Potentiometric determination of the concentration of active subtilisin

Elizabeth Daugherty, The College of Wooster

Abstract

Subtilisin is a proteolytic enzyme which attacks internal peptide bonds, and hence it tends to undergo autodigestion (13). Further impurities in commercial preparations result from contamination with another hydrolase (20). The concentration of subtilisin cannot, therefore, be determined from the amount of enzyme weighed into solution, but must be measured by some other method. Generally, it is determined from ultraviolet absorbance at 278 mu using a molecular weight of 27,600 and an A1%278 of 11.7 (20). Erlanger and Sack have employed an alternative method for measuring the concentration of the related enzyme chymotrypsin (6). They have used an inhibitor which liberates fluoride ion in the course of binding the active serine residue and have followed the reaction with a fluoride sensitive electrode. This method can hopefully be adapted for use with other serine proteases such as subtilisin. This suggestion forms the basis for the project discussed here. Subtilisin is rapidly inhibited at serine 221 by phenylmethanesulfonylfluoride (PMSF), with the liberation of fluoride ion in solution (18): INSERT FORMULA HERE. Since virtually complete inhibition takes place in several minutes, the amount of fluoride ion is equivalent to the amount of active enzyme originally present in the sample. A potentiometric measurement of the fluoride concentration could be made with an electrode sensitive specifically to the fluoride ion, using a calibration curve to translate millivolts into molarity of active enzyme. Results could be compared to those obtained spectophotometrically. Once subtilisin concentration could be measured rapidly and accurately, other enzyme studies involving the fluoride electrode might be attempted.