Purification and Properties of Neuraminidase from Vibrio cholerae

Abstract

A method is described for the purification of neuraminidase from culture fluids of Vibrio cholerae. Five steps are involved: fractionation with methanol, adsorption to and elution from human red cells, fractionation with ammonium sulphate, chromatography on columns of hydroxyl apatite, crystallization. From 35 1 of culture filtrate, an average yield of 21% of the original enzyme activity was obtained as crystals. The degree of purification was about 5000 fold. Purified neuraminidase possessed 12.6x106 units of biological activity/mg. dry weight, and gave a value for E 2hq m[mu] of 8.96, measured at 0.025% (w/v). Enzyme activity was stimulated by calcium ions and inhibited by ethylenediaminetetra-acetate. In the presence of 0.001 [image]-CaCl2, neuraminidase showed maximum activity at pH 5.6. With sially lactose as substrate, a value of 1.2 x 10-3 [image]_ was found for the Michaelis constant. At an enzyme concentration of 0.16 [mu]g./ml., V was 0.021 [mu][image]_ N-acetylneuraminic acid/min./ml. The enzyme was stable when dried from the frozen state and stored under vacuum at 0[degree]. A suspension of crystals in water also retained activity when stored at 0[degree]. Solutions of crystalline neuraminidase showed a small increase in activity when stored at 0[degree] for several weeks. This effect was greatest at pH 6.7 and 8.5 but was barely detectable at pH 4.6. At pH 5.6 or 6.7, the enzyme lost about 20% of its activity over a period of 2 hr. at 37[degree] (concentration = 15 [mu]g./ml.) No proteolytic activity nor N-acetylneuraminic acid aldolase activity was detected in the crystalline preparation.