Isolation of Vitamin B12-binding Proteins Using Affinity Chromatography

A vitamin Blz-binding protein has been isolated from human granulocytes derived from patients with chronic granulocytic leukemia. Utilizing affinity chromatography as the sole purification technique, the protein was purified 9,860-fold with a yield of over 90% and was homogeneous based on polyacrylamide disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein binds 34.9 pg of vitamin BIG per mg of protein and has a single vitamin Blz-binding site. The molecular weight determined by sedimentation equilibrium ultracentrifugation was 56,000, whereas that determined by amino acid and carbohydrate analysis was 58,200. The protein contains 33 % carbohydrate which accounts for the elevated molecular weight values (121,000 to 138,000) obtained using gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

II has been isolated from Cohn Fraction III derived from 1,400 liters of pooled human plasma, using affinity chromatography on vitamin B+Sepharose and several conventional purification techniques. The final preparation was purified 2 million-fold relative to human plasma with a yield of 12.8% and was homogeneous based on polyacrylamide disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, and chromatography on Sephadex G-150. Transcobalamin II binds 28.6 pg of vitamin Blz per mg of protein and contains one vitamin Bla-binding site per 59,500 g of protein as determined by amino acid analysis. The molecular weight determined by sedimentation equilibrium ultracentrifugation was 53,900 and by gel filtration on Sephadex G-150 was 60,000.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis disclosed two peptides with molecular weight values of 38,000 and 25,000 which suggests that transcobalamin II contains 2 subunits. When vitamin Blz binds to transcobalamin II there is a shift in the peak of vitamin Blz absorption from 361 nm to 364 nm. Analysis of transcobalamin II for carbohydrate content using gas-liquid chromatography and amino sugar analysis by the amino acid analyzer suggest that this trace plasma protein is not a glycoprotein.
Human plasma contains two vitamin Biz-binding proteins in approximately equal concentration which can be distinguished from each other on the basis of a number of physical and functional parameters.
The first of these proteins, transcobalamin I, has a molecular weight of approximately 120,000 as determined by gel filtration and does not bind to Cm-Sephadex at pH 6.0. The second major plasma vitamin Biz-binding protein, trans-* This work was si:pportcd by Grants AM 10550 and TIE 00022 from the National Institutesof Health, PRA-33 from the American Cancer Society, and Special Research Fellowship AM 51261 from the National Institutes of Health.
This work was presented in part, at the Meeting of the American Society of Clinical Investigation, At.lantic City, New Jersey, May, 1972 (1).
cobalamin II, has a moIecular weight of 36,000 to 38,000 by gel filtration and does bind to Cm-Sephadex at pH 6.0 (2).
Several investigators have recently postulated the existence of a transcobalamin III based on the finding of two peaks of 120,000 molecular weight vitamin Biz-binding protein when human plasma is fractionated on DEAE-cellulose (3,4). It has not been established whether the heterogeneity observed for the 120,000 molecular weight vitamin Biz-binding protein is a result of microheterogeneity of transcobalamin I or whether it reflects the existence of separate protein species with major structural differences.
Questions of this type are difficult to resolve using crude human plasma.
Transcobalamin I contains approximately 80y0 of the vitamin Blz found in normal plasma (5) and vitamin Blz bound to this protein has a plasma half-life of approximately 9 days (6). A specific transport function for transcobalamin I has not been defined.
Transcobalamin II is postulated to function in vitamin B12 transport.
It has been observed in viva that after physiological levels of [57Co]vitamin-B12 are absorbed from the ileum, the vitamin appears in the plasma bound almost exclusively to transcobalamin II (7). Vitamin Blz bound to transcobalamin II in plasma is cleared primarily by the liver and has a plasma half-time of 12 hours (8). Studies conducted in vitro have demonstrated that crude preparations containing transcobalamin II facilitate the cellular uptake of vitamin Blz by human reticulocytes (9, lo), HeLa cells (11,12) and Ehrlich ascites tumor cells (11). Significantly greater amounts of vitamin B12 are taken up from culture media by these cells when vitamin Blz is bound to transcobalamin II than when vitamin Blz is present in unbound form or is bound to other vitamin B12-binding proteins such as transcobalamin I or intrinsic factor. Additional studies concerning the plasma vitamin Bn-binding proteins have been limited by the fact that the vitamin Blzbinding capacity of human plasma is less than 2 pg per liter (3,4). Using the molecular weight values obtained by gel filtration and assuming one vitamin Bn-binding site per molecule of vitamin B12-binding protein, 1 liter of human plasma contains less than 100 pg of either transcobalamin I or transcobalamin II. Purification in excess of a million-fold would be required to achieve homogeneity for either of the proteins, and this has been beyond the limits of conventional purification techniques.
