Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

Abstract

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.

The Synthesis of Several 8-Substituted Derivatives of Adenosine 5'-Monophosphate to Study the Effect of the Nature of the Spacer Arm in Affinity Chromatography

“…Yeast alcohol dehydrogenase displays little affinity for either adsorbent, supporting the contention that this dehydrogenase may prefer 6-linked rather than derivatives linked through other positions [I 7, 291. ~-Glyceraldehyde-3-phosphate dehydrogenase is known to demonstrate a considerable degree of nonbiological adsorption on binding to a NAD + derivative linked through a hydrocarbon spacer arm [9,30] but that this could be alleviated by introduction of a more hydrophilic spacer arm [17]. Fig.4 (F) confirms these observations ; the enzyme interacts very weakly with the more hydrophobic adsorbent and is completely unretarded by the hydrophilic adsorbent.…”

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

“…It has been suggested that this strengthening is due to a 'compound affinity' effect in which the threshold salt concentration allows the operation of marginal non-biospecific interactions with the spacer arm which synergistically reinforce the weak bioaffinity to produce a relatively strong retardation, without being sufficient to cause any significant retardation on their own [8,9] .…”

Complete purification of the acetylcholine receptor protein from mammalian muscle

“…When no more protein could be removed by the salt wash given, 2.1 mM Flaxedil (gallamine triethiodide) solution was used to achieve a bio-specific [9] elution of ACh.R. This eluent also displaced a small amount of other protein.…”

“…For muscles, quantitation and localisation of ACh.R, by exploitation of its essentially irreversible binding of labelled cu-bungarotoxin (BuTX), was initially described [3], and solubilisation of this ACh.R in detergents and some fractionation has been reported [4-71. Subsequently [S] , we reported upon a preparation, partly purified by affinity chromatography, of the form of ACh.R which is responsible for the extrajunctional ACh sensitivity of denervated mammalian muscle. We have further applied affinity chromatography in a more bio-specific form (in the sense in which this technique has been critically reviewed by Barry and O'Carra [9] ), and describe here the complete purification of this muscle ACh.R. [S] , as were materials not separately specified here.…”