It has been established that three binders of vitamin B,, can be separated by DEAEcellulose column chromatography following addition of labelled vitamin to normal serum (1-4). All 3 binders are present in trace amounts. They are known as transcobalamin I (TC I ) , transcobalamin I1 (TC 11), and transcobalamin I11 (TC 111) or "main protein peak binder" (MPPB) . TC I1 and TC I11 have an electrophoretic mobility of a-globulin, while TC I has the mobility of ,&globulin (1, 2).Labelled vitamin B,, is cleared rapidly from intravenously administered TC I1 5 7 C~ B,, but much more slowly from TC I 5 7 C~ B,, (2,5,6). The differences in clearance are less marked, however, when the transcobalamins are labelled with orally administered labelled vitamin BI2. The rapid clearance of 57C0 B12 from intravenously administered B,,--TC I1 has been attributed to partial denaturation of the protein during its purification in vitro (7). Finkler and Hall (8) reported that HeLa cells take up TO B,, from TC I1 in vitro but not from TC I while Retief et al. (9) observed that TC I1 delivers vitamin B,z to reticulocytes at a faster rate than TC I. Uptake by perfused rat liver of labelled vitamin B,, from 5 7 C~ B,, labelled TC I1 and TC I11 from serum of normal donors and pernicious anemia patients was substantially greater than from 5 7 C~ B,, labelled TC I (10). The exact role of TC 111 vita-1In receipt of a grant from the Leukemia Research Fund.747 min B,, in plasma is still uncertain. Chanarin and co-workers have recently shown that following oral administration of 5 7 C~ BIZ, the labelled vitamin is taken up simultaneously by all 3 serum binders (7, 11). These authors also showed immunological identity of TC I and TC 111. However, the ability of TC I11 to deliver vitamin B,, to cells is still unknown.H o a r a n d et al. (12) used phytohemagglutinin (PHA) -transformed lymphocytes as a model cell system to investigate uptake of serum bound radioactive vitamin B,, by normal proliferating human hemopoietic cells. In the present study we have used this system to compare the ability of human TC I11 with that of TC I and TC I1 to deliver labelled vitamin B,, to human cells. Materials and Methods. Preparation of vitamin B,, binders.This has been reported in detail elsewhere (13). Thirty ml of normal serum were used in each column chromatographic separation, Serum used in each experiment was drawn from an individual donor considered to be hematologically normal. Five hundred pg of 5 7 C~ B,, (specific activity 120 mCi/mg; purchased from the Radiochemical Centre, Amersham, Amersham, England) were added per ml to 30 ml of serum. The solution was allowed to stand at 37" for 20 min, then dialysed at 4" against 4 litres of 0.0175 M sodium phosphate buffer, pH 6.3, for 48 hr. The buffer was changed twice during this period.DEAE-cellulose ( Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 and 0.95 mEq/g) was packed after

It has been reported that three binders of "7Co BIZ are separated by DEAE-cellulose column chromatography following addition of labeled vitamin to normal serum (1, 2). These binders are known as transcobalamin I1 (TCII), transcobalamin I (TCI) and main protein peak binder (MPPB) . TCII and MPPB have an electrophoretic mobility of ,%globulin, while TCI has the mobility of a-globulin ( 2 ) . The molecular size of MPPB is approximately 120,000, similar to that of TCI, and larger than TCII (36,OOO) ( 2 ) .Further observations reveal that intravenously administered TCII 57C0 BIZ and MPPB 57C0 B12 are cleared rapidly from plasma while T C I 57C0 B12 shows a much slower pattern (2-4). Finkler and Hall (5) reported that HeLa cells take up TCII but not TCI bound TO BIZ. Retief et al. (6) observed that TCII delivers TO BIZ to erythrocytes at a faster rate than TCI. Haught et al. ( 7 ) recently reported that rat T C I I has no function in B12 uptake by perfused rat liver.In this study uptake by isolated perfused rat liver of VO B12 bound to the three binders was investigated. Naterials and Methods. Preparation of L 7 C~ B1! binders. The binder used in each perfusion experiment was prepared from 30 ml of serum, as follows. Serum from normal subjects or pernicious anemia (PA) patients was kept a t -20" before use. 300 pg of TO BIZ (sp act around 200 mCi/mg; purchased from Philips-Duphar, Holland) were adtded/ ml to 30 rnl of thawed serum. The solution was allowed to stand at 37" for at least 15 min, then was dialyzed in the cold room against 0.0175 M sodium phosphate buffer pH 6.3, for 24 hr.DEAE-cellulose, Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 to 0.95 mEq/g, was packed after proper preparation, into 3 X 6-cm columns ( 2 ) .The following buffers were used for elution a t 4 O and flow rate of 30 ml/hr: 0.0175 AL? sodium phosphate buffer, pH 6.3 (600 ml) ; 0.04 M sodium phosphate buffer, p H 5.9 (1000 m l ) ; 0.1 M sodium phosphate buffer, pH 5.8 (500 ml); and 0.4 M sodium phosphate buffer, p H 5.2 (700 ml). Buffer sohtions were made in distilled water containing 0.09% methylparaben and 0.01 % propylparaben (Tenneco Chemicals). TCII was eluted with the 0.04 M buffer, MPPB with the 0.1 M buffer and TCI with the 0.4 M buffer (2). Eluates containing binders were dialyzed and freeze dried, No further attempt a t purification was made.Liver perfusion. Rat liver perfusions were carried out as 2 hr experiments according to the procedure described by Miller et al.

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Three vitamin B12 binders have been isolated from normal serum by DEAE-cellulose column chromatography utilizing 3 X 60 cm columns (1-3). These binders have been designated as transcobalamin I1 (TCII) , main protein peak binder (MPPB) , and transcobalamin I (TCI) ( 2 , 3 ) . TCII has a molecu-_ _ _ _~

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