Ureidoglycolate Synthetase of Streptococcus allantoicus I. Measurement of Glyoxylate and Enzyme Purification

Abstract

Gaudy, Elizabeth T. (University of Illinois, Urbana), R. Bojanowski, R. C. Valentine, and R. S. Wolfe . Ureidoglycolate synthetase of Streptococcus allantoicus . I. Measurement of glyoxylate and enzyme purification. J. Bacteriol. 90: 1525–1530. 1965.—A new spectrophotometric method for the determination of glyoxylate is described. The technique is based on measurement of the initial rate of formation of glyoxylic acid phenylhydrazone in neutral solution. Its advantages include rapidity and convenience, suitability for use with mixtures containing acid-labile substrates, and elimination of possibly inhibitory reagents from the enzyme incubation mixture. Ureidoglycolate synthetase, which cleaves ureidoglycolate to glyoxylate and urea, was purified from crude extracts of Streptococcus allantoicus grown on allantoin-containing medium. The purification procedures include treatment with MnCl 2 , fractionation on calcium phosphate gel, fractional precipitation with ammonium sulfate, and column chromatography on diethylaminoethyl cellulose. The final enzyme preparation was purified 77-fold and contained 35% of the total activity of the extract.