Scand J Haematol(l977) 18, 403-412

Comparison of Biochemical Properties of Human Serum B I ~

Binders Derived from Sephadex and DEAE-Cellulose

Chromatography

SUDHIR KUMAR, LEO M. MEYER & RICHARD A. GAMS

Hematology Section, Veterans Administration Hospital, Brooklyn, New York and

Hematology Division, Department of Medicine, University of Alabama Medical School, Birmingham, Alabama, USA

Vitamin Biz binding proteins were separated into 2 peaks corresponding to small (TCS) and large (TCL) molecular weight fractions by gel-filtration on Sephadex G-200 using 0.005 M-sodium phosphate buffer, pH 7.4 containing 1 M-NaCI. Each peak, after dialysis and concentration, was chromatographed on DEAE-cellulose. 3 peaks of radioactivity were recovered from both TCS and TCL. Peaks from TCS had an apparent mol. wt. of about 40,000 and thme from TCL about 11O,OOO, as determined by gel-filtration on Sephadex G-200. On electrophoresis, peaks eluted with 0.06 M- phosphate buffer, pH 5.85 from both TCS and TCL moved as ,@-globulins; those eluted with 0.1 M buffer, pH 5.8 between p- and a2-globulin and those eluted with 0.25 M buffer, pH 5.4 between q- and a2-globulin. In our assay system, TCS d e livered 57CoB1~ to L-1210 leukaemic lymphoblasts while TCL had no such activity. Of the 6 binders from DEAE-cellulose, only peaks eluted with 0.06 M and 0.1 M buffers from TCS delivered labelled Biz to these cells. Antisera prepared against TC I1 BIZ reacted only with TC I1 BIZ from serum, TCS, and fractions D and E obtained from TCS which were eluted with 0.06 and 0.1 M buffers, respectively.

Key words: DEAE-cellulose - human serum BIZ binding proteins - Sephadex - vitamin Biz binders

Accepted for publication January 20, 1977

Correspondence to: Dr. S. Kumar, Perinatal Laboratory, Christ Hospital, 4440 West 95th Street, Oak Lawn, Illinois 60453, USA

Many dif€erent methods have been de- techniques of protein separation which have scribed for the measurement of vitamin been used include paper and starch-gel elec- BIZ binding proteins in human serum. The trophoresis (Miller & Sullivan 1959a/b,

A part of this paper was presented at the 59th Annual FASEB Meetings at Atlantic City, Apr, 1975

404 SUDHIR KUMAR, LEO M. MEYER & RlCHARD A. GAMS

1961), Geon-block electrophoresis (Hall & Finkler 1962), column chromatography and immunodiffusion technique of Ouchterlony (Hall & Finkler 1963), DEAE-cellulose (Hall & Finkler 1966, Retief et a1 1967, Gizis et a1 1970, Gizis & Meyer 1971, 1972, Kumar et a1 1974), and CM-cellulose ion exchange chromatography (Hall & Finkler 1965, Hom et a1 1966), Sephadex G-200 (Hom et a1 1966, Lawrence 1969), affinity chromatography (Allen & Majerus 1972 a/b/c), and more recently isoelectric fo- cusing (Stenman & Grasbeck 1972, Sten- man 1974). It is, however, of interest that no two laboratories have used identical procedures for isolating the vitamin Biz binding proteins from human serum.

The presence of at least 3 vitamin Biz binding proteins in human serum has been demonstrated (Hall & Finkler 1966, Retief et al 1967, Lawrence 1969, Gizis et a1 1970, Gullberg 1970, Bloomfield & Scott 1972, Gizis & Meyer 1971, 1972, Chanarin et a1 1972, Kumar et a1 1974). These binders have been designated as TC I, TC I1 and TC 111. In earlier studies, B12 binding pro- teins from normal serum were first sepa- rated into 2 fractions by chromatography on a DEAE-cellulose column, using a 2- buffer system for elution (Lawrence 1969, Chanarin et a1 1972). These fractions were termed TC I and TC 11. This was followed by gel-filtration of the TC I1 fraction on a Sephadex G-200 column to separate low mol. wt. TC I1 from the higher mol. wt. TC 111, which was eluted along with TC I1 in the first separation on DEAE-cellulose (Lawrence 1969, Chanarin et a1 1972). The 2 fractions thus obtained on gel-filtration were designated as TC I1 and TC I11 on the basis of mol. wt. and electrophoresis (Bloom- field & Scott 1972). In a previous study, Gullberg (1970) subjected whole serum to

gel-filtration with separation into 2 major fractions, transcobalamin large (TCL) and transcobalamin small (TCS), but no further fractionation was performed.