Using affinity chromatography in addition to ion exchange chromatography and gel filtration, we have succeeded in isolating transcobalamin II. This report is concerned with the purification and physical properties of this protein.

Materials
Cohn Fraction III was obtained from the American Red Cross National Fractionation Center.
Other materials were obtained as described in the first two papers in this series (13,14).

Methods
Vitamin BrZ-binding assays were performed using a modification (13,14) of the method of Gottlieb et al. (15). Solutions containing radioactive and nonradioactive vitamin B12 were assayed as described in the first paper in this series (13). The isolation of monocarboxylic acid derivatives of vitamin Biz and their covalent attachment to 3,3'-diaminodipropylamine-substituted Sepharose using a carbodiimide was performed as described in the first paper in this series (13). The content of covalently bound vitamin Bla was 0.68 pmole per ml of packed Sepharose.
Protein assays, polyacrylamide disc gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis, sedimentation equilibrium ultracentrifugation, molecular weight determinations by gel filtration, amino acid analysis, assay of sulfhydryl group content, ca.rbohydrate analysis, and absorption and difference spectra were all performed as described in the second paper in this series (14).

Purijication of Transcobalamin II
Step I: Cohn Fraction III of Human Plasma-Transcobalamin II was purified starting with Cohn Fraction III of human plasma. All procedures were performed at 4". Each lot of Cohn Fraction III (72 kg) was derived from approximately 3000 liters of pooled human plasma.
A typical purification using 34 kg of this material is described below. The frozen material was chopped with an ice pick into pieces weighing less than 500 g and 8.5 kg of these frozen pieces of Cohn Fraction III were placed in each of four new loo-liter plastic trash containers which contained the following: 90 liters of Hz0 at 4", 124.2 g of NaH2P04.H20, 29.8 g of NazHP04.7 HzO, and 526 g of NaCl. After the addition of Cohn Fraction III each container was stirred continuously for 4 hours using a motor-driven propcllor type mixer. At the end of this time the pH of the Cohn Fraction III suspension was approximately 5.8.
Step 2: Cm-Sephadex-Sixty-three grams of dry, unprocessed Cm-Sephadex-C50 were added to each container and stirring was continued for an additional 4 hours. After the addition of the Cm-Sephadex, the pH of the suspension rose to 5.9. Stirring was stopped and the Cm-Sephadex was allowed to settle overnight.
The upper 85 liters of each container were next removed through a siphon and discarded.
The Cm-Sephadex was collected from the 8 liters of suspension remaining in each container by suction filtration using a Buchner funnel and 24.cm diameter circles of S & S filter paper No. 585. Approximately 2 kg of Cm-Sephadex, wet weight, were recovered from each of the large plastic containers, and the Cm-Sephadex from each container was suspended in 4 liters of the original Cohn Fraction III suspension solution.
After stirring for 5 min each suspension of Cm-Sephadex was again collected by suction filtration. Each batch of Cm-Sephadex was then suspended in 2 liters of 0.1 M sodium phosphate, pH 5.8, containing 1.0 M sodium chloride and stirred with a magnetic stirrer for 30 min. Each suspension was suction-filtrated on a Buchner funnel containing a 24.5-cm diameter circle of S & S glass wool No. 24 on top of a 24-cm diameter circle of S & S filter paper No. 585. The filter cake was then washed directly on the Buchner funnel with an additional 2 liters of the same eluting solution.
A combined total of 19,700 ml of this elution filtrate was obtained and contained 78% of the vitamin Blrbinding activity present in the initial Cohn Fraction III suspension.
The elution filtrate contained only a faint turbidity and was used directly for affinity chromatography on vitamin Br,-Sepharose.
