THE MECHANISM OF UPTAKE OF VITAMIN B₁₂ IN A B₁₂-REQUIRING LACTOBACILLUS

III. ISOLATION AND PURIFICATION OF A B₁₂-COMPLEX FROM THE CELL WALL

Abstract

A vitamin B₁₂-complex has been isolated from Lactobacillus delbrueckii No. 1 walls by treatment with 0.2N HCl. This was purified by means of repeated CM-cellulose column chromatography. The complex was obtained as red powder with a yield of 0.43mg per gram of cells (dry wt.). Ultracentrifugal analysis showed a single peak with the sedimentation coefficient of 1 S_{20, w}. From the absorption at 361mμ and the dry weight of the complex and consideration of the sedimentation coefficient, the molecular weight of the complex is deduced to be approximately 15, 000. Absorption spectra and chemical analysis showed that the complex is composed of B₁₂ and peptide. Using excess cyanide ions in alkaline pH, B₁₂-moiety is suggested to combine with peptide at the position of cobalt which is ordinarily co-ordinated with the N 3 of benzimidazole-moiety The intact complex can be used as well as heat- and/or KCN-treated ones for the growth of the organism.