Six-step Scheme Research Articles

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, lactate dehydrogenase (LDH) and pyruvate kinase (PK), from rabbit muscles. The purification scheme was specifically designed so that both purified enzymes were comparable, in terms of specific activity and impurity content, to commercial high-purity analytical grade. The proposed protocol comprises two dye-columns (dye-sorbents), Cibacron blue 3GA and Procion yellow MX-4G both immobilised on CL-Sepharose 6B, which were integrated in the following six-step scheme: (i) muscle extract preparation, (ii) 0–45% ammonium sulphate fractionation, (iii) blue-column chromatography where the two enzymes were separated and LDH was purified, (iv) blue-column chromatography, (v) thermal treatment, and (vi) yellow-column chromatography where PK was purified. This protocol affords LDH of specific activity 470 units/mg (no detectable PK) at 65% overall yield, and PK of specific activity 251 units/mg (no detectable LDH and enolase) at 40% overall yield.