Step 3: Afinity Chromatography on Vitamin Bls-Sepharose-A column 2.5 cm in diameter and 2 cm tall of vitamin Br-Sepharose was prepared and washed with 100 ml of 0.1 M glycine-NaOH, pH 10.0, followed by 100 ml of 0.1 M sodium phosphate, pH 5.8, containing 1.0 M NaCl.
This procedure served to remove traces of vitamin Br2 which had become hydrolyzed from covalent linkage to Sepharose.
The entire 1.0 RI NaCl elution filtrate from t,he previous Cm-Sephadex batch step was then applied to the column of vitamin B&Sepharose with a gravity head of approximately 250 cm of water. The flow rate was approximately 500 ml per hour. Only 7.70/, of the vitamin Brs-binding activity applied to the vitamin B&Sepharose column was recovered in the total effluent.
Small aliquots of the effluent were collected directly from the vitamin B&Sepharose column at various times during the sample application.
These aliquots were also assayed for vitamin Br-binding activity. They indicated that early in the sample application greater than 99% of the vitamin Bi*-binding activity was adsorbed to the vitamin B&Sepharose and that this level of adsorption had fallen to 90% near the end of the sample application.
After the entire sample had been applied, the column was then washed with different volumes of a variety of solutions in the following order. The flow rate during the first six column washes was 200 ml per hour and that of Wash 7 was 100 ml per hour.
The effluent from each wash was collected separately.
At the completion of Wash 7, the flow rate was decreased to 20 ml per hour and a solution of 0.1 M potassium phosphate, pH 7.5, containing 7.5 M guanidine IICl was applied. The first 43.0 ml of column effluent were collected in their entirety and were designated as column Wash 8a. The next 5.3-ml effluent from Wash 8 was collected separately and designated as column Wash 8b. At this point the column was clamped and allowed to stand for 18 hours. At the end of this time the column was unclamped and the first 13.0 ml of effluent were collected and were designated as column Wash 8c. Each of the column effluents mentioned above was assayed for vitamin B!,-binding activity and, except for those fractions containing guanidine, was also assayed for protein content.
The results are presented in Table I. Eluate 8a from vitamin Blz-Sepharose affinity chromatography was mixed with [57Co]vitamin-B!z (1320 pg, 0.0034 /.&I per pg) in a final volume of 44 ml. This mixture was dialyzed against 6 liters of 0.1 hf Tris-HCl, pH 8.9, contain- ing 0.2 M NaCl. After 4 hours the dialysate was changed and after an additional 24 hours the dialysate was changed to 6 liters of 0.1 M Tris-HCI, pH 8.9, without NaCl and dialysis was continued for an additional 45 hours. Greater than 99c/, of unbound vitamin BIB was removed by dialysis under these conditions. The transcobalamin II-vitamin Brz fraction was centrifuged at 50,000 x g for 30 min to remove denatured protein prior to chromatography on DEAE-cellulose.
Step 4: Chromatography on DEAE-Cellulose-A column (0.9 X 12 cm) of DEAE (Whatman DE 52) equilibrated with 0.1 M Tris-HCl, pH 8.9, was first washed with 60 ml of 0.1 M Tris-HCl, pH 8.9, containing 0.0111 pg of [57Co]vitamin-Blz per ml (0.0034 $Zi per pg) before the transcobalamin II-vitamin Blz fraction from Step 3 was applied to the column at a flow rate of 25 ml per hour. The column was washed with 10 ml of the [S7Co]vitamin-13!z containing equilibrating solution and then eluted with a linear gradient in which the mixing chamber contained 225 ml of 0.1 M Tris-HCl, pH 8.9, and the reservoir contained 225 ml of 0.1 M Tris-HCl, pH 8.9, containing 0.5 M NaCl. All of these eluting solutions contained [57Co]vitamin-Blz as described above. Fractions were assayed for AZQ vitamin Brz content, and conductivity.
The results are presented in Fig. 1. Fractions 50 through 65 were pooled.
Step 5: Chromatography on S ,S'-Diaminodipropylamine-substituted Sepharose-A column (0.9 X 6 cm) of 3,3'-diaminodipropylamine-substituted Sepharose was equilibrated with 100 ml of 0.1 M Tris-HCl, pH 8.9, containing 0.2 M NaCl. The DEAE-cellulose pooled fractions 50 to 65 of transcobalamin II-vitamin Blz were applied to the column at a flow rate of 50 ml per hour and the column was eluted with this same buffer. The first 82 ml of effluent from the column contained greater than 99% of the transcobalamin II-vitamin Brz applied. The transcobalamin II-vitamin Blz solution was adjusted to contain 0.75 hf NaCl and was then concentrated to approximately 1 ml using an hmicon ultrafiltrator equipped with a Diaflo UM-10 membrane.