MATERIAL AND METHODS

Venous blood from normal volunteers was col- lected in tubes containing no preservative and kept at room temp. for 30 min followed with 2 h in a refrigerator (W0 C), before centrifugation in the cold to obtain the sera. Sera thus obtained were stored in 2 ml aliquots at -20O C. Once thawed, samples were not re-used. Radioactive cyanocobalamin ( 5 7 C o B ~ ~ , sp. act. 182 mCi/mg) was obtained from AmershadSearle.

Gel-filtration 011 Sephadex G-200 colunin

(a) 2 ml of serum were incubated with 1,000 pg of 57CoB1~ at 37OC for 20 min and the mixture dialysed against 4 1 of 0.005 M phosphate buffer, pH 7.4 containing 1 M NaCl overnight at 4' C. Dialysed serum was placed on a Sephadex G-200 column (2.6 x 33 cm, void vol 50 ml; prepared according to the manufacturer-Pharmacia), pre- equilibrated with 0.005 M phosphate buffer, pH 7.4 containing 1 M NaC1. Elution was performed with the same buffer. 2 ml samples were collected at a flow rate of 10 ml per h. The fractions were counted for 2 min each in an automatic well- type scintillation counter (Nuclear-Chicago, model 1185). 3 peaks of radioactivity were obtained, al- though peaks 2 and 3 contained more than 98 % of total radioactivity recovered. The first peak, obtained in the void vol, constituted about 1-2 % of total radioactivity and was not used. This peak seems to be similar to the one designated as TC-0 by Hom & Ahluwalia (1968). Only fractions containing maximum counts corresponding to peak 2 (TCL; elution vol 65 to 75 ml) and peak 3 (TCS; elution vol 103 to 117 ml) were collected, dialysed overnight against 4 1 of distilled water, and concentrated on a Bl5 microconcentrator (MiniconTM concentrator, Amicon). More than 95 % of the applied radioactivity of TCL and TCS were recovered as TCL and TCS, re- spectively, when the pooled, concentrated mate-

HUMAN SERUM BIZ BINDERS 405

rial was resubjected to gel-filtration on the same Sephadex column.

(b) In another set of experiments, fractions TCS and TCL were obtained as follows: 2 ml of serum were incubated with 1000 pg of 57CoB12 at 37O C for 20 min; mixture, after dialysis against 0.005 M phosphate buffer, pH 7.4 containing 1 M NaC1, was placed on a Sephadex G-200 column (2.6 x 60 cm, void vol 118 ml) and elution per- formed with the same buffer. The 2 main peaks of radioactivity corresponding to TCL and TCS, were collected, dialyzed and concentrated, as de- scribed before.

DEA E-cellulose chromatography on ‘small columns’

DEAE-cellulose was prepared according to Gizis & Meyer (1972). Concentrated fractions (TCL and TCS), obtained from either small (a) or large (b) Sephadex columns, were applied on DEAE-cellu- lose columns (5 ml pipette plugged with glass wool) and elution performed with step-wise gra- dient of 5 sodium phosphate buffers, as reported by Kumar et a1 (1974). Percentages of each binder, with respect to total radioactivity recovered, were calculated by combining counts for each peak of radioactivity (Kumar et a1 1974).

Molecular weight determination using Sephadex G-200 colurizns

Molecular weights of the 6 vitamin Biz binding protein fractions obtained on DEAE-cellulose column chromatography of TCL and TCS, were determined by gel-filtration on Sephadex columns similar to those utilized for initial separation of TCL and TCS fractions from whole serum.

