme assays

Enzyme activity follow bell curve

Two aspects are responsible for pH dependent activity:

• State of protonation of functional group of amino acids and co-factors involved in the catalytic reaction.
• The native, 3-D Protein structure of enzyme.

Protonation is reversible, but damage to the protein structure is irreversible.

The Inflexion points of the curve at half maximum velocity (Vmax/2) indicate the pKa value approximately. pH <3 or="">11 even for short time should be avoided except for the enzymes like trypsin which is tolerant to acidic pH.

Influence of Buffer Ions on Enzyme activity

Buffers helps to adjust and stabilize pH during enzyme assay.

pH = pKa - log [HAc] / [Ac-]

pH = -log[H+]; pKa = -log Ka

Two criteria for buffers:

• Ionic Strength and Concentration : generally 0.05 - 0.2 M used.
• Nature of buffer components

Influence of Temperature on Enzyme activity

Typical dependence of the enzyme activity on the temperature.

 (A)Direct plotting and(B)Arrhenius diagram.

The green lines represent the range of the increase of the reaction velocity with the temperature;its continuation(dotted violet line) is interrupted by progressive inactivation (redlines).

Inactivation is forced by pre-incubation of the enzyme at the high temperature,causing a decrease and shift of the temperature maximum to the lower range (black arrows).In(A)the three most commonly used assay temperatures are indicated.

Enzyme Denaturation Mechanism

Enzyme denaturation is the unfolding of enzyme tertiary structure to a disordered polypeptide in which key residues are no longer aligned closely enough for continued participation in functional or structure stabilizing interactions.

Invitro Protein Stability:

1. Thermodynamic Stability (Kinetic Stability)
2. Long term stability (Conformational Stability)

Enzyme Inactivation Studies:

• Biochemical / Structural - Effect of temperature and other reagents on secondary and tertiary structure.

• Mathematical Simulations - Effects of agents on enzyme activity.

Enzyme Inactivation:

Enzyme inactivation is a two step Process

1. Reversible unfolding of original enzyme followed by

2. Kinetically irreversible steps: Aggregation or covalent changes in the enzyme.

Factors Affecting Enzyme Stability / Inactivation

• Temperature
• Pressure
• Salt

Enzyme Stabilization: In aqueous environment

• Screening enzymes from extremophiles & their isolation.
• Production of stable enzyme in mesophilic organisms.
•  Stabilize unstable enzyme by protein engineering, chemical modification, Immobilization and medium engineering by additives.

Extremophiles: Eg: Taq Polymerase in thermus aquaticus

Observation in thermophilic enzymes: 

In thermophilic enzymes lysine is replaced by arginine, Thermophilic enzymes generally have high proline content and aspargine/glutamine content is low.

Thermozymes have more interactions:

• Hydrogen bonds
• Disulfide bonds
• Hydrophobic Interaction
• Electrostatic Interaction
• Superior conformational structure.

Kinetic Stability is expressed as half life at defined temperature.

dG is 5-20 kcal/mol.

Modification of Mesophilic Enzymes

Introduction of mutation: Entropic Stabilization by introduction of prolines or disulfide bridges.

Thermal stability due to rigid conformation and higher number of hydrophobic interactions.

Aspargine is thermoliable, threonine or isoleucine have similar geometry to aspargine but are moste heat stable.

Chemical Modification:

Chemical modification of the amino acid side chain can yield stability.

• Monofunctionally substituted proteins:

Thermostability can be achieved by replacing lysine or histidine by arginine resulting in enhanced intramolecular or inter-subunit salt bridges.

• Grafting to Polysaccharides
• Grafting to Polymers

Enzyme Immobilization

Thermal Stability from molecular rigidity introduced by attachment to rigid support and protection by microenvironment.

• Adsorption
• Covalent Bonding
• Entrapment
• Membrane Confinement

Additives:

Addition of ligands, salts, etc can give enzyme stability.

Salts:

• Ion Effect of divalent cations on thermo-stability. Ions employed in concentration ≤ 0.1 M.
• Non- Specific ion effect:  Salts employed at higher concentration ≥ 0.1 M. 

Non-specific ions bind to charged groups or dipole leading to salting out resulting in the compression of the enzyme.

References:

Enzyme stability and stabilization—Aqueous and non-aqueous environment Padma V. Iyer, Laxmi Ananthanarayan.

Enzyme assays Review, Hans Bisswanger