Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

✍ Scribed by Peter C. Farley; John T. Christeller; Michelle E. Sullivan; Patrick A. Sullivan; William A. Laing

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 281 KB
• Volume: 15
• Category: Article
• ISSN: 0952-3499
• DOI: 10.1002/jmr.568

✦ Synopsis

Abstract

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10^4^M
^−1^s^−1^), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI‐pepsin complex was much slower (dissociation rate constants ca 10^−4^s^−1^), especially at low pH values. Similar results were obtained with a His‐tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans‐secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4‐inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata‐secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant. Copyright © 2002 John Wiley & Sons, Ltd.