ABSTRACT
Lactate dehydrogenase (LD) isoenzymes 1, 2, and 3 were prepared from human erythrocytes by sequential ion-exchange chromatography followed by general-ligand (AMP analog) affinity chromatography. Respective yields, purification factors, and specific activities (kU per gram of protein) were 25%, 4394-fold, and 209.7; 40% 4385-fold, and 199.1; and 18%, 7565-fold, and 192.9. The respective preparations contained less than 0.5% of contaminating LD isoenzyme activity as judged from electrophoresis on thin-layer agarose, were homogeneous as judged by electrophoresis on polyacrylamide gel (both in the presence and absence of sodium lauryl sulfate), and showed minor contamination by other LD isoenzymes as judged by analytical isoelectric focusing. We think that these preparations would be useful as human-based calibrating or reference materials. Their purity is such that these preparations could also be used as antigens for the development of suitable antisera.
Subject(s)
Erythrocytes/enzymology , L-Lactate Dehydrogenase/isolation & purification , Chromatography, Affinity , Chromatography, Ion Exchange/methods , Electrophoresis, Polyacrylamide Gel , Humans , Isoelectric Focusing , Isoenzymes , L-Lactate Dehydrogenase/standardsABSTRACT
We have further assessed the accuracy of the thin-layer agarose fluorescent technique of Elevitch et al. [Am J Clin Pathol 46, 692 (1966)]. Previously, we used semi-purified human lactate dehydrogenase isoenzyme-1 and -5 [Clin Chem 22, 1995 (1976)] and isoenzyme-1 and -2 [Clin Chem 27, 1708 (1981)] to show that this assay accurately measures the proportions of these binary mixtures. In the present study, using ternary and quaternary mixtures of isoenzyme- 1, -2, -3, and -5, we show that the assay gives accurate estimations of all of these isoenzymes, within the errors of the techniques used. We also show that peak area (integration) is more nearly accurate, but less precise, than peak height (amplitude) measurements.