Enzyme assay
The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively.
It is the micro moles of product formed by an enzyme in a given amount of time (minutes) under given conditions per milligram of total proteins.
An active site titration process can be done for the elimination of errors arising from differences in cultivation batches and/or misfolded enzyme and similar issues.
The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L−1 s−1).
An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.
Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction.
These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required.
Some enzyme reactions produce light and this can be measured to detect product formation.
Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.
Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution.
Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.
Microscale thermophoresis (MST)[8] measures the size, charge and hydration entropy of molecules/substrates at equilibrium.
[9] The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme.
[10] The material consumption of the all optical MST method is very low, only 5 μl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition.
They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).
Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography.
Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag.
There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates.
At the saturation point, the reaction will not speed up, no matter how much additional substrate is added.
Large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.
