Inactivation of cathepsin B by active site-directed disulfide exchange. Application in covalent affinity...

The ability of cystamine, bis-N-aminoethyl disulfide, to inactivate cathepsin B by disulfide exchange is considerably enhanced by the addition of hydrophobic residues which apparently occupy secondary binding sites in the extended active center of the protease. For example, a peptidyl cystamine derivative such as symmetrical Gly-Phe-cystamine is substrate-like and promotes the disulfide exchange by affinity labeling. The effectiveness of this type of reagent is dependent on the peptidyl sequence. The inactivation is more rapid with increasing pH in the narrow range studied, pH 5-7, and is completely reversed by added thiols. Immobilization of the peptidyl cystamine derivatives provided an effective and rapid procedure for the covalent chromatography of cathepsin B following ammonium sulfate fractionation of beef spleen or pork liver homogenates, replacing the need for ion exchange chromatography. Pork liver cathepsin B thus obtained was a mixture of single chain and double chain forms as found by other methods.