SHORT COMMUNICATIONS Purification of Swine Haptoglobin by Affinity Chromatography Thomas E. Eurell, William F. Hall and David P. Bane
ABSTRACT
INTRODUCTION
Swine haptoglobin is a plasma A globin-agarose affmity chromatography technique was used to purify alpha2-glycoprotein which is analoswine haptoglobin. This technique gous to human haptoglobin type 1-1 provides a highly specifilc, single-step (1). Previously reported techniques purification method without the have produced isolates of swine contamination of extraneous serum haptoglobin contaminated by albuproteins reported by previous studies. min, albumin dimer (2), several preComplex formation between the albumin components (1), and hemohaptoglobin isolate and swine hemo- pexin (3). The present study was globin confirmed that biological designed to evaluate whether an activity was maintained during the affinity chromatography technique purification process. Immunoelectro- developed for the isolation of human phoretic and Ouchterlony immunodif- haptoglobin could be used to purify fusion methods revealed that the swine swine haptoglobin. haptoglobin isolate cross-reacted with polyvalent antisera against human MATERIALS AND METHODS haptoglobin.
graphy, the swine serum was mixed with loading buffer (75% serum/25% buffer) to minimize nonspecific binding of serum proteins to the affinity matrix. Twenty mL of the serum/ buffer solution were added to a 1 cm x 10 cm bed of the globin-agarose matrix. Chromatography was performed at room temperature using gravity flow elution with a constant hydrostatic pressure of 20 cm. Elution fractions corresponding to the major chromatography peaks were pooled and dialyzed against 0.01 M Tris-HCl containing 0.1 M NaCl (pH = 7.4). CHARACTERIZATION OF SWINE HAPTOGLOBIN
Polyacrylamide gel electrophoresis A 10% polyacrylamide gel was ISOLATION OF SWINE HAPTOGLOBIN used with a nondissociating TrisSwine haptoglobin was isolated HCl-glycine buffer system (5). The from the serum of healthy donor gel was maintained at 40 C and animals following the procedure of electrophoresis performed with a Javid and Liang (4). The globin- constant electromotive force of 50 agarose affinity matrix (prepared watts for 3 h. The resulting protein from bovine hemoglobin which was bands were stained with Coomassie chemically modified to remove the blue (5). Complex formation between heme group) was obtained from a the swine haptoglobin isolate and commercial source (Sigma Chemical, swine hemoglobin was induced in St. Louis, Missouri). The affinity vitro by mixing swine haptoglobin chromatography was performed in with a swine erythrocyte lysate. The three stages using 0.01 M Tris-HCl swine erythrocyte lysate was made containing 5 M NaCl (pH = 7.0) as the using 1 mL of a 10% packed cell sample loading buffer and 1.6 M volume of swine erythrocytes in guanidine-HCl (pH = 7.0) and 3.5 M 10 mL of distilled water. The guanidine-HCl (pH = 5.0) as the haptoglobin-hemoglobin complex elution buffers. Prior to chromato- was visualized using a commercially -
RESUME L'haptoglobine porcine a ete purifiee par chromatographie d'affiniti sur gel d'agarose-globine. Cette technique a permis, son isolement, en une seule etape sans contamination par les proteines seriques. La formation d'un complexe entre l'haptoglobine et l'hemoglobine a confirme le maintien de l'activite biologique au cours du processus de purification. Deux techniques dlimmunodiffusion revelent une reaction croisee contre un anticorps polyvalent anti-haptoglobine humaine. (Traduit par Dr NormandLarivi&re).
Department of Veterinary Biosciences (Eurell) and Department of Veterinary Clinical Medicine (Hall, Bane), College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801. Source of support: Illinois Department of Agriculture. Submitted December 18, 1989.
