Biochimica et Biophysica Acta, 709 (1982) 353-357 353 Elsevier Biomedical Press

BBA 31409

INTERACTION OF GROUP-SPECIFIC COMPONENT (VITAMIN D-BINDING PROTEIN) WITH IMMOBILIZED CIBACRON BLUE F3-GA *

COLETTE CHAPUIS-CELLIER **, ELISABETTA GIANAZZA *** and PHILIPPE ARNAUD

Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29425 (U.S.A.)

(Received June 14th, 1982)

Key words: Group-specific component," Dye binding," Vitamin D-binding protein; (Human plasma)

Group-specific component (vitamin D-binding protein) was purified to homogeneity from human plasma by a three-step procedure involving pseudo-ligand affinity chromatography on immobilized Cibacron blue F3-GA followed by gel filtration and ion-exchange chromatography. Upon pseudo-ligand chromatography, Gc globulin was separated into two peaks. The first, which represented approx. 4% of the total Gc globulin, was eluted together with other a-globulins of similar M r and/or pl, and the second (96% of Gc globulin) was clearly retarded. Collection of the latter provided a fraction 10-fold enriched in Gc globulin, with yields higher than 90%. Incubation of plasma with trace amounts of radioactively-labeled 25-OH vitamin D3 showed that the radioactivity cocluted with the first peak. In addition, after saturation with 25-OH vitamin D3, all the Gc globulin was eluted in the first peak. This indicates that the two peaks correspond to the holo and the apo forms of the protein, respectively, and suggests that either the interaction of the apo form with the Cibacron blue dye involves the binding site for vitamin D metabolites, or that the holo-protein undergoes a conformational change as a consequence of formation of the complex.

Introduction

Group-specific component [1] is present in hu- man plasma at a" concentration of 20-50 mg/dl [2]. This protein is highly polymorphic, and more than 30 different alleles have been described [3]. Its biological function has been shown to be the transport of vitamin D and its metabolites in plasma [4]. In contrast, few structural data on Gc

* Publication No. 537 from the Department of Basic and Clinical Immunology and Microbiology, Medical Univer- sity of South Carolina.

** Present address: Department de Biochimie et d'im- munochimie, Universite Claude Bernard 69374, Lyon, France.

*** Present address: Department of Biochemistry, University of Milano, Via Celoria 2, 20133 Milano, Italy.

Abbreviation: Gc globulin, group-specific component.

0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

globulin were available until recently [5]. The basis for its extensive polymorphism is still not known, probably because of the difficulty inherent in the purification of the protein and the very low yields reported by several authors [6,7].

The use of immobilized blue F3-GA in a pre- parative protocol has been reported for enzyme purification [8]. Although most of the enzymes studied were shown to possess the dinucleotide fold [9], recent data have restricted the specificity of the binding site(s) of the dye [10]. Cibacron blue F3-GA is also able to bind to a series of plasma proteins. This property has been used to remove albumin from human plasma [11], and to provide substantially enriched fractions for the purifica- tion of a2-macroglobulin [12,13], a~-anti- chymotrypsin [14] and other plasma proteins [15]. In the present report, we show that during chro- matography on Blue A, most of the Gc globulin is

354

clearly retarded in contrast with other plasma pro- teins of similar molecular weight and/or isoelec- tric point. This results in a 10-fold purification of the protein, which can then be purified to homo- geneity by two additional steps of gel filtration and ion-exchange chromatography. We have also analyzed the mechanism of the interaction be- tween Gc globulin and Blue A.

Experimental Procedures

Materials Human plasma was obtained from healthy

volunteers with the Gc globulin l-1 phenotype [3]. Blood was collected in plastic containers on a solution of sodium citrate/soybean trypsin inhibi- tor (0.5 mg/ml inhibitor in 3.8% sodium citrate) in a ratio 9 : I. After centrifugation at 200 X g at 4°C for 15 rain, the supernatant was decanted and dialyzed overnight at 4°C against 0.03 M sodium phosphate buffer, pH 7.0. Radioactively-labeled 25-OH vitamin D3 (25-OH-26(27)-methyl-aH- cholecalciferol, spec. act. 10 Ci/mmol) was ob- tained from Amersham. When the radioactively- labeled product was to be used as a tracer, a molar ratio of 25-OH vitamin D3 to Gc globulin of 1/2500 was employed. Saturation of the plasma with 25-OH vitamin D3 (Hoffman-LaRoche) was performed by incubation with a 5-fold molar ex- cess over Gc globulin concentration, as determined by rocket immunoelectrophoresis.

