Isoelectric focusing studies of A2/1957 influenza neuraminidase and its subunits

Abstract

Purified A2/1957 influenza neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18) and its subunits were examined by isoelectric focusing (`electrofocusing') in sucrose gradients. Native neuraminidase contained enzymically active components with isoelectric points (pI) of about 5.2, 5.35, 5.5, 5.8, 6.2 and 6.5. The major components were at about pI 5.5 and 5.8. Neuraminidase was dissociated into subunits, whose sulfhydryl groups were blocked with iodo[14C]acetamide. 80% of isotope label incorporated was present in a single size of subunits with a molecular weight (Mr) of 51 000 as determined by sodium dodecyl sulfate acrylamide gel electrophoresis. The pI of denatured subunits was about 3.6 to 4.4. 14C-labelled peptides of tryptically digested neuraminidase had predominantly acidic isoelectric points. Results are consistent that the pI of native neuraminidase is about 1.5-2 pH units higher than the pI of its structural subunits, suggesting that side chain carboxyl groups are conformationally masked in the native enzyme, and that isoelectric heterogeneity of neuraminidase may result from conformation-dependent variations in the acid-base dissociation of these groups.