Radioactive fractions eluted from DEAE-cel- lulose columns with phosphate buffers 0.06 M , pH 5.85; 0.1 M , pH 5.8; and 0.25 M , pH 5.4, were dialysed and concentrated on a minicon Bis mi- croconcentrator (Amicon) and subjected to gel- filtration. Elution vol for each binder were deter- mined on the same Sephadex column and mol. wt.’s determined by locating points on a standard selectivity curve prepared by determining elution vol for proteins of known mol. wt.’s, correspond- ing to their Kav values.

Paper electrophoresis of sub f ructions obtained from two Sephadex peaks

Electrophoresis of concentrated peaks of radio- activity obtained after chromatography of the Sephadex fractions (TCL and TCS) on DEAE- cellulose columns, were performed on a Durrum- type of electrophoresis cell (BeckmadSpinco, model R, Series C). Samples of each of three peaks from TCL and TCS in a normal serum control were applied to strips of paper and elec- trophoresis performed at 75 V for 18 h in 0.075 M Barbital buffer, pH 8.6. One strip of each binder and normal serum was stained with bromophenol blue (0.1 % in methanol). Unstained strips of binders were cut at 0.5 cm intervals, location of major serum protein fractions identified, and radioactivity counted in a well-type scintillation counter.

Uptake of B12 binding proteins by ascites tumor L-1210 cells

In earlier studies, we reported that only TC II- 57CoB12 derived from normal human serum has the capacity to deliver labelled vitamin to lym- phoblasts obtained from BDFl mice transplanted with L-1210 ascites tumor cells, while Biz from T C I and TCIII was not taken up by these cells (Meyer et a1 1974). We repeated these studies using TCL and TCS fractions plus six binders derived from the above Sephadex peaks to de- termine delivery of 57C0B12 to L-1210 cells.

Lymphoblasts were collected in normal saline from abdominal cavities of BDFl leukaemic mice as previously described (Meyer et a1 1974). A sus- pension containing lo7 cells in 250 ji1 aliquots was prepared in 0.02 M tris-normal saline, pH 7.4, containing 0.01 M CaCIz. Frozen transcobalamins were thawed and suspended in tris calcium saline buffer, centrifuged at 1,650 g for 30 min, and supernatant retained. Supernatants (each contain- ing about 5,000 cpm) and suspension of lympho- blasts were incubated at 37O C for 2 h, centri- fuged, pellets washed with normal saline 3 times, and counted in an automatic scintillation counter. All uptake studies were performed in duplicate.

Immune reactions against antibodies for TCII B12

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HUMAN SERUM Biz BINDERS 407

(a) subfractions obtained on DEAE-cellulose col- umns, (b) TCL and TCS, and (c) TC I, TC 11 and TC 111 obtained by DEAE-cellulose chroma- tography of normal serum, these binding pro- teins were tested for their immune reactions against antibodies for TC 11-Biz prepared in rabbit (kindly given to us by Dr. C. Hall, VA Hospital, Albany, New York). TCII-Biz used for preparing antibodies in rabbit was obtained by chroma- tography of 57CoB1~ labelled normal human se- rum on DEAE-cellulose columns. Immune reac- tions were demonstrated by the change in elution vol of gel-filtration on Sephadex G-200 due to the formation on an antigen-antibody complex, which was eluted with the void vol (Imrie & Schilling 1965).

To about 200 pg each of 57CoB1~ labelled binder, 0.2 ml of diluted antisera were added. The mixture was incubated at 37OC for 1 h and then refrigerated at 4O C for 16 h. Mixture was dialyzed against 0.005 M-sodium phosphate buffer, pH 7.4, containing 1 M-NaCI, placed on Sephadex G-200 column, and elution performed with the same buffer.

RESULTS

serum was chromatographed on either a small or large Sephadex G-200 column (Figures 1 and 3). More than 98 % of total radioactivity was recovered in 2 main peaks, with about 1-2 % of radioactivity in a peak eluted in void volume. Distribution of 57CoB1~ in peak corresponding to TCL was 203 pg and that in peak corresponding to TCS was 698 pg (Table 1). Although only fractions containing maximum counts cor- responding to peak 2 (TCL) and peak 3 (TCS) were used for further fractionation on DEAE-cellulose, results in Table 1 are expressed after extrapolating the data to the total amount of TCL and TCS to represent total activity.