Methods Chromatography on immobilized Blue A. Pre-

parative isolation of Gc globulin was performed at room temperature. 80 ml plasma were deposited on a column (2.5 x 100 cm, Pharmacia, Piscata- way, N J) containing 400 ml cross-linked agarose gel (bead diameter 150-300 pm) with covalently coupled reactive Blue A dye (Affigel Blue, color index 61211, a gift from BioRad Laboratories, Richmond, Ca). The gel was equilibrated in 0.03 M sodium phosphate buffer, pH 7.0, I = 0.05. The column was eluted with the equilibration buffer, at a flow rate of 20 ml/h, and 3-ml fractions were collected at 4°C. For analytical studies of Gc globulin before and after saturation with 25-OH vitamin D3 a smaller column (1.0 X 20 cm) was used containing 50 ml gel. The fractions were

analyzed for Gc globulin content by fused rocket immunoelectrophoresis.

Gel filtrations. A column (2.5 x 100 cm) was filled with Sephadex G-100 superfine (Pharmacia Fine Chemicals, Piscataway, N J), swollen and equilibrated with 0.1 M H3PO4/sodium buffer, pH 6.5. Fractions containing Gc globulin from the Blue A column were pooled, concentrated to 5 ml, dialyzed against the phosphate buffer, and applied to the column. The flow rate of the elution was 10 ml/h, and 2.0-ml fractions were collected. Frac- tions containing Gc globulin were detected by fused rocket immunoelectrophoresis, pooled, con- centrated and dialyzed against the ion-exchange chromatography buffer.

Ion-exchange chromatography. The experiments were run on a column (0.9 x 15 cm) of DEAE-Af- figel Blue (BioRad Laboratories) equilibrated in 0.02 M HaPO4/potassium buffer, pH 8.0. After application of the sample, the gel was washed with 60 ml of the equilibration buffer and then eluted at a flow rate of 10 ml /h with a linear gradient (from 0.0 to 0.5 M; total volume of the gradient, 150 ml) of NaC1 in the buffer using a gradient mixer (GM1, Pharmacia). 2-ml fractions were col- lected and monitored for Gc globulin content by fused rocket immunoelectrophoresis.

Other methods. Fused rocket immunoe- lectrophoresis, immunoelectrophoresis using total antiserum against human plasma proteins (Meloy) or specific antisera against Gc globulin (Dako), rocket immunoelectrophoresis, and SDS-poly- acrylamide gel electrophoresis were performed according to standard procedures [16-20]. Total protein measurement was performed according to Bradford [21], using bovine serum albumin as standard.

Results and Discussion

When plasma proteins were run through a Blue A column with a low molarity buffer, the elution profile showed a major peak and a trailing peak. Several proteins (cq-acid glycoprotein, aranti- trypsin, prealbumin, transferrin, ceruloplasmin, IgG, IgA), eluted with significant overlap [15]. Using a specific antiserum, Gc globulin was iden- tified in the trailing part of the curve (Fig. 1A). In the pool of the positive fraction, Gc globulin was

355

I

, r o ~ t b ° . . u t a h * ,

Fig. 1. Chromatography steps for the purification of Gc globulin. The bar indicates the elution of Gc as detected by fused rocket immunoelectrophoresis [15]. A: Affigel blue (pH 7.0), I = 50 mEquiv. B: Sephadex G-100. C: DEAE Affigel blue, pH 8.

contaminated mostly by IgG, ceruloplasmin and transferrin (Fig. 2). This step resulted in a purifi- cation of approx. 10-fold (Table I).

Gc globulin was further purified by two addi- tional steps of gel filtration and ion-exchange chromatography (Fig. 1B and C). The total yield was higher than 70% (Table I), which is to be compared with 1.5-13% yields with classical tech- niques [6,7] and, more recently, 10-20% [22]. In particular, the first step clearly separated Gc globulin from al-antitrypsin, which has been re- ported as a major contaminant in previous purifi- cation procedures [23].

It appeared intriguing that upon chromatogra- phy on immobilized Blue A, Gc globulin was retarded in comparison with proteins of similar isoelectric points and/or molecular weight. This observation suggested the possibility of an interac- tion between Blue A and Gc globulin. In addition

to most enzymes containing the dinucleotide-fold [23,24], other proteins possess the ability to inter- act with Blue A, including albumin [11] and a number of other plasma proteins [12-15].

