Enzyme assay

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

Enzyme units

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.

Enzyme activity

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s⁻¹, but this is an excessively large unit. A more practical and commonly used value is enzyme unit (U) = 1 μmol min⁻¹. 1 U corresponds to 16.67 nanokatals.

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.

K-Casein

K-Casein (or Kappa-casein, k casein, kappa casein) is a mammalian milk protein involved in a number of important physiological processes. In the gut, the ingested protein is split into an insoluble peptide (para kappa-casein) and a soluble hydrophilic glycopeptide (caseinomacropeptide). Caseinomacropeptide is responsible for increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens.

Structure

Caseins are a family of phosphoproteins (αS1, αS2, β, κ) that account for nearly 80% of bovine milk proteins and that form soluble aggregates known as "casein micelles" in which κ-casein molecules stabilize the structure. There are several models that account for the special conformation of casein in the micelles. One of them proposes that the micellar nucleus is formed by several submicelles, the periphery consisting of microvellosities of κ-casein Another model suggests that the nucleus is formed by casein-interlinked fibrils. Finally, the most recent model proposes a double link among the caseins for gelling to take place. All 3 models consider micelles as colloidal particles formed by casein aggregates wrapped up in soluble κ-casein molecules. Milk-clotting proteases act on the soluble portion, κ-casein, thus originating an unstable micellar state that results in clot formation.