Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Four isozymes of β-N-acetylhexosaminidase (β-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated β-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-β-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. β-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to β-NAHAs from other plant sources.