Abstract
The identity of the major cobamide in liver has not been satisfactorily established. Cyanocobalamin (vitamin B12, 5,6dimethylbenzimidazolylcobamide cyanide), which has been obtained in crystalline form with liver as a source material (1, 2), is certainly a very minor component of the cobalamins in liver. Analyses of liver concentrates, both by a paper chromatographicbioautographic method (3) and by a chemical method (4), have shown that cyanocobalamin is usually not present in detectable amounts and seldom constitutes as much as 10% of the total cobalamin. The preparation of cyanocobalamin from liver probably was made possible by the presence of cyanide in the charcoal used in the isolation process. Examination of a liver extract by the chromatographic-bioautographic method (3) indicated that hydroxocobalamin (vitamin B12b, 5,6-dimethylbenzimidazolylcobamide hydroxide) was the most abundant cobalamin. This compound was also obtained in pure form from liver (1, 5). However, the recent discovery of the cobamide coenzymes in bacteria (6,7) and the demonstration that they constitute a large part of the total cobamides in these organisms (8,9) raised the possibility that coenzyme Bit (5,6-dimethylbenzimidazolylcobamide coenzyme, DMBC coenzyme, DBC coenzyme) may be a constituent of liver. Since coenzyme B12, like the adenylcobamide coenzyme (lo), is relatively unstable and is readily converted to hydroxocobalamin or cyanocobalamin by treatment with light or cyanide,’ respectively, the inadvertent decomposition of the coenzyme, if present in liver extracts, could account for previous observations on the occurrence of the two cobalamin vitamins. The present paper reports some observations on the occurrence and abundance of a cobamide coenzyme, which has been identified as coenzyme Blz, in livers of rabbit, chicken, sheep, and man. A brief report of the occurrence of coenzyme Blz in rabbit liver has already appeared (7).
Highlights
MethodsMethod of Isolation and Identijication ofDimethylbenzimidazole-A sample containing approximately 0.09 pmole of coenzyme from human liver was made 0.1 M with potassium cyanide and left at room temperature for 2 hours
A small amount of Co% appeared in the solution passed through the column. This represents a neutral or acidic decomposition product of the labeled coenzyme BY& No red color was detected in this region, indicating that no substantial amount of cyanocobalamin was present in the preparation
Cobamide coenzymes have been isolated in moderate purity from sheep, rabbit, chicken, and human livers
Summary
Method of Isolation and Identijication ofDimethylbenzimidazole-A sample containing approximately 0.09 pmole of coenzyme from human liver was made 0.1 M with potassium cyanide and left at room temperature for 2 hours. The absorption spectrum of the solution was identical with that of vitamin BIz. The solution was made 6 N with hydrochloric acid, sealed in a vacuum in a glass tube, and heated at 150’ for 20 hours. After removal of excess hydrochloric acid by evaporation to dryness over sodium hydroxide in a vacuum at 37”, the residue was taken up in 2.0 ml of 0.1 N hydrochloric acid and extracted twice with 2-ml volumes of chloroform. The water phase was made 0.1 N with respect to sodium hydroxide by addition of 0.15 ml of 2.5 N NaOH and was extracted six times with l-ml volumes of chloroform. The chloroform was evaporated in a stream of nitrogen and the residue was taken up in 1.0 ml of 0.1 N hydrochloric acid
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