Can J Vet Res 1990; 54: 501-503
501
2
Cl a)
0
1
0
agarose gels following the procedures reported by Nilsson (6,7). A polyvalent human haptoglobin preparation (containing types 1-1, 2-1 and 2-2) and polyvalent antiserum against human haptoglobin (types 1-1, 2-1 and 2-2) were obtained from a commercial source (Sigma Chemical, St. Louis,
Missouri). RESULTS
Fraction Number Fig. 1. Densitometry scan (280 nm) of swine serum proteins eluted from globin-agarose affinity column. Peak #1 was recovered in sample loading buffer, peak #2 was recoverd in 1.6 M guanidine-HCI (pH = 7.0), and peak #3 was recovered in 3.5 M guanidine-HCI (pH = 5.0). Fractions were collected in 5 mL volumes.
available 0-dianisidine stain (Isolab, Akron, Ohio) which detects its peroxidase activity.
Immunoprecipitation analysis Immunoelectrophoresis and Ouchterlony analysis were performed in
Three peaks were obtained following affinity chromatography of the swine serum (Fig. 1). The peak eluted by 3.5 molar guanidine-HCl (peak 3) contained a single protein band analyzed using a 10% polyacrylamide gel (Fig. 2, band Bi). 0-dianisidine was used to confirm the complex formation of the affinity purified isolate with porcine hemoglobin (Fig. 2, band G3). Several components of the swine erythrocyte lysate were detected using the Coomassie blue stain (Fig. 2, E*). The hemoglobin component of the erythrocyte lysate was specifically detected using the 0dianisidine stain (Fig. 2, lane F). Immunoprecipitation analysis was used to further identify the affinity chromatography isolate (Peak 3) as haptoglobin (Fig. 3). Ouchterlony immunodiffusion using a polyvalent rabbit antihuman haptoglobin antiserum revealed a reaction of partial identity between swine haptoglobin and a polyvalent human haptoglobin preparation (Fig. 3, panel 1). The globin-affinity isolate of swine haptoglobin migrated as an alpha2 globulin and had an immunoelectrophoretic pattern similar to the polyvalent human haptoglobin preparation (Fig. 3, panel 2).
DISCUSSION Fig. 2. Native polyacrylamide gel electrophoresis of swine serum proteins. Lanes A through E were stained using Coomassie Blue. Lane A = whole swine serum, lane B = swine haptoglobin isolated by globin-affinity chromatography, lane C = whole swine serum + swine erythrocyte lysate, lane D = swine haptoglobin isolate + swine erythrocyte lysate, lane E = swine erythrocyte lysate (*) = protein components released by lysis of swine erythrocytes. Lanes F and G were stained using 0-dianisidine. Lane F = swine hemoglobin and lane G = swine haptoglobin + swine hemoglobin.
502
Globin-agarose affinity chromatography is simple to perform and results in a swine haptoglobin isolate which maintains the ability to react with hemoglobin and lacks the contaminants of previously reported methods. The globin-agarose affinity matrix used in the present study was chemically modified to remove the heme group from the hemoglobinagarose ligand. This modification improves the ligand specificity of the
Fig. 3. Immunoprecipitation in gel techniques. Panel 1: Ouchterlony immunodiffusion-well A = polyvalent rabbit anti-human haptoglobin, well B = polyvalent human haptoglobin preparation, well C = swine haptoglobin. Panel 2: Immunoelectrophoresis-well A = polyvalent human haptoglobin preparation, well B = swine haptoglobin, trough C = polyvalent rabbit antihuman haptoglobin.
affinity matrix by utilizing a feature of the haptoglobin-hemoglobin interaction. The binding of haptoglobin to the hemoglobin molecule is solely dependent on the globin portion of the hemoglobin molecule, whereas hemopexin binds to the heme moiety of hemoglobin (8). Thus, the globinagarose matrix provides: (A) a simple method to separate haptoglobin from all contaminant plasma proteins and (B) a specific method to separate haptoglobin from hemopexin. Immunoelectrophoresis and Ouchterlony immunodiffusion techniques were used to demonstrate that swine haptoglobin cross-reacts with a polyvalent antiserum against human haptoglobin types 1-1, 2-1 and 2-2 (Fig. 3). This immunological crossreactivity is consistent with the biochemical profile of swine haptoglobin. Amino acid analysis, polypeptide mapping and swine haptoglobin/ human hemoglobin complex formation indicate a high degree of homology between swine haptoglobin and human haptoglobin type 1-1 (9). The reaction of partial identity in the Ouchterlony immunodiffusion (Fig. 3, panel 2) indicates that there are shared binding sites for the polyvalent
antiserum between the human haptoglobin preparation (Well B) and the swine haptoglobin isolate (Well C).
