Kritischer Vergleich der Lactat-Dehydrogenase-Isoenzym-Fraktionierung mit Isoelektrischer Fokussierung und Diskelektrophorese in Polyacrylamid-Mikrogelen

Abstract

On fractionation of lactate dehydrogenase [LDH-EC 1.1.1.27] isoenzymes by isoelectric focusing in microgels, the most anodic LDH 1 is largely inactive and the cathodic fractions are predominant in the isoenzyme pattern, even of organs such as the heart and brain, which are reported to contain mainly LDH 1, LDH 2 and LDH 3. On focusing of isolated rat LDH 1 and LDH 5, an inactivation of the anodic isoenzyme of 86% relative to LDH 5 was observed. This inactivation is due to the acid denaturation of the enzyme protein at its isoelectric point as shown by incubation experiments with pure isoenzymes in test tubes. Excellent resolution of the isoenzymes can also be achieved with a newly developed micro-disc-electrophoretic technique. The isoenzyme patterns compare favorably with the results reported in the literature, although in this case the most cathodic isoenzyme LDH 5 is partially inactivated. Triton X-100 increases the color intensity of the bands 2-4-fold after enzyme staining with tetrazolium blue. This effect is independent of the fractionation technique used. The detergent does not influence the enzymatic reaction but promotes the formazan development, thus acting as an unspecific amplifier which further increases the sensitivity of enzyme detection in microgels. A relevant interpretation of isoenzyme patterns is only possible if the principle hazards of the separation technique applied are thoroughly considered.