Crystalline anti-pernicious anemia factor from liver.

Abstract

A red crystalline substance, intensely active against pernicious anaemia, has been isolated in very small yield from ox liver. It is probably identical with vitamin B 12 isolated a few weeks previously in America. The procedures employed were almost all physical rather than chemical. They included repeated adsorption on charcoal and elution with hot 65% alcohol, adsorption chromatography on alumina, silica and charcoal, and partition chromatography between butanol or other solvents and moist silica; proteolysis to break down peptide impurities, fractional salting-out with ammonium sulphate and precipitation with phosphotungstic acid were generally also necessary before the substance could be crystallized from aqueous acetone. A second red factor, also active both clinically and microbiologically, has been separated by partition chromatography on starch or filter paper, but has not yet been crystallized. A number of physical measurements have been made. These include crystallography and X-ray crystallography, measurement of refractive indices, absorption spectrum, optical rotation, electrometric titration, electrical conductivity and mobility, and polarography. Chemical analyses have been made for the elements C, H, N, P and Co. The presence of 4·0% of cobalt leads to a molecular weight of 1500 for the dried substance, in agreement with the values from X-ray crystallography. The anti-pernicious anaemia factor is slowly destroyed by cold dilute acid or alkali, by light and by strong oxidizing or reducing agents. Attempts to induce chemical exchange with radioactive phosphate or radioactive cobaltous chloride, or with a radioactive cobaltammine, were unsuccessful.