Despite stirring during the concentration procedure, a red film was observed on the Diaflo membrane at the completion of the concentrating procedure. The Amicon concentrate 7711 was removed and the concentrating vessel was rinsed repeatedly with 0.05 M potassium phosphate, pH 7.5, containing 0.75 M NaCl until the red fihn on the membrane went into solution. The final concentrate was slightly turbid and this precipitate was removed by centrifugation at 10,000 X g for 10 min. Approximately 4yo of the total vitamin Blz present was present in the small pink precipitate, with the remaining 96% being present in the 6.0 ml of red supernatant solution. This supernatant solution was immediately subjected to chromatography on Sephadex G-150.
Step 6: Chromatography on Xephadex G-150-A column (2.0 X 90 cm) of Sephadex G-150, fine grade, was equilibrated with 0.05 M potassium phosphate, pH 7.5, containing 0.75 M NaCl and [57Co]vitamin-Blz (0.0111 pg per ml, 0.0034 &i per I.cg). The transcobalamin II-vitamin Blz fraction from the preceding step was applied directly to the top of the column and the column was eluted with the equilibrating solution at a flow rate of 20 ml per hour. Fractions of 3.3 ml were collected and assayed for vitamin B12 content and for absorption at 280 nm (Fig. 2). Fractions 51 to 64 were pooled and concentrated using an Amicon ultrafiltrator as described in Step 5. A red film also observed on the Diaflo membrane at the end of this concentration procedure was dissolved as described in Step 5. Less than 1% of the vitamin Blz placed in the Amicon ultrafiltrator passed through the UM-10 membrane.
The Amicon concentrate and the rinses were combined, centrifuged at 10,000 X g for 10 min, and the red supernatant decanted. A small dark red pellet containing 2% of the total vitamin Br, present was discarded.
The red supernatant, containing 98% of the vitamin B12, was divided into 1.5-ml aliquots, quick-frozen in a Dry Ice-acetone bath, and stored at -70".
A summary of the purification procedure is presented in Table II.  Cohn Fraction III, supplied as a frozen wet paste in X-kg lots derived from 3000 liters of pooled human plasma, was used as the st.arting material.
We have tested five separate lots of Cohn Fraction III and have observed vitamin Blz-binding ackities ranging from 12 to 18 ng of vitamin Blz bound per g of frozen, wet paste. No loss of activity has been noted after storage of Cohn Fraction III at -20" for several months.
Based on the binding of the vitamin B12-binding protein in Cohn Fraction III to Cm-Sephadex as well as its elution profile on Sephadex G-150 we conclude that greater than 95% of the vitamin B,a-binding activity in Cohn Fraction III is attributable to transcobalamin II (2). Assumin g that pooled human plasma contains I ng per ml of transcobalamin II-vitamin &-binding activity, 27 "/i to 40 "vc of the transcobalamin II present in plasma is recovered in Cohn Fraction III.
As shown in Table II transcobalamin II is partially purified by batch chromatography on Cm-Sephadex before it is further purified by affinity chromatography on vitamin B&epharose. 'The Cm-Sephadex step results in a 20.fold reduction in volume and a 5-fold purification, but its major advantage is that transcobalamin II is obtained in a solution that is capable of passing over a column of vitamin B&epharose without clogging the column.
Initial attempts to suspend Cohn Fraction III in rarious buffers followed by centrifugation failed to produce solutions that were suitable for direct application to vitamin Bls-Sepharose because of protein precipitation.  (14) where no detectable contaminating protein is present after affinity chromatography.
The most likely reason for this difference resides in the fact that 98% of the granulocyte vitamin Blz-binding protein remained adsorbed to vitamin B&3epharose when the column was washed with 5.0 M guanidine HCl while significant amounts of transcobalamin II are eluted with 5.0 M guanidine HCl and this washing procedure could not be employed for transcobalamin II. The comparative ease of elution of transcobalamin II is also demonstrated by the fact that only several hours of incubation with 7.5 M guanidine are required for elution (see Table I) while 41 hours are required for the granulocyte vitamin B12-binding protein (14). Transcobalamin II has been purified 2 million-fold relative to plasma with a recovery of 12.8yc (Table II).
