PURIFICATION OF REACTIVE SULFHYDRYL ENZYMES BY BIOSELECTIVE ADSORPTION ON MONOMERIC AVIDIN: PURIFICATION OF SULFHYDRYL OXIDASE

A biotinylation-bioselective adsorption procedure was developed for single-step purification of bovine sulfhydryl oxidase from solubilized skim milk membrane vesicles. Sulfhydryl oxidase was specifically biotinylated by reaction of its chemically reactive sulfhydryl group with a disulfide-containing biotinylation reagent giving a mixed disulfide between the enzyme and the biotinyl moiety. The biotinylated enzyme was then selectively adsorbed on a monomeric avidin matrix. After washing all nonspecifically bound proteins from the matrix, the enzyme was released by reduction of the disulfide with dithiothreitol. Both the biotinylation and the isolation are performed at pH 7.0 under extremely mild conditions. The resulting enzyme preparation appeared to be homogeneous by gel electrophoresis and the specific activity increased over 3000 fold in comparison to whey. The monomeric avidin matrix can be regenerated and used indefinitely. This purification procedure should be generally applicable to proteins with a chemically reactive sulfhydryl group.