Requirement for a sulfhydryl group for sulfhydryl oxidase activity

Sulfhydryl oxidase was isolated from bovine skim milk membranes using a transient covalent affinity chromatographic method. This preparation exhibited two chemically reactive sulfhydryl groups in the native enzyme and three in the denatured form, based on a subunit weight of 85 kdaltons. The kinetics of inactivation by carboxymethylation with iodoacetate indicated that modification of one sulfhydryl group per enzyme subunit caused complete loss of activity. These results, together with the enzyme's attachment to cysteinylsuccinamidopropyl-glass and the observed initial rate enzyme kinetics, strongly implicate a substituted-enzyme kinetic mechanism with a mixed disulfide as the intermediate enzyme form.