Purification of rat liver asparagine synthetase by affinity chromatography on reactive blue 2-agarose

Abstract

An affinity chromatographic method for the high purification of asparagine synthetase from rat liver was demonstrated. A partially purified enzyme was bound to reactive blue 2-agarose column in a buffer containing magnesium ion and was specifically eluted from the column with the buffer added with both ATP and l-aspartic acid. In this affinity elution step 65-fold purification was obtained and overall 2500-fold purification was obtained. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band, which corresponded to a molecular weight of 62,000. In urea-polyacrylamide gel electrophoresis the enzyme gave again a single band, but in dise gel electrophoresis the enzyme revealed two major species, which had molecular weights of about 60,000 and 120,000. It is possible that the enzyme consists of two identical subunits.