Crystalline l-Asparaginase from Escherichia coli B

Abstract

Abstract Crystalline l-asparaginase from Escherichia coli B has been shown to consist of four subunits (mol wt 33,000) by the methods of sedimentation velocity and equilibrium, viscosity, and electrophoresis in sodium dodecyl sulfate on polyacrylamide gels. The dissociation-reconstitution behavior of the enzyme has been characterized. The native enzyme does not dissociate into subunits in neutral aqueous solutions at protein concentrations as low as 15 µg per ml. No evidence for an intermediate form between the native protein (mol wt 133,000) and its 33,000 molecular weight subunits is found. The enzyme can be dissociated into its subunits in 7 m urea and 5 m guanidine and then its disulfides can be reduced. No enzymatic activity remains. Upon reoxidation and reconstitution the enzyme regains at least 85% of its original activity. The physical properties of the native and reconstituted enzyme are virtually identical. The enzyme appears to be essentially globular in shape in aqueous solution. Approximately 30% of the tyrosine residues titrate readily. Titration of the remaining tyrosine residues results in the dissociation and denaturation of the enzyme.