45] Affinity chromatography of cysteine peptidases

Abstract

Publisher Summary Cysteine peptidases rely on the thiol group of a cysteine residue for catalytic activity. To obtain fully active preparations of cysteine peptidases, it is necessary to separate active and activatable (reversibly oxidized) forms of the enzyme from the irreversibly oxidized sulfinic acid, and from other contaminating proteins. This chapter describes affinity chromatography of cysteine peptidases. A preparation of active cysteine peptidase is passed through a column comprising an immobilized organomercurial compound. Thiol-containing proteins are retained by the column. The extremely low pK of the active site cysteine of cysteine peptidases is taken as an advantage and if the sample is applied at a mildly acidic pH, most thiol-containing compounds will not bind to the column but cysteine peptidases will. These are subsequently eluted with buffer containing a mercurial salt or a thiol compound. In disulfide exchange, a thiol-containing reagent is immobilized on a chromatography matrix and a mixed disulfide is subsequently formed, possibly by use of a thiol containing spectrophotometric reporter group.