Isoelectric Focusing of N-Acetyl-α-D-Galactosaminidase from Clostridium Perfringens
Freeman, Susan L.
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The separation of N-acetyl- α -D-galactosaminidase (Azyme.) from known contaminants (β -galactosidase and β-N-acetylglucosaminidase) is of interest because it enzymatically cleaves the terminal N-acetylgalactosamine from type A red blood cells, giving them type O(H) properties. Analytical isoelectric . focusing in polyacrylamide and Sephadex gels was employed as a means of purifying the Azyme. In gels containing wide range carrier ampholytes, the Azyme.ahd contaminants co-focused but not at the Azyme isoelectric point. Modifications of focusing conditions were undertaken in order to change the behavior of Azyme such that it would separate from the contaminants. The apparent isoelectric point (apI) of Azyme shifted between pH 4.0 and pH 5.0 with alternations in focusing conditions. Variations in gel and carrier ampholyte types and concentrations caused only minor Azyme apI shifts. Addition of different amounts of product inhibitor to the gel caused a slight acidic shift. In each case examined the apI was independent of the duration of focusing. Refocusing Azyme did not alter the apI. The Azyme remained contaminated to some degree in all cases. The anomalous behavior of Azyme under different conditions suggested that the Azyme became insoluble before it reached its pl. Addition of 0.25 KC1 to the electrolytes kept the Azyme solubilized throughout the focusing procedure, allowing it to migrate to its pl. At its pl. however, the Azyme was irreversibly inactivated.