Purine nucleoside phosphorylase: Immobilization by covalent chromatography and a study on its sulfhydryl groups

Covalent chromatography is used for studying the role of sulfhydryl groups in pig brain purine nucleoside phosphorylase (PNP). This enzyme has been immobilized on an insoluble polymeric reagent (thiol-Sephnrose 4B) by a thiol-disulfide interchange reaction between the disulfide groups of the gel and some of its thiol groups. The immobilized enzyme retained its activity and the coupling was reversible under reducing conditions, allowing the recovery of the enzymic activity. These results suggest that PNP contains nonessential sulfhydryl groups that can react with the thiol-Sepharose. On the other hand, inactivation with some thiol reagents shows that thiol groups directly involved in the catalytic activity are present at or near the active site. The technique described should be generally useful in the immobilization of thiol-containing proteins and in the characterization of these thiol groups.