Downstream processing of diagnostic enzymes: Optimised protocols for the simultaneous separation and purification of lactate dehydrogenase and pyruvate kinase from rabbit muscle

Abstract

Cibacron Blue 3GA-Sepharose CL6B was used to design two optimised, inexpensive and easy to scale-up processes for the simultaneous separation and purification of l-lactate dehydrogenase (LDH) and pyruvate kinase (PK) from rabbit muscles. The tissue was homogenised, filtered, and the liquid treated by DEAE-cellulose and Sephadex-G25 gel to obtain the “pre-treated” extract which was used in the dye-column. The first process, involving two identical dye-columns (1 ml each), afforded from the first column 2.3 mg PK-free LDH of specific activity (S.A.) 470 units/mg with 73% yield, and from the second column 1.9 mg LDH-free PK of S.A. 66 units/mg with 63% yield. The second process, involving only one dye-column (1 ml), afforded both enzymes in good yield (65–67%) but with less purity: S.A. 360 units/mg for LDH (0.1% PK) and 44 units/mg for PK (0.01–0.04% LDH). In both processes LDH was eluted biospecifically from both columns with NADH (5 mM), whereas, PK was eluted with KCl (0.15 M). Biospecific elution of PK from the blue adsorbent resulted in poor enzyme recovery (25%). The following factors were proven to be important: Extract pretreatment, ionic strength and pH, amount loaded on the adsorbent, and elution conditions.