Proteases are enzymes that facilitate proteolysis, or the breakdown of protein and peptide molecules into smaller polypeptides and/or amino acids. These enzymes do their job by cleaving the peptide bonds linking the amino acids together in the polypeptide chains.
Virtually all living organisms have proteases in their system. These enzymes are involved in many physiological reactions such as breaking down proteins in food during digestion to allow for the absorption of amino acids. They also play a crucial role in bone formation, blood coagulation, programmed cell death (apoptosis), the maturation of prohormones and in regulating the immune function. In addition, proteases have also been identified as good drug development targets.
Proteases can also become unavoidable contaminants during protein purification. When present in high levels, it can significantly impact the yield, stability, and activity of purified proteins. Due to the versatility and diverse effects of these enzymes, most researchers find it necessary to monitor their activity in the laboratory. To accomplish this goal, researchers use non-specific protease activity assays which use casein as a substrate as a standardized procedure.
However, since these assays involve multiple steps and long incubation times, several protease assays such as fluorometric and bioluminescent assays have been developed to overcome these limitations.
Universal Protease Activity Assay with Casein as a Substrate
When the protease digests casein, amino acids (including the amino acid tyrosine) and other peptide fragments are liberated. Folin-Ciocalteus Phenol reagent (also known as FCR or Folin’s reagent) then reacts with the free tyrosine to produce a blue colored chromophore, which is measured as an absorbance value on the spectrophotometer. Basically, the amount of tyrosine released from casein is directly proportional to the amount of chromophore generated. Thus, the more tyrosine released, the stronger the activity of the protease in the sample.
Absorbance values are then compared to a standard curve, which is generated by reacting known quantities of tyrosine with Folin's reagent. The absorbance values for the standards, the test samples and the blanks (for both standard and test samples) are recorded and the difference between the absorbance of the test sample and the test blank is calculated. The standard curve is then created by plotting the change in absorbance of the standards (Y axis) and the amount in micromoles for each of the standards (X axis).
Fluorometric Protease Assay
For the accurate quantitative determination of proteases present in the solution (up to picogram level), researchers now rely on newer fluorometric protease assays to do the job. These assays use highly quenched fluorescein isothiocyanate (FITC) labeled casein as substrate.
Since the fluorescence of the substrate is increased upon the digestion of the substrate into smaller peptide fragments, the protease activity can then be measured and quantified directly. The fluorescence intensity is directly proportional to protease activity and is measured with excitation at 485nm and emission at 530nm. Chemically stabilized trypsin is usually used as a general protease standard although other specific protease standards can also be used.
Fluorescent protease assay kits can also help you assess the functional integrity and progress of protease isolation procedures, and quantify protease contamination in biological samples. In addition, they can help characterize the activity of unusual proteases in your protein sample.
In addition, you can also identify the presence of specific proteases in the sample solution by using a sensitive colorimetric protease screening assay and protease inhibitors. This procedure is of utmost importance since it can help researchers formulate a highly specific protease inhibitor cocktail using the minimal number of protease inhibitors required to test their samples. It can also be used to test existing protease inhibitor cocktails and identify their limitations.
Image source: Raymond Bryson