The final preparation is homogeneous based on results of disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, gel filtration on Sephadex G-150, and the ratio of total amino acid content to bound vitamin B~z. Based on the pooled Sephadex G-150 fractions, 1 mg of protein contains 28.6 pg of bound vitamin and has an Azso of 1. 5  (i.e. vitamin Bls-binding ability) of transcobalamin II is greater when guanidine is removed by dialysis in the presence of a a-fold excess of vitamin B12 than when aliquots of the protein in guanidine are diluted 1:5,000 and assayed for vitamin Blz-binding activity directly. This is illustrated in Table I where the initial 7.5 M guanidine HCI eluate from vitamin B,,-Sepharose bound 353,000 ng of vitamin BL2 by the former method and only 198,000 ng by the latter method.
Similar results are obtained using the final preparation of transcobalamin II. Thus, when a a-fold excess of vitamin Blz is added prior to dialysis, transcobalamin II binds 27-30 pg vitamin Bit per mg of protein compared to a value of 14 to 18 pg of vitamin Bla per mg of protein when the vitamin is added after dialysis or after a 5,000 to 75,000 dilution of a solution containing the protein and guanidine. These results indicate that the presence of the vitamin is an important factor in the renaturation process. A similarly increa.sed yield of native protein after renaturation in the presence of vitamin Blz was also observed for the granulocyte vitamin Brz-binding protein (14).
In other studies variation in protein concentration, temperature, pH, and salt concentrations as well as the addition of EDTA, sulfhydryl compounds, and glycerol have not resulted in any significant increase in the renaturat,ion (i.e. vitamin B,zbinding activity) of transcobalamin II when guanidine is removed in the absence of vitamin 13,~. The presence of 0.02 M 2-mercaptoethanol and dithiothreitol both cause a marked decrease in the degree of transcobalamin II renaturation. Polyacrylamide Disc Gel Eleclrophoresis-When 30 pg of the transcobalamin II-vitamin B12 complex were subjected to polyacrylamide disc gel electrophoresis and stained for protein the pattern presented in Fig. 3 was obtained.
Unstained gels had a faint red color that was localized to the entire region of the gel that stained for protein.
Unstained gels were cut into l-mm sections and the distribution of vitamin Blz was determined by measuring the radioactivity of the individual gel slices. A single broad peak of radioactivity was observed that coincided with the gel region that stained for protein.
The reason for the failure to obtain a sharper band of either protein or vitamin Bn has not been determined but may be related to the limited solubility (see above) of the transcobalamin II-vitamin Blz complex since high protein concentrations are achieved during the stacking period of disc gel electrophoresis. Plots of In A280 versus R2 and In A360 versus R2 gave straight lines in all three experiments.
The plot of In 4~80 versus R2 obtained at 0.136 mg of protein per ml is shown in Fig. 4. The values for the slopes of the straight lines obtained by plotting In absorbance versus R2 were the same when cells were scanned at 280 nm and 360 nm indicating correspondence between protein and vitamin B12. No dependence on protein concentration was observed. Using the partial specific volume of 0.747 calculated from the amino acid analysis (see below) a molecular weight of 53,900 & 2,360 SD. was obtained for the transcobalamin II-vitamin Blz complex using the data from the 280 nm scans. When data from one of the 360 nm scans were used to calculate the molecular weight, a value of 52,800 was obtained.
Amino Acid Analysis of Transcobalamin II-The amino acid composition of transcobala.min II is presented in Table III. When sulfhydryl groups were assayed in 7.5 M guanidine HCl containing 0.1 M potassium phosphate, pH 7.5, no sulfhydryl residues were detected ( < 0.1 residue per mole of transcobalamin II).
This finding indicates that any cysteine residues in transcobalamin II are involved in disulfide bands.
Based on the molecular weights of the individual amino acids determined, transcobalamin II contains 59,500 g of amino acid per mole of bound vitamin B12. This value is close to the respective molecular weights of 53,900 and 60,000 determined for the transcobalamin II-vitamin B12 complex by sedimentation equilibrium ultracentrifugation (see above) and gel filtration on Sephadex G-150 (see below).
