Purification and its Problem of Human Lactate Dehydrogenase Isozymes by Affinity Chromatography

Abstract

A simple and rapid method for purification of LDH (L-lactate: NAD⁺ oxidoreductase; ECI.1.1.27) isozymes in human tissues was established by the affinity chromato -graphy employing N⁶(6-aminohexyl)-5'-AMP-Sepharose as bed material. The results were shown as follows:
1. Crude extract (hemolysate) or ammonium sulfate fractionates (heart, liver) were applied directly to the column. The adsorbed LDH was eluted with a solution of NADH. After removing NADH by dialysis, each LDH isozyme was isolated with a common ion 0-exchange chromatography.
2. Sodium dodecylsulfate polyacrylamide gel electrophoresis for LDH preparations from three different human tissues (erythrocyte, heart, liver) revealed homogeneous as protein in each case and also gave that the molecular weights of LDH A-and B-subunits were 34,500 and 35,000-daltons, respectively. Specific activities of the purified enzymes were 730IU/mg (liver, A₄), 700IU/mg (erythrocyte, B₄) and 678IU/mg (heart, B₄).
3. The rate of purification for the LDH in erythrocyte, heart and liver were 4120, 234, 429-folds and yields were 84 (B₄), 63 (B₄), 49 (A₄) percents, respectivery.
4. On the tion-exchange chromatography, some LDH fractions having electrophoretically abnormal behavior were isolated in each tissue preparation. The elimination of unknown protein (FX) having similar properties to LDH A₄ was also problematical in case of purification of LDH from liver.