The Determination of Dissociation Constants by Affinity Chromatography on an Immobilized Adenosine Monophosphate Analogue

Within a few years affinity chromatography has become a widely used technique for the separation and purification of enzymes. The development and advantages of general ligands in affinity chromatography have previously been reported (Mosbach et al., 1972; Ohlsson et al., 1972). Thus Sepharose-bound N6-(6-aminohexyl)-AMP has been used for the separation and purification of NAD+-dependent enzymes (Ohlsson et al., 1972, Lowe et al., 1974) as well as for the separation of the isoenzymes of lactate dehydrogenase (Brodelius & Mosbach, 1973). The method has, however, been used almost exclusively for the purification of enzymes. I t is also possible to use the method for kinetic investigations. Thus the formation of ternary complexes with a number of dehydrogenases (Ohlsson et al., 1972) and the order of binding of substrates to lactate dehydrogenase (O’Carra & Barry, 1972) has been indicated. The five isoenzymes of bovine lactate dehydrogenase were resolved with a gradient of NADH into five distinct peaks by affinity chromatography on a column containing the Sepharose-bound AMP analogue. The separation was interpreted as reflecting the differences in the dissociation constant (Kdlss.) for the enzyme-NADH binary complex. In the present investigation a number of lactate dehydrogenases from different sources whose Kdlss. values were known from the literature were applied to such columns of Sepharose-bound AMP analogue and subsequently eluted with linear gradients of NADH. Fig. 1 shows a direct proportionality between these Kdlar. values and the eluting concentration of NADH. This linearity indicates that the other factor involved in affinity chromatography, i.e. the Kdiss. for the complex between enzyme and immobilized ligand, is in this case of minor importance for the elution process. The