Enzyme assays 

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Enzyme assays are mainly used for determination of body status of vitamins. As vitamins usually function either as coenzymes or building blocks of coenzymes, the activity of the vitamin-dependent enzymes is a measure of vitamin status.

Usually, the assay is carried out by determining the enzyme activity with and without activation by added coenzyme. The activity can be monitored by measuring changes in concentration of substrates or products during the reaction. An activation coefficient can be deduced, which reflects the status of the enzyme investigated, and thus the vitamin status.

Most assays are conducted with whole blood or the separated erythrocyte fraction. They can be automated with clinical analysers. Disadvantages include difficulties in assay standardization, instability of the enzymes during storage, and misleading results, e.g., due to conditions other than vitamin deficiency leading to low Apo enzyme concentrations.

Enzyme Structure

Enzymes are a linear chain of amino acids, which give rise to a three-dimensional structure. The sequence of amino acids specifies the structure, which in turn identifies the catalytic activity of the enzyme. Upon heating, enzyme’s structure denatures, resulting in a loss of enzyme activity that typically is associated with temperature.

Compared to its substrates, enzymes are typically large with varying sizes, ranging from 62 amino acid residues to an average of 2500 residues found in fatty acid synthase. An only small section of the structure is involved in catalysis and is situated next to the binding sites. The catalytic site and binding site together constitute the enzyme’s active site. A small number of ribozymes exist which serves as an RNA-based biological catalyst. It reacts in complex with proteins.

Types of Enzyme Assays

All enzyme assays measure either the consumption of substrate or production of product over time. Biochemists usually study enzyme-catalysed reactions using four types of experiments:

• Initial rate experiments: When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
• Progress curve experiments: In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. Progress curve experiments were widely used in the early period of enzyme kinetics, but are less common now.
• Transient kinetics experiments: In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require specialist techniques (such as flash photolysis of caged compounds) or rapid mixing (such as stopped-flow, quenched flow or continuous flow).
• Relaxation experiments: In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored.. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.

On the occasion of its 10 years, Successful Journey, Journal of Clinical and Experimental Pathology decided to provide a partial waiver on its article processing charges to promote quality research from across the nations of the globe to encourage the latest research in the field of Infections, Diseases and Medicine. Journal of Clinical and Experimental Pathology also planning to release a special issue on its new approaches.

Regards,

Robert Solomon

Editorial office

Journal of Clinical and Experimental Pathology

E-mail: pathol@eclinicalsci.com

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