SIMULTANEOUS SEPARATION AND PURIFICATION OF PYRUVATE-KINASE AND LACTATE-DEHYDROGENASE BY DYE-LIGAND CHROMATOGRAPHY

This work describes an improved downstream processing protocol for the simultaneous separation and purification of two diagnostic enzymes, l actate dehydrogenase (LDH) and pyruvate kinase (PK), from rabbit muscl es. The purification scheme was specifically designed so that both pur ified enzymes were comparable, in terms of specific activity and impur ity content, to commercial high-purity analytical grade. The proposed protocol comprises two dye-columns (dye-sorbents), Cibacron blue 3GA a nd Procion yellow MX-4G both immobilised on CL-Sepharose 6B, which wer e integrated in the following six-step scheme: (i) muscle extract prep aration, (ii) 0-45% ammonium sulphate fractionation, (iii) blue-column chromatography where the two enzymes were separated and LDH was purif ied, (iv) blue-column chromatography, (v) thermal treatment, and (vi) yellow-column chromatography where PK was purified This protocol affor ds LDH of specific activity 470 units/mg (no detectable PK) at 65% ove rall yield, and PK of specific activity 251 units/mg (no detectable LD H and enolase) at 40% overall yield.