A useful method to assay many hydrolytic enzymes is to follow enzymatic activity by determining production or uptake of hydrogen ions during the reaction. For example, consider a protease that catalyzes cleavage of internal peptide binds in a protein:

Assume the enzyme is fully active at pH 8.8, that the pKa of an a-carboxyl group in a peptide is 3.5, and that the pKa of an a-amino group in a peptide is 8.5. How will the action of this protease on a protein affect the pH of an unbuffered reaction mixture (starting at pH 8.8)? Explain your answer.

The protease reaction is run in a pH-stat, a device that maintains a constant pH in a reaction mixture by continuous, automatic addition of acid or base. The instrument has a recorder that plots the volume of acid or base added versus time of the reaction. If the action in (a) is run in a pH-stat maintained at pH 8.8, calculate the millimoles of titrant that must be added per millimole equivalent of the peptide bonds hydrolyzed by the enzyme.