The agreement among these studies indicates that transcobalamin II contains a single vitamin B12-binding site and that the final preparation of transcobalamin II is devoid of major contamination by denatured transcobalamin II or other proteins.
Carbohydrate Analysis-No carbohydrate residues were detected in the final preparation of transcobalamin II by gasliquid chromatography and no amino sugar residues were detected on the amino acid analyzer.  b Accurate quantitation was not possible since ninhydrinpositive material was present in the cysteic acid posit.ion in the absence of performic acid oxidation.
If one assumes that all of the material in this region is cysteic acid, then 9 residues were present in the standard analysis and 13 residues were present after performic acid oxidation.
c Determined spectrophotometrically. analyzed was such that 1 mole of fucose, galactose, glucose, mannose, N-acetylgalactosamine, N-acetylglucosamine, or sialic acid per mole of bound vitamin Brz would have been detected. Thus, transcobalamin II is not a glycoprotein.

Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis-When 30 pg of the transcobalamin II-vitamin Blz complex were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, two protein bands were observed that stained with Coomassie brilliant blue with equal intensity (Fig. 5). The molecular weights estimated for these polypeptides were 38,000 and 25,000.
The sum of the molecular weights of these two components is 63,000 which is similar to the molecular weight estimates for transcobalamin II, obtained by sedimentation equilibrium, amino acid analysis, and gel filtration, and suggests that transcobalamin II consists of one 38,000 molecular weight subunit and one 25,000 molecular weight subunit. Molecular Weight Determination by Gel Filtration-Gel filtration was the final step used in the purification of transcobalamin II. This was performed on a column (2.0 x 90 cm) of Sephadex G-150 equilibrated with the same solution used for the sedimentation equilibrium studies, i.e. 0.05 M potassium phosphate, pH 7.5, 0.75 M NaCl.
Transcobalamin II was eluted as an isolated peak with correspondence between the amount of Azso and vitamin Brz as shown in Fig. 2. Based on the empirically determined relationship between K,, and log molecular weight (see Fig. 6 Fig. 2). The apparent molecular weight obtained for the transcobalamin II-vitamin Bx~ complex from this experiment was 60,000. @ indicates the value of K,, obtained when 40 pg of the final preparation of transcobalamin II were applied to the same column of Sephadex G-150 in the presence and absence of [Wolvitamin-Bn. An apparent molecular weight of 38,000 was obtained in both of these experiments.
(See text for additional details and comment.) subunits of 38,000 and 25,000 molecular weight, and it is important to note that during the final purification on Sephadex G-150 no shoulder of A%0 or vitamin Br2 content was observed at the 38,000 molecular weight position and almost no A%,, or vitamin Br2 was present at the 25,000 molecular weight position. This observation suggests that the 2 transcobalamin II subunits were associated during the Sephadex G-150 final purification step.
Other gel filtration experiments were performed on the same Sephadex G-150 column using samples of transcobalamin II which were 250-fold less concentrated with respect to protein than in the experiment described above. Eighty micrograms of the isolated protein were dialyzed against 7.5 M guanidine HCl to remove greater than 99% of the bound vitamin Blz. Half of this protein solution was then dialyzed against 300 volumes of 0.05 M potassium phosphate, pH 7.5, containing 0.75 M NaCl for 72 hours with changes at 24 and 48 hours. The other half was dialyzed in an identical manner except that 3.4 pg of [Wolvitamin-Blz were added to the protein-guanidine solution prior to dialysis.
Each of the two dialyzed protein solutions was adjusted to a volume of 6.0 ml containing 10 mg of blue dextran 2000 and 2 mg of dinitrophenyl alanine and applied separately to the Sephadex G-150 column.
