Rapid Purification of the Unstable Enzyme Carbamoyl Phosphate Synthase by High Pressure Liquid Chromatography

Abstract
Extracts of soluble proteins obtained from rat liver mitochondria by freeze-thawing and subsequent diafiltration were fractionated by HPLC on a I 250 protein column. The column was eluted either with 0.05 M phosphate buffer pH 6.85 or 0.1 M acetate buffer pH 7.15. Specific fractions obtained by elution with either phosphate or acetate buffer showed a 6.1-fold or 5.5-fold increase in the specific activity of Carbamoyl phosphate synthase when compared with that of crude mitochondrial preparations. The purification and the molecular weight of carbamoyl phosphate synthase were verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.