SIMULTANEOUS PURIFICATION OF L-MALATE DEHYDROGENASE AND L-LACTATE DEHYDROGENASE FROM BOVINE HEART BY BIOMIMETIC-DYE AFFINITY-CHROMATOGRAPHY

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-li gand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purifica tion protocol exploited the biomimetic affinity adsorbent, in combinat ion with a cross-linked agarose DEAE anion-exchanger. The procedure co mprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the fi rst step, was purified by specific elution with NAD(+)/sulphite (22.5- fold purification, 55% step-yield). The procedure afforded mMDH prepar ation of specific activity approx. 1,300 u/mg (25 degrees C) at 45% ov erall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminas e (GOT) and fumarase. The LDH activity, which, bound to the anion-exch anger during the first step, was recovered from the adsorbent in 200 m M KCl, and finally purified by biomimetic-dye affinity chromatography (NAD(+)/sulphite elution) and a second ion-exchange chromatography ste p (elution with 200 mM KCl). The LDH preparation exhibited specific ac tivity approx. 500 u/mg at 25 degrees C (content of impurities: pyruva te kinase and GOT were not detected; MDH, 0.01%).