A native cobalamin–polypeptide complex from liver: isolation and characterization

Abstract

A cobalamin-polypeptide complex has been isolated from ox liver by a technique involving essentially physical methods. The complex was obtained as a pink amorphous powder in a yield of 3.8%, representing a purification of more than 13,000-fold. The substance behaved as a single component when examined by chromatography and electrophoresis on paper. After acid hydrolysis the following amino acids were identified: glycine, alanine, threonine, valine, leucine, isoleucine, aspartic acid, glutamic acid, tyrosine, histidine, cysteine, proline and lysine. The absorption spectrum has characteristic peaks at 274, 350 and 523 m[mu]. After addition of cyanide the absorption changes to a cyanocobalamin type of spectrum with maxima at 274, 361 and 550 m[mu]. The infrared spectrum of the complex shows no absorption in the 4.5 [mu] region. This indicates the absence of cyanide. Calculations of the cobalamin content from the absorption spectrum at different wavelengths gave somewhat divergent results. After addition of cyanide more consistent results of 14.7% were obtained. The molecular weight, as determined by cobalt analyses and estimated from sedimentation and diffusion data, was found to be about 9100.