The purification and properties of a factor containing vitamin B12 concerned in the synthesis of methionine by Escherichia coli

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Abstract
Escherichia coli strain PA 15 contains, when grown in the presence of cobalamin, a factor which enables tetrahydropteroylglutamic acid to serve as the C1-transfer cofactor for methionine synthesis by extracts of acetone-dried organisms grown without cobalamin. A method is described for the assay of this factor and the preparation of extracts rich in it. The purification (150-fold) of the factor is described. It includes removal of nucleoprotein with calcium chloride, pH fractionation, column chromatography on Celite in the presence of protamine and chromatography on diethylaminoethylcell-ulose. The factor is non-diffusible and when purified is unstable to heating and to storage even at low temperatures. It is most stable at pH 8.0-8.5. The protein nature of the factor is indicated by the 280 m[mu]/260 m[mu] absorption ratio of 1.6 and the loss of activity on digestion with trypsin and papain. During the later stages of purification the factor is closely associated with bound vitamin B12. Chromatography on diethylaminoethylcellulose produces an apparently homogeneous peak with a constant ratio of factorprotein:vitamin B12. The vitamin B12 of the factor is largely unavailable for Escherichia coli strain 113/3 (a cobalamin or methionine auxotroph) but can be released by heating, digestion with papain or extraction with hydrogen cyanide. The purified factor is destroyed by cyanide. Irradiation with UV light inactivates the factor, but irradiation with visible light or treatment with activated charcoal is without effect. Since the factor is a protein and it acts catalytically and contains vitamin B12, it is suggested that it is an enzyme with a prosthetic group related to cobalamin and concerned in the conversion of homocysteine into methionine under the conditions studied.