P~ 58

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SUMMARYA method is described for the purification of neuraminidase from culture fluids of Vibrio cho2erae. 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 yo of the original enzyme activity was obtained as crystals. The degree of purification was about 5000 fold. Purified neuraminidase possessed 12.6 x los units of biological activity/mg. dry weight, and gave a value for Eigmp of 8.96, measured a t 0.025 yo (w/v). Enzyme activity was stimulated by calcium ions and inhibited by ethylenediaminetetra-acetate. I n the presence of 0.001 M-CaCl,, neuraminidase showed maximum activity a t pH 5.6. With sialyl lactose as substrate, a value of 1.2 x 1 0 -3~ was found for the Michaelis constant. At an enzyme concentration of 0.16 pg./ml., V,,,. was 0.021 ,UM N-acetylneuraminic acid/min./ml. The enzyme was stable when dried from the frozen state and stored under vacuum a t 0". A suspension of crystals in water also retained activity when stored at 0". Solutions of crystalline neuraminidase showed a small increase in activity when stored at 0" for several weeks. This effect was greatest at pH 6.7 and 8.5 but was barely detectable a t pH 4.6. At pH 5.6 or 6.7, the enzyme lost about 20 yo of its activity over a period of 2 hr. at 37' (concentration = 15 pg./ml.) No proteolytic activity nor N-acetylneuraminic acid aldolase activity was detected in the crystalline preparation.