Isolation and purification of aspartate aminotransferase isoenzymes from human liver by chromatography and isoelectric focusing

Abstract

To purify cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from human liver, we used heat treatment, ammonium sulfate precipitation, anion- and cation-exchange chromatography, affinity chromatography, and isoelectric focusing. Final preparations of the isoenzymes were homogeneous, with specific activities of 198 and 208 kU/g for the cytoplasmic and the mitochondrial enzymes, respectively. The mitochondrial isoenzyme focused as a single band with a pl value of 9.60, whereas the cytoplasmic isoenzyme had subforms with pl values of 5.22, 5.42, and 5.62 at 4°C. In Tris . HCl buffer, both isoenzymes have an activity maximum at pH 7.8. In [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)-methane (Bistris) buffer, however, the mitochondrial isoenzyme also showed an optimum pH of 6.7.