In fact, a small percentage of Gc globulin (from 3 to 5% of the total, Fig. 3A) was eluted from the Blue A column together with the other plasma proteins. It is known that Gc globulin represents the carrier of vitamin D metabolites in the plasma [4,7] and that it is normally only approx. 3-4% in the holoform [25,26]. Labeling of plasma with a trace amount (1:2500 molar ratio) of radioac- tively-labeled 25-OH vitamin D showed (Fig. 3A) that a single peak of radioactivity coeluted with the first small peak of Gc globulin, indicating that this fraction corresponded to the holoprotein. After saturation of plasma with 5-fold molar excess 25- OH vitamin D3, Gc globulin was eluted as a single peak whose maximum corresponded to the posi-

TABLE I

PROTEIN CONCENTRATION, GC GLOBULIN CONCENTRATION AND YIELDS AFTER THE DIFFERENT PURIFICA- TION STEPS

Total protein concentration was measured by the technique of Bradford [21]. GC globulin was measured by Laurell rocket' immunoelectrophoresis [19] by comparison with a protein standard (batch 75425) obtained from Calbiochem Behring (La Jolla, CA).

Material Volume Total protein Gc globulin Yield Purifi- (ml) concentration concentration (%) cation

(mg/dl) (mg/dl) factor

Plasma 80 6.150 43.0 Affigel blue

pH 7.0 (fractions 96-154) 177 242.9 17.6

Sephadex G- 100 (fractions 113-137) 50 222 53.0

DEAE-Affigei blue pH 8.0 (fractions 129-144) 32 81.2 78.4

93 9.6

77 34.16

73 138.5

356

Fig. 2. A. In'ununoelectrophoresis indicating the results of the different purification steps. I, after Affigel blue; II, after gel filtration; and III, after ion-exchange chromatography, a-S, antitotal human serum, a-Gc, antiserum to Gc globulin. B. SDS-polyacrylamide gel electrophoresis of Gc globulin after ion-exchange chromatography. Lane A; high molecular weight standards, Lane B, low molecular weight standards (both from Pharmacia), Lane C, the protein preparation (2.1 ~g). Experimental: 10~ polyacrylamide gels with 5~ stacking gel were made in the discontinuous buffer system of Laemmli [19]. The silver stain technique of Merril [30] was used for protein coloration.

o o 3

x IK

'~ t u b e n u m b e r

Fig. 3. Comparison of fused rocket immunoelectrophoresis (gel stain) and radioactive counts (dotted line) following chromatography of human plasma on immobilized Cibacron Blue F3-GA. A, plasma incubated with radioactively-labeled 25-OH vitamin D3 (molar ratio of vitamin D3 to Gc globulin, 1/2500). B, plasma incubated with unlabeled and radioactively-labeled 25-OH vitamin D3 (molar ratio of vitamin D3 to Gc globulin, 5/1).

tion of the holoprotein (Fig. 3B). These results indicate that the fraction of Gc

globulin that is retarded upon chromatography on immobilized Blue A corresponds to the apoprotein, whereas the holoprotein is eluted together with plasma proteins of similar molecular characteris- tics. Furthermore, the results of saturation with the specific ligand suggest that the interaction with Blue A involves the binding site of the protein for vitamin D metabolites or the region in its vicinity. It is interesting to note that other transport pro- teins, such as haptoglobin, hemopexin, thyroxine- binding globulin [15], and transcobalamin [27], bind to the dye both in the holo and apo forms. In contrast, the binding of human and animal al- bumins to the dye is greatly reduced by previous incubation with such ligands as bilirubin and palmitate, a phenomenon which led Leatherbar- row and Dean to propose that the ligands compete with the dye at the binding site(s) of the protein [28]. The fact that Gc globulin is retarded rather than bound on the Blue A column could indicate that its binding site has a relatively weak affinity for the dye. On the other hand, the binding of vitamin D metabolites to Gc globulin affects its net charge [22,26,29]. Indeed, the association be- tween a neutral molecule such as vitamin D3 and a protein with a p l at 4.86 and 4.93 for the two bands of the major Gc globulin phenotype leads to a complex whose heterogeneity is preserved, but whose pI is decreased by approx. 0.03 pH units [29]. To explain this intriguing finding, Svasti and Bowman [22] suggested that the holo form of Gc globulin had undergone a change in its conforma- tion. It is therefore possible that the different behavior of holo- and apo-Gc globulin on Cibacron blue is related to the modification of the net charge of the complex.

Acknowledgments

We thank David L. Emerson for performing SDS-polyacrylamide gel electrophoresis and Charles L. Smith for editorial assistance. Research supported in part by USPHS Grant CA-25746 and by General Medical and Faculty Research Ap- propriation Grant 22300-A-091, Medical Univer- sity of South Carolina. C.C.-C. and E.G. were postdoctoral fellows of the College of Graduate Studies, Medical University of South Carolina.

357

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