Separation of vitamin B 12-binding proteins of serum, gastric juice and saliva by rapid DEAE cellulose chromatography.

By FRANCOIS P. RETIEF, CHESTER W. GOTFLIEB, SHAUL KOCHWA, PETER W. PRATF AND VICTOR HERBERT N ORMAL SERUM contains at least two binders of vitamin B12, one migrating electrophoretically as an alpha-globulin2’3 and the other as a beta-globulin.46 There is evidence that these substances have different metabolic functions. Whereas native B12 is bound to alpha-globulin,2 the betaglobulin binder probably acts as a temporary B12 carrier.6’7 Certain disease states, and chronic myeloid leukemia (CML) in particular, are characterised by a striking increase of alpha-globulin binder.8 This may be of some diagnostic value in differentiating between the myeloproliferative syndromes.9 Separation of the B12-binders thus has clinical implications in addition to its obvious value in the study of B12 kinetics. We wish to describe a separation technic based on DEAE cellulose column chromatography which can be completed in approximately 2 hours. This “baby column” method is a modification of the procedure reported for isoagglutinin separation by Kochwa et al.1#{176} Briefly, it consists in ( 1 ) adding an excess of Co57B12 to 1 ml. of serum to saturate B12 binders; (2) removing free Co57B12 with hemoglobin-coated charcoal;1’ (3) adding 2 ml. distilled water to the 1 ml. serum; (4) passing the 3 ml. sample through a 5 ml. pipette packed with cellulose, collecting 6 samples (of 2 ml. each) using a buffer eluant and 5 more using 1 M NaC1 as eluant. The 0.06 M, pH 6.3, phosphate buffer elutes gamma globulin, /32A, /3k, B12-binding /3, and