Potentiometric determination of ionizations at the active site of papain

Publisher Website
Google Scholar
Abstract
The ionization behavior of groups at the active site of papain [from papaya latex] was determined from the pH dependence of the difference in proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by 2 independent methods. One method involved potentiometric measurements of the protons released on demthylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3-7.6 on deprotonation of His-159 at 29.degree. C, T/2 0.05. Similarly, the pK of His-159 shifts from 4.3-8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate the equilibrium constant for transfer of a proton from Cys-25 to His-159 is 8-12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.

This publication has 4 references indexed in Scilit: