Chemistry in a microenvironment of low pH, generated with the aid of an immobilized proteinase

alpha-Chymotrypsin, when immobilized in a collodion membrane, exhibits high activity and remarkable stability. When the immobilized proteinase is exposed to 15 mM ethyl N-acetyl-L-tyrosinate in dilute pH 8.5 buffer it generates a microenvironment which, indicator studies suggest, has an effective pH of approximately 4. The presence of this locally highly acidic region produces a marked increase in the rate of hydrolysis of BzPheal = Ala dissolved in the buffer solution (BzPheal = Ala is the acylhydrazide obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide). The observed rate is 10-times greater than in comparable control experiments incorporating a concentrated buffer solution, in which a pH-gradient does not form. The enhanced hydrolysis rate is quantitatively explained if it is attributed to the approximately 20 microliters of pH 4 solution within the membrane. Other experimental data are also consistent with this hypothesis.