Pressure effects on the Raman spectrum of proteins: stability of the salt bridge in trypsin and elastase

Abstract

Pressure induced changes in trypsin and elastase have been studied with Raman spectroscopy. The secondary structure of the enzymes has been calculated by a singular value analysis of the amide I band. The pressure titration of trypsin indicates a conformational transition. The salt bridge between Asp(194) and Ile(16) is disrupted and the enzyme becomes inactive. No such changes are observed for elastase. It is concluded that the presence of the bulky Val(216) and Thr(226) in the substrate binding site prevents the enzyme changing its conformation as a function of pH and pressure. Starting from a simple electrostatic model, an estimate is made of the dielectric constant of the environment of the salt bridge in trypsin.