Affinity chromatography of nucleotide - dependent enzymes

Clonis, John D (1980) Affinity chromatography of nucleotide - dependent enzymes. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The synthesis, characterisation and immobilisation to CNBr-activated Sepharose of two IMP analogues, 8-(6-aminohexyl)-amino-IMP (C8-IMP) and inosine 2',3'-[I-(6-aminohexyl)-levulinic acid amide]-acetal 5'-monophosphate (R-IMP), is described. The immobilised IMP analogues displayed specificity for the IMP-dependent enzyme E.coli IMP dehydrogenase and this enzyme could be purified to homogeneity on a large scale(20 1) with good yield. The free IMP analogues, C8-IMP and R-IMP, were enzymically inactive and did not bind to the IMP-binding site of E.coli IMP dehydrogenase.Immobilised triazine dyes were studied as alternatives to nucleotide ligands and show a varying degree of affinity to nucleotide-dependent enzymes. Adenylosuccinate synthetase from E.coli could be partially purified on Sepharose-bound Procion Red H-3B and Procion Blue H-B columns 0 producing enzyme with the highest specific activity (508 units/mg at 25 C) reported so far. Free Procion Red H-3B and Procion Blue H-B were able to discriminate between the nucleotide-binding sites of E.coli adenylosuccinate synthetase. The binding strength of various Sepharose-immobilised triazine dyes for nucleotide-dependent enzymes could be altered by simple chemical treatment of the dye-gels. In particular, yeast hexokinase displayed a unique behaviour versus the monochlorotriazinyl dye Procion Green H-4G. The adsorption of this enzyme to Sepharose-immobilised Procion Green H-4G was contingent on the presence of Mg ++ in the column irrigants. Reactive triazinyl dyes were shown to irreversibly inactivate typical nucleotide-dependent enzymes at sites competitive with NAD+, NADP+ and ATP, suggesting that these reactive molecules may be used as general affinity labels for nucleotide-dependent enzymes. The use of immobilised triazine dyes as chromatographic media in enzyme purification is compared with classical immobilised nucleotides and coenzymes.

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