Affinity chromatography of lactate dehydrogenase Model studies demonstrating the potential of the technique in the mechanistic investigation as well as in the purification of multi‐substrate enzymes

Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortive complex formation

“…It therefore required a higher 'threshold' salt concentration, 0.5 M KC1 being found to give the best results. As noted previously [6] the bulk of the protein in the crude extract is washed straight through the column after application of the sample in NADH, and any non-LDH protein which becomes adsorbed remains adsorbed after subsequent elution of the LDH. Peak I, eluted after the change-over to NADC ( fig.…”

“…the affinity chromatographic procedure in general rather than to the modification allowing specific separation of the M4 isoenzyme. The M4 enzyme can also be isolated as a stable preparation and in good yield by first isolating the total LDH by the affinity chromatographic method as previously described [6] and subsequently seperating the isoenzymes by ionexchange chromatography [ 131. Fig.…”

“…We have since found that the method as originally described [6] gives somewhat erratic results and relatively low substitution levels owing, apparently, to decomposition of a proportion of the carbodiimide-activated oxalate. This has also been recently observed by Spielmann et al [7].…”

“…In a previous paper [6] we described an effective affinity chromatographic system for LDH based on the affinity of the enzyme for immobilized oxamate, a pyruvate analogue. Owing to the ordered kinetic mechanism, adsorption of the enzyme by the immobilized oxamate gel does not occur unless NADH is included in the applied enzyme samples and in the irrigating buffer.…”

The interaction of some dehydrogenases with N6‐(6‐aminohexyl)‐adenosine 5′‐monophosphate sepharose

“…The general utility of the technique has been greatly enhanced by the introduo tion of immobilised coenzymes as 'general ligand' adsorbents for binding a wide range of complementary enzymes [3,4]. More recently, however, a number of analytical applications of affinity chromatography have been proposed and applied to the exploration of binding mechanisms and kinetics [5], the investigation of enzyme-substrate interactions [6] and the determination of dissociation constants [7]. However, despite general appreciation of the utility of these applications of affinity chromatography, little is known about the effect of immobilisation of the ligand on subsequent interactions with the complementary macromolecule.…”