INTRODUCTION OF COB(Ⅰ)ALAMIN AS AN ANALYTICAL TOOL: APPLICATION TO REACTION-KINETIC STUDIES OF OXIRANES

摘要

The strongly nucleophilic cob(Ⅰ)alamin, i.e. Vitamin B_(12) with Co(Ⅲ) reduced to Co(Ⅰ), is introduced as a trapping agent in the determination of concentrations of electrophilic reagents. This compound was applied, in comparison with the previously used moderately reactive nicotinamide (H.J.C.F. Nelis and J.E. Sinsheimer (1981). Anal. Biochem., 115, 151.). Oxiranes, metabolites of 1-alkenes, were chosen as model electrophiles. The reagents (nicotinamide and cob(Ⅰ)alamin) were evaluated in the determination of the rates of reaction toward valine methylamide, a model of N-terminal valines in hemoglobin often used for monitoring of doses in vivo of genotoxic carcinogens. The rate constants for reaction at 37℃ with valine methylamide (k_(VMA)) determined by the cob(Ⅰ)alamin and nicotinamide procedure, respectively, were for ethylene oxide (1.6, 1.7), propylene oxide (0.9, 1.1), 1,2-epoxybutane (0.7, 0.8) and 1,2-epoxyoctane (0.5, 0.6) M~(-1) h~(-1), decreasing with increasing number of carbons of the oxirane. Concentrations of oxiranes trapped with nicotinamide are underrated in reaction mixtures containing valine methylamide due to consumption by reaction with the competing nucleo-phile, a disturbance that is not observed in trapping with cob(Ⅰ)alamin which reacts about 10~5 times faster than nicotinamide. Cob(Ⅰ)alamin which was demonstrated to be an efficient nucleophile for trapping of electrophiles, also in the presence of competing nucleophiles, is promising as an analytical tool in toxicological studies of reactive compounds. Furthermore, cob(Ⅰ)alamin can be used to detect, measure and compare electrophilic reactivity of chemical substances, a property that is associated with genotoxic potency.