In both of these experiments, a single symmetrical peak of vitamin B12binding activity (or [57Co]vitamin-Br2) was observed at an elution position corresponding to a molecular weight of 38,000 (see Fig. 6). These two results suggest the possibility that the transcobalamin II subunits were not associated under the conditions in which these experiments were performed and that the 38,000 molecular weight subunit contains the binding site for vitamin Bl2. It is also possible that transcobalamin II interacts with Sephadex at low protein concentrations with a resulting retardation of the protein. --, transcobalamin II (409 pg per ml) containing 11.7 pg per ml of bound vitamin B12; ---, vit,amin Blz (11.7 pg per ml). Absorption and Difference spectra-The spectrum of the transcobalamin II-vitamin Blz complex is presented in Fig. 7 together with the spectrum of an equal concentration of unbound vitamin BU. When vitamin BJ2 is bound to transcobalamin II there appears to be a general enhancement of the vitamin I& spectrum above 300 nm since the spectrum of transcobalamin II devoid of vitamin B12 in 7.5 M guanidine HCl, 0.05 M potassium phosphate, pH 7.5, is that of a typical protein with insufficient absorption above 300 nm to account for the difference between the two spectra presented in Fig. 7. When vitamin Blz binds to transcobalamin II, there is a shift in the 361 nm spectral maximum for unbound vitamin El2 to 364 nm for the transcobalamin II-vitamin Ulz complex. The difference spectrum between the transcobalamin II-vitamin B12 complex and a concentration of unbound vitamin BE containing equal absorption at 361 nm is presented in Fig. 8.

DISCUSSION
Tra,nscobalamin II has been isolated in homogeneous form for the first time after being purified 2 million-fold relative to human plasma.
Affinity chromatography on vitamin B&epharose was the crucial purification technique employed and resulted in a 24,000-fold purification of transcobalamin II. The fact that this technique has been applicable to the isolation of the granulocyte vitamin B12-binding protein (14) as well as transcobalamin II suggests that it may be of general value in isolating other vitamin B12-binding proteins.
Plasma fractions containing transcobalamin II facilitate the uptake of vitamin B1:: by a number of different types of cells (g-12).
The availability of homogeneous transcobalamin II allows for new experiments to elucidate the mechanism of protein facilitated cellular uptake of vitamin B1?. Preliminary experiments' indicate that our final preparation of transcobalamin II does facilitate vitamin Blz uptake by confluent cultures of human diploid fibroblasts.
Thus, vitamin Ulz bound to transcobalamin II is taken up by fibroblasts in significantly greater amount than either unbound vitamin Blz or vitamin BE bound to the granulocyte vitamin B12-binding protein.
This result indicates that our final preparation of homogeneous transcobalamin II retains its functional ability as well as its ability to bind vitamin Blz.
Studies using crude preparations of transcobalamin II have indicated that this protein contains a single vitamin BIT-binding site (16) and has a molecular weight of 36,000 to 38,000 (2,17) when determined by gel filtration.
Our studies using homogeneous transcobalamin II also indicate that the protein has a single vitamin Bjz-binding site, but our studies demonstrate a molecular weight of approximately 60,000 when measured by gel filtration, sedimentation equilibrium ultracentrifugation, and amino acid analysis.
We have determined that transcobalamin II is a dimer consisting of 1 approximately 38,000 molecular weight subunit and 1 approximately 25,000 molecular weight subunit. Bdditional gel filtration experiments suggest that the 2 subunits may dissociate under certain conditions or that the protein interacts with Sephadex thus resulting in an apparent molecular weight of 38,000 based on the elution profile of protein-bound vitamin BE. It is of interest that studies using crude transcobalamin II yield gel filtration molecular weight values greater than 38,000 for this protein under certain conditions (E-20) and that partial purification or high salt concentrations, or bot.h, are required to obtain transcobalamin II in its 36,000 to 38,000 molecular weight form (2). Transcobalamin II has a number of properties in common with the granulocyte vitamin Blz-binding protein (14), but the two proteins also differ in a number of respects. Similar properties include: (a) both proteins appear to have single vitamin B12binding sites and have molecular weights close to 60,000.
(b) The presence of vitamin HI2 is required to obtain maximal vitamin Blz-binding activity when the proteins are renatured from 7.5 M guanidine HCl.
(c) Sulfhydryl compounds are deleterious to the renaturation of both proteins. (d) Neither protein contains any demonstrable free sulfhydryl groups.
(e) When vitamin B,z is bound to either protein there is a general enhancement of the vitamin B12 spectrum above 300 nm.
Differences between transcobalamin II and the granulocyte vitamin Bin-binding protein include: (a) transcobalamin II contains one 38,000 molecular weight subunit and one 25,000 molecular weight subunit, whereas the granulocyte vitamin B12-binding