Chromatography of TCL and TCS on D E A E-cellulose columns

A summary of the isolation procedure and results is presented in Table 1. 2 main peaks of radioactivity obtained from Sephadex columns subjected to further chroma- tography on DEAE-cellulose each dis- closed 3 peaks of radioactivity. Peaks A and D were eluted with 0.06 M phosphate buffer, pH 5.85, peaks B and E with 0.1 M ,

Gel-filtration on Sephadex G-200 columns

As reported by earlier workers (Lawrence 1969, Chanarin et a1 1972) 2 main peaks of radioactivity were observed when normal

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408 SUDHIR KUMAR, LEO M. MEYER & RICHARD A. GAMS

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pH 5.8 and peaks C and F with 0.25 M, pH 57C~B12 was recovered with peak D, 60 pg 5.4. Results of 8 experiments are presented with peak E and 152 with peak F. in Table 1. Both TCL and TCS contained On re-chromatography on DEAE-cellu- all three binders in varying amounts: 128 lose columns, all fractions were recovered pg of 57CoB1~ in TCL was recovered in as single peaks at identical positions where peak A; 46 pg with peak B and 29 pg they were obtained on initial chroma- with peak C; while in TCS, 484 pg of tography.

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HUMAN SERUM B12 BINDERS

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Repeated gel-filtration o f D E A E - cellulose column fractions on Sephadex G-200

On repeating gel-filtration of B12 binding proteins obtained on DEAE-cellulose col- umn chromatography of TCL and TCS fractions, 3 binders obtained from TCL were recovered at the original positions of elution of TCL and 3 binders obtained from TCS were recovered at the same position of elution as TCS. The 2 sets of binders obtained from TCL and TCS correspond to apparent mol. wt. of about 110,000 L- 10,000 and 42,000 k 1,500, respectively.

Paper electrophoresis

Results of electrophoresis of fractions ob- tained from both TCL and TCS are shown in Figure 2. The major portions of frac- tions A and D from TCL and TCS, respec- tively, as determined by counting radio- activity in different areas, moved as p-glo- bulin (Rf = 0.24), fractions C and F moved between ol- and az-globulins (Rf = 0.67), and fractions B and E between p- and a2- globulins (Rf = 0.45).

Uptake of B I Z binding proteins by L-1210 cells

Results of a representative experiment on delivery of radioactive B12 binding proteins

(X-----X) Normal radioactivity pattern when serum bound 57CoB1~ is chromatographed on Sephadex. First peak in void vol (105-135 ml); second peak - TC'L' (148-176 ml); third peak - TC'S' (187-235 ml). (X- X) Elution of Biz binding proteins af- ter reaction with T C I I BIZ antibodies. I. Fraction 'D' from TCS; 11. Fractions A, B, or C from TCL; 111. Fraction E from TCS.

410 SUDHIR KUMAR, LEO M. MEYER & RICHARD A. GAMS

introduced to the L-1210 cell system are presented in Table 1.

While there was no appreciable uptake of 5 7 C 0 B ~ ~ and its complex with TCL (0.3 %>, 8% of total radioactivity of TCS was taken up by L-1210 cells. As expected, there was no uptake of the individual binders derived from TCL. On the other hand, binders D and E obtained from TCS were taken up by these cells (E = 4.7 % and D = 2.2 %). No uptake of Biz from fraction F of TCS was observed. Results indicate that of all six binders, only fractions D and E from small mol. wt. Sephadex peak (TCS) were capable of mediating B12 uptake by L-1210 cells in vitro.

Immune reaction against TC I I B12 antibodies

Anti-TC I1 Biz serum reacted only with TC I1 B12 from serum, TCS, and fractions D and E obtained from TCS. It did not react with any of the other binders (Figure 3). These results codinn that sub-fractions A, B and C obtained on chromatography of TCL are not a contamination from TCS.

