In many articles I found this description for protease enzyme assay using culture supernatant: "One unit of protease activity was defined as that amount of enzyme causing an increase in absorbance of 0.01 at 280 nm".
How actually is it calculated?
For example, culture supernatant is added to a solution containing casein or gelatin as the protease-substrate and then at 0 min and 60 min of incubation, OD measured at 280 nm against a reaction blank where supernatant is replaced by (suppose) water. From these two OD values at two time points, how is the protease activity calculated?
Optical measurements can give you a tolerably good measurement of activity, with the right substrate. Chymotrypsin, for example, can be measured by observing the hydrolysis of N-Benzoyl-L-tyrosine ethyl ester (BTEE), which changes absorbance strongly at 256nm when ethanol is hydrolyzed from it. I presume your substrate has a strong change at 280nm, due to the peptide itself...? Exposure of a tryptophan or tyrosine, or strong change to its electronic environment...? (https://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/learning-center/concentration-calculation.html). Not knowing the specific substrate you are using makes it hard to say why you are monitoring at 280nm. Even not knowing the specific substrate, however, the principle at work is the same: you are selecting the wavelength that gives the maximal difference in absorbance between the substrate and the product. If the substrate absorbs more at that wavelength than the product, then you monitor loss of absorbance, which is directly proportional to the loss of substrate. If the product absorbs more at that wavelength than the substrate, then you monitor gain of absorbance which is directly proportional to the gain of product. Either one allows you to calculate your protease activity.
Activity = [(Change of Abs) * Dilution Factor]/[Extinction Coefficient*pathlength*(Change in time)]
I will only add that the optical approach has certain limitations, and isn't always the best. Among other factors, for example, it does require that you not have concentrations be very high, that there is no adhesion of substrate to the optical cuvette, and that the cuvette not absorb light strongly at your analytical wavelength. Don't use glass cuvettes or plastic ones at this wavelength, for example. If you need information on various correction techniques that you will have to do, let me know. Good luck.
In the assay, a substrate protein is incubated with the protease and then precipitated with TCA. Amino acids and small peptides formed by the action of protease on the substrate protein are soluble and stay in solution. If the protein contains amino acids absorbing at 280 nm, these can be measured. The absorption coefficients (L mol-1 cm-1) are 1280 for Tyr, 5690 for Trp and 120 for Cys-Cys (doi:10.1016/0003-2697(89)90602-7, similar values also in doi:10.1111/j.1432-1033.1986.tb09653.x). Thus, the rate of increase of absorbance (dA/dt) is proportional to the rate at which product is formed, that is, the enzymatic activity. Unfortunately, it also depends on the amino acid composition of the substrate protein, hence the enzymatic activity is expressed as dA/dt rather than d[P]/dt.
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