Purification and characterization of isoforms of β-N-acetylhexosaminidase from mungbean seedlings

Three isoforms of beta-N-acetylhexosaminidase (beta-NAHA), named beta-NAHAs I, II and III, were isolated from six-day-old etiolated mungbean (Vigna radiata) seedlings. beta-NAHA I was purified to apparent homogeneity by a procedure involving Con A-Sepharose chromatography, chromatofocusing, and gel filtration. beta-NAHAs II and III were highly purified. beta-NAHAs I, II and III had molecular masses of 135,127 and 110 kDa, respectively. beta-NAHA I was dissociated into a single 67 kDa protein band. II was dissociated into two protein bands corresponding to 60 and 48 kDa, and III was dissociated into a single 48 kDa protein band in SDS-polyacrylamide gel electrophoresis. The results suggest that isoforms I and III are homodimeric enzymes, each comprising two identical subunits with molecular masses of 67 kDa and 48 kDa, respectively, while isoform II is a heterodimeric enzyme, comprising two non-identical subunits with molecular masses of 60 kDa and 48 kDa. All the enzymes were active against paranitrophenyl-beta-N-acetylglucosaminide (PNP-beta-N-acetylglucosaminide) and PNP-beta-N-galactosaminide. The enzymes were inhibited by 5, 5'-dithiobis (2-nitrobenzoic acid)(DTNB), Ag+, Hg2+, and N, N'-diacetylchitobiose. Km values for isoforms I, II and III were 0.67 mM, 1.04 mM and 1.76 mM, respectively, using PNP-beta-N-acetylglucosaminide as a substrate. These three isoforms had acidic pI values (1, 6.3; II, 6.1; and 111, 5.9). Their optimal pH in the reaction towards PNP-beta-N-acetylglucosaminide was 5.4, 4.7 and 5.7, and optimal temperatures were 65degreesC, 65degreesC and 50degreesC for isoforms I, II and III, respectively.