DISCUSSION

In earlier studies, B12 binding proteins from human serum were separated by either chromatography on DEAE-cellulose col- umns or by gel-filtration on Scphadex G- 200. From the study of the properties of these binders obtained from DEAE-cellu- lose columns, it has generally been assumed that the peak which comes out as T C I on DEAE-cellulose is similar to the high mol. wt. peak obtained by gel-filtration of serum on Sephadex, because both peaks have a mol. wt. of approximately 140,000; both move between al- and a2-globulin on elec-

trophoresis on paper; and both binders are incapable of mediating BIZ uptake by trans- formed human lymphocytes or lympho- blasts obtained from L-1210 cells (Meyer et a1 1974ah). Similarly, TCI I binder iso- lated by DEAE-cellulose column chroma- tography of the whole serum using a 2 buffer system, and the TCS peak from Sephadex column, were also considered to be identical proteins. At the same time, it was also reported that while TC I was made up of only 1 binder, TC I1 contained 2 binders, which could be separated by gel- filtration into a low mol. wt. binder, TC 11, and a high mol. wt. binder, TC 111, which eluted with TC I1 on DEAE-cellulose (Law- rence 1969, Chanarin et a1 1972). However, isolation of the large and small mol. wt. binder peaks by gel-filtration on Sephadex followed by chromatography of these frac- tions on DEAE-cellulose column to verify foregoling assumptions had not been carried out. Results of our present study indicate, that both large (TCL) and small (TCS) mol. wt. fractions of Biz binding proteins ob- tained by gel-filtration of normal serum on Sephadex G-200 can be separated into at least 3 different B12 binding proteins by chromatography on DEAE-cellulose col- umns. The fractions obtained from both TCL and TCS correspond in elution pat- terns to TC 11, TC I11 and TC I when nor- mal serum is chromatographed directly on DEAE-cellulose.

While fractions A and D, B and E, and C and F from both Sephadex peaks have similar electrophoretic properties, they are characteristically different in their mol. wt.; the binders from TCL having an apparent mol.wt. of 100,000-120,000 and those from TCS about 40,000, as determined by gel- filtration on Sephadex G-200.

In recent studies, Stenman (1974) has

HUMAN SERUM Biz BINDERS 411

shown that the a-globulin fraction (corre- sponding to TC I) contains 3 vitamin B12 binding components isoelectric at pH 2.0, 3.3 and 3.6 respectively. The components isoelectric at pH 3.3 and 3.6 are also present in the @-globulin fraction of high mol. wt. binder (possibly TC 111) which in addition also contain components which are isoelec- tric at pH 3.9 and 4.2. Similarly, the low mol. wt. @-globulin fraction, TC 11, is also focused at pH 5.4, 6.1 and 6.6 giving 3 iso- electric components.

Our results also rule out the possibility of these fractions being contaminated with each other since (1) all the fractions ob- tained from TCL are recovered as ‘large’ mol. wt. peak, and the fraction from TCS are recovered as ‘small’ mol. wt. peak, on repeating the gel-filtration on Sephadex G- 200; and (2) only the fractions obtained from TCS are capable of mediating B12 uptake by L-1210 cells, whereas fractions corresponding in elution to TC I, TC I1 and TC I11 from TCL do not mediate Biz up- take by these cells in vitro. The reasons for the inability of ‘fraction F’ obtained from TCS in mediating uptake of Biz by L-1210 cells are not clear, It is possible that it might be a ‘large’ md. wt. TC but without a sialic acid molecule, thus behaving as a ‘TCS’. This however, needs further confir- mation. While confirming that only the small mol. wt. binders are capable of me- diating BIZ uptake by L-1210 cells whereas the binders from the high molecular peak (TCL) are not, the results also suggest that all the 3 binders known heretofore, may exist as multiple molecular forms in human serum. These studies make no attempt to isolate TC I, TC I1 and TC I11 in pure form as performed by Allen & Majerus (1972

In view of the present study, the inter- a/b/C).

pretation of previous results using Sephadex and/or DEAE-cellulose need to be recon- sidered.

ACKNOWLEDGEMENT

The authors are grateful to Dr. C. Hall for his suggestions and for supplying TC I1 BIZ anti- bodies.

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Bloomfield F J & Scott J M (1972) Identification of a new vitamin Biz binder (Transcobalamin 111) in normal human serum. Br I Haematol 22, 3342.

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412 SUDHIR KUMAR, LEO M. MEYER & RICHARD A. GAMS

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