Purification of human lactate dehydrogenases by general ligand affinity chromatography

Bachman, B.K.; Lee, C.Y.

Analytical Biochemistry 72: 153-160

1976


ISSN/ISBN: 0003-2697
PMID: 942045
DOI: 10.1016/0003-2697(76)90517-0
Accession: 041138578

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Abstract
Lactate dehydrogenases were highly purified from three different human tissues by employing the techniques of general ligand affinity chromatography. After passing the crude extracts through an 8-(6-aminohexyl)-amino-5′-AMP-Sepharose column, the adsorbed enzymes were readily eluted from the column with a solution of 0.2 mm of reduced NAD-pyruvate adduct. The eluted enzymes in the peak fractions appeared to be homogeneous from the results of the SDS-polyacrylamide gel electrophoresis. With an 8-(6-aminohexyl)-amino-NAD-Sepharose column, the adsorbed enzymes could not easily be eluted with the aduct under the same experimental conditions. As a result, the purity of the eluted enzymes obtained from 8-substituted-NAD-Sepharose column is much lower than that obtained from the corresponding 8-substituted-5′-AMP-Sepharose column. Compared to the conventional lengthy procedures, selective purification of human lactate dehydrogenases by affinity chromatography provides not only a fast and efficient means of obtaining the enzymes but also a high degree of recovery.