Separation, purification and some properties of pneumococcal neuraminidase isoenzymes

Abstract

1. 1. Neuraminidase from a rough strain of Diplococcus pneumoniae has been partially purified by a series of steps involving (NH 4) 2SO 4 fractionations, ultrafiltration through a Diaflo XM-50 filter, heat treatment at 55–56° for 4 min, and batchwise absorption and desorption from DEAE-cellulose. The most variable stages in this procedure involved the heat and DEAE-cellulose treatments. 2. 2. Enzyme purified in this manner was resolved into two peaks of activity by chromatography on DEAE-cellulose and into three populations of neuraminidase activity upon CM-cellulose chromatography. Both discontinuous and linear gradient elutions with phosphate-citrate buffer (pH 6.4) range 0.01–0.10 M were used. 3. 3. The initially eluted CM-cellulose enzyme fraction (Fraction I) was in turn resolved into two activity peaks upon sequential chromatography on CM-Sephadex with the same developing buffer system. Comparable fractions, modified in their origins only insofar as deletion of the heating and batchwise DEAE-cellulose steps, provided 5 isoenzyme peaks on CM-Sephadex chromatography. It is presumed that these results reflect associated-dissociation phenomena due to temperature and monovalent cation exposure. 4. 4. The second CM-cellulose peak of enzyme activity (Fraction II) was additionally resolved into three isoenzymes by serial CM-Sephadex chromatography. Two of these neuraminidase forms appeared at elution volumes sufficiently different from those which were derived from CM-cellulose Fraction I or modified CM-cellulose Fraction I so as to indicated their non-identity. Furthermore, the CM-cellulose Fraction II isoenzymes possessed much lower specific activities than those which were similarly obtained from CM-cellulose Fractions I. 5. 5. Confirmation of the existence of multiple forms of pneunomococcal neuraminidase came from polyacrylamide gel electrophoresis experiments. The appearance of at least five zones of enzyme activity was found on electrophoretic zymograms of the partially purified material. CM-cellulose Fraction I preparations exhibited a minimum of three zones of activity, but each zone gave indications of heterogeneity. Correlations between isoenzyme zone as found above and activity peaks resultant from ion-exchange chromatography have not yet been carried out. 6. 6. Further enrichment of pooled CM-cellulose Fractions II was effected by precipitation with 0.65 saturated (NH 4) 2SO 4. This procedure led to a 400-fold purification, and an apparently crystalline enzyme. Polyacrylamide gel electrophoresis of the crystalline enzyme showed the presence of at least six protein staining bands. Zymograms of unstained replicate disc gels indicated that the enzymatic area, which encompassed only two of the protein species present, was now apparently somewhat more homogeneous. Whether the residual neuraminidase-negative proteins represent inactive forms of the enzyme, or impurities with almost identical salt solubility and ion-exchange properties, remains to be determined.