THE CATALYSIS OF THE AUTO-OXIDATION OF 2-MERCAPTOETHANOL AND OTHER THIOLS BY VITAMIN B12 DERIVATIVES. POLAROGRAPHIC AND OTHER INVESTIGATIONS

Abstract

At neutral pH, vitamin B12 derivatives catalyse the auto-oxidation of 2-mercaptoethanol to the disulphide form. The effect can be readily demonstrated manometric-ally but this method does not give relibale rate measurements bacause of side reactions. Apolarographic apparatus suitable for measuring changes in the partial pressure of oxygen of a few minutes'' duration is described and has been used to study mercaptoethanol oxidation. At pH 7[center dot]1 and with all vitamin Bl2 derivatives tested except Factor A, the fall in oxygen concentration is linear with time for the greater part of the reaction. The final oxygen pressure reached is less than 2 mm. Hg. With Factor B as catalyst, the oxidation rate increases with pH from nearly zero at pH 6[center dot]0 to a maximum at pH 8[center dot]0, and increases approximately fourfold between 25[degree] and 37[degree]. The rate shows a Michaelis-type dependence on mercaptoethanol concentration (half-maximum velocity at about 30 m[image]) and is directly proportional to catalyst concentration. At 37[degree] and pH 7[center dot]1 with Factor B as catalyst, activity with different thiols increased in the order; glutathione; cysteine; thioglycollate; 2-mercaptoethanol; 2,3-dimercaptopropanol; dihydro-6-thioctic acid; dihydro-6-thioctic acid amide. With mercaptoethanol, the approximate molar catalytic activities of different vitamin B12 derivatives relative to cyanocobalamin (1[center dot]0) are: dimethylbenzimidazolylcobamide coenzyme, 0[center dot]3; hydroxocobalamin, 10; sundry cobalamin analogues, 7-50; Factor B, 4000; Factor D, 2500. Activities of all the cobamide derivatives are greatly enhanced by brief heating with acid. Co2+, Ni2+ and Cu2+ ions catalyse the oxidation of mercaptoethanol; yeast extract has slight catalytic activity. EDTA (1 m[image]) completely abolishes the effect of Co2+ or Ni2+ ions, but not that of Cu2+ ion. Catalysis by Factor B is unaffected by 1 mM-EDTA, but is completely inhibited by 0[center dot]1 m[image] -potassium cyanide. Large concentrations of yeast extract interfere with the reaction. Spectral studies with hydroxocobalamin and mercaptoethanol indicate that vitamin B12r is a catalytic intermediate in thiol oxidation at pH 11. At neutrality the situation is obscured by side reactions. The implications of these reactions in vitro and in vivo and their possible use for establishing anaerobic conditions and in the determination of vitamin B12 derivatives are discussed.