RESUMO
For affinity labeling of NAD-dependent dehydrogenases, dinucleotide analogs were prepared by connecting nitrobenzene or nitrobenzimidazole systems with adenosine diphosphate. The distance between the two parts of the molecule was varied by insertion of propyl, butyl and pentyl chains or ribose. Reduction of the nitro group with hydrazine/Raney nickel yielded the corresponding amino derivatives which were converted to the diazonium salts by nitrous acid. Due to specific linking of ADP moiety to dehydrogenases, the reactive diazonium group combines with nucleophilic amino acid side chains in the active centre of dehydrogenases, the enzymatic activity of which was protected by NAD and NADH. Fluorescence titration experiments proved a linear correlation between incorporation of nucleotide anhydride, residual activity and remaining NADH capacity of the enzymes. The different modified amino acids showed characteristic absorption bands which allowed the identification of the reacting group as well as the estimation of the stoichiometry of the reaction. The latter could be estimated by titration of the enzyme with the diazonium salt. Only in a few cases was the spectrophotometric identification of the modified amino acid side chain uncertain. This fact required enzymatic degradation of the protein followed by electrophoresis and amino acid analysis.
