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Abstract Crystalline preparations of l-amino acid oxidase, which contained the three electrophoretic components, A, B, and C, were separated further into multiple components by isoelectric fractionation in a pH gradient. At least 18 peaks of coincident enzymatic activity and protein were observed when electrofocusing was carried out in a sucrose density gradient according to Vesterberg and Svensson (Acta Chem. Scand., 20, 820 (1966)). The components ranged in isoelectric point from pH 5.2 to 8.4 and differed in amino acid composition. A new technique of electrofocusing in polyacrylamide gels yielded even greater resolution. A two-dimensional separation, utilizing both electrophoresis and electrofocusing in gel, showed that A, B, and C each consisted of five or more components having different isoelectric points. This was confirmed by a study of fractions obtained by ion exchange chromatography of the enzyme and by the study of venoms from individual snakes possessing only Component A, only C, or A, B, and C. The blood, the venom, and extracts of the venom gland of a single snake exhibited identical isozyme patterns. For comparison, gel electrofocusing of d-amino acid oxidase was also carried out. Only two active components, identified as the holoenzyme and the apoenzyme, were observed.
Hayes et al. (Mon,) studied this question.