Formation of a vitamin B-12-serum complex on heating at alkaline pH

The binding of vitamin B-12 to serum proteins during heating at alkaline pH was investigated by gel filtration of serum supplemented with cyano[57Co]-cobalamin. Heating for 5 min at 100 degrees C destroyed most of the vitamin B-12 binding activity of serum but, with further heating, the vitamin B-12 became incorporated into a complex that did not correspond in molecular size to the original vitamin B-12 binding proteins. Radioassay of vitamin B-12 in heated serum showed correspondingly first an increase then a progressive decrease in the apparent vitamin B-12 level suggesting that, on heating, vitamin B-12 was initially released then subsequently complexed by the serum. The formation of complexed vitamin B-12 was abolished by the presence of the reducing agent dithiothreitol during the heating step.