Separation of isozymes of horse liver alcohol dehydrogenase and purification of the enzyme by affinity chromatography on an immobilized AMP-analogue

Andersson, L.; Jörnvall, H.; Akeson, A.; Mosbach, K.

Biochimica et Biophysica Acta 364(1): 1-8

1974


ISSN/ISBN: 0006-3002
PMID: 4373066
DOI: 10.1016/0005-2744(74)90126-0
Accession: 044296470

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Abstract
A mixture of the two homogeneous isozymes Ee and Ss of horse liver alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, Ec 1.1.1.1) was separated on a column of N6-(6-aminohexyl)-Amp substituted Sepharose with a linear gradient of NAD+ at p H 7.5 in the presence of 1.5 m M cholic acid. Ss was eluted first at 0.4 m M NAD+, and Ee later at 3.4 m M NAD+. This separation is due to true biospecific adsorption since both isozymes showed no affinity to a gel to which n-hexylamine had been covalently bound. From a horse liver crude extract alcohol dehydrogenase could be purified in a one-step procedure using a pulse of 0.1 m M NAD+ plus 1.1 m M pyrazole resulting in 22 times purification with a yield of 36%. The enzyme obtained was homogeneous on dodecylsulphate-gel electrophoresis and showed a specific activity of about 3.1 units/mg when measured at p H 10.0.