Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography

Abstract

1. 1.Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoly- and NAD +-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the α-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2. 2.Hydrophobic chromatography on a homologous series of alkyl-Sepharoses leads to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C 5–C 6). The second fraction is reversibly bound on Sepharose-NC 3 and -NC 4 but binding becomes irreversible at higher chain lengths. 3. 3.Aminoalkyl-Sepharoses behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. 4.Matrices which are negatively charged (Sepharose-NC n COOH, n = 3–7) have no affinity at pH 7.2. 5. 5.The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC 6OH, Sepharose-NC 6NH 2 and Sepharose NC 6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC 6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.