The Nature of the Vitamin B₁₂ Binding Protein in Human Serum*

Abstract

IT HAS been known for some years that the vitamin B12 circulating in the plasma is bound to protein. When this plasma binding capacity is exceeded, by the oral or intravenous administration of large doses of the vitamin, the excess unbound vitamin is rapidly excreted in the urine (Mollin and ROSS, 1953). The exact nature ofthis binding protein has yet to be identified. hi normal subjects and in patients with myeloproliferative disorders it migrates electrophoretically with the speed of an a,-globulin, and is present in that fraction of the serum proteins which is not precipitated by 0.6 M-perchloric acid, the seromucoid fraction (Weinstein, Weissniaii and Watkin, 1959). This serum fraction contains a number of antigenically distinct proteins (Burtin, 1964)~ two of which have been identified respectively as Orosomucoid and the a,-acid-glycoprotein of Schultze (Hardwicke and St. Cyr, 1961).In this paper the binding of vitamin B,, to the constituent fractions of a seromucoid, obtaincd by (NH4)z SO4 fractionation of serum, has been investigated by chromatography on DEAE-cellulose. Binding into this seromucoid fraction of the serum proteins is confirmed, in iiorinals and in myeloproliferative disorders, but this binding appears to be onto two separable serum proteins which are neither the Orosoniucoid nor any other recognized a1glycoprotein. METHODSScrurn was separated from venous blood samples and stored at -zoo C. until used.Vitaniin B, , was estimated by micro-biological assay with Euglenagracilir Z strain. Radioactive Biz. Cyanocobalamin 5 8 Co (specific activity > I pC./mg.) and Cyanocobalaiiiin 7 c~ (specific activity> 10 pC./nig.) were obtained from the Radiochemical Centre, Amersham, England. Estimations of radioactivity were made on a well-type sodium iodide scintillation counter.St.romucoid fractions were prepared by the addition to whole serum of an equal volunie of saturated ammonium sulphate. A first precipitate ('A') was harvested. The pH was then reduced slowly to 3.8 by the addition of 2 N-HC~, and a further precipitate ('B') was formed. The first of these contains mainly globulin fractions and the second mainly albumin (Winzler, Devor, Mehl and Smyth, 1948). The supernatant is analogous to the seroinucoid fraction of serum. This method of preparation is preferred since the addition of strong acid to reduce the pH to 1-2, as is found in the perchloric method, will remove a substantial proportion of the a-acetyl-neuraminic acid from the glycoproteins, with consequent alterations in the physical characteris tics (Hardwicke, personal observation).Additioii ofB,, to seritnz, OY tojactions. When radioactive vitamin was added to a protein