ACKNOWLEDGMENTS We are grateful to Mses. Janet Sinn and Lisa Graning for technical assistance.
REFERENCES 1. SHIM B, YOON C, OH S, LEE T, KANG Y. Studies on swine and canine serum haptoglobins. Biochim Biophys Acta 1971; 243: 126-136.
2. TERPSTRA FA, SMITH DB. The purification of porcine haptoglobin by affinity chromatography with concanavalin ASepharose. Can J Biochem 1976; 54: 999-1001. 3. GRUNDER AA. Distinction between heme and hemoglobin-binding proteins of swine and rabbits by electrophoreis. Vox Sang 1968; 14: 236-240. 4. JAVID J, LIANG JC. The hemoglobinhaptoglobin bond. I. Dissociation of the complex and recovery of the native haptoglobin in an affinity chromatography system. J Lab Clin Med 1973; 82: 991-1002. 5. HAMES BD. An introduction to polyacrylamide gel electrophoresis. In: Hames BD, Rickwood D, eds. Gel Electrophoresis of Proteins: A Practical Approach. Oxford, England: IRL Press, 1981: 1-86.
6. NILSSON LA. Immunoelectrophoresis. In: Nils HA, ed. Handbook of Immunoprecipitation in Gel Techniques. Oxford, England: Blackwell, 1983: 71-76. 7. NILSSON LA. Double diffusion in gel. In: Nils HA, ed. Handbook of Immunoprecipitation in Gel Techniques. Oxford, England: Blackwell, 1983: 57-68. 8. PUTNAM FW. Haptoglobin. In: Putnam FW, ed. The Plasma Proteins: Structure, Function, and Genetic Control. Vol. 2. New York: Academic Press, 1975: 2-46. 9. FRASER IH, SMITH DB. Studics on porcine haptoglobin and its complex with human hemoglobin. Can J Biochem 1971; 49: 141-147.
503
ABSTRACT
INTRODUCTION
Swine haptoglobin is a plasma A globin-agarose affmity chromatography technique was used to purify alpha2-glycoprotein which is analoswine haptoglobin. This technique gous to human haptoglobin type 1-1 provides a highly specifilc, single-step (1). Previously reported techniques purification method without the have produced isolates of swine contamination of extraneous serum haptoglobin contaminated by albuproteins reported by previous studies. min, albumin dimer (2), several preComplex formation between the albumin components (1), and hemohaptoglobin isolate and swine hemo- pexin (3). The present study was globin confirmed that biological designed to evaluate whether an activity was maintained during the affinity chromatography technique purification process. Immunoelectro- developed for the isolation of human phoretic and Ouchterlony immunodif- haptoglobin could be used to purify fusion methods revealed that the swine swine haptoglobin. haptoglobin isolate cross-reacted with polyvalent antisera against human MATERIALS AND METHODS haptoglobin.
graphy, the swine serum was mixed with loading buffer (75% serum/25% buffer) to minimize nonspecific binding of serum proteins to the affinity matrix. Twenty mL of the serum/ buffer solution were added to a 1 cm x 10 cm bed of the globin-agarose matrix. Chromatography was performed at room temperature using gravity flow elution with a constant hydrostatic pressure of 20 cm. Elution fractions corresponding to the major chromatography peaks were pooled and dialyzed against 0.01 M Tris-HCl containing 0.1 M NaCl (pH = 7.4). CHARACTERIZATION OF SWINE HAPTOGLOBIN
Polyacrylamide gel electrophoresis A 10% polyacrylamide gel was ISOLATION OF SWINE HAPTOGLOBIN used with a nondissociating TrisSwine haptoglobin was isolated HCl-glycine buffer system (5). The from the serum of healthy donor gel was maintained at 40 C and animals following the procedure of electrophoresis performed with a Javid and Liang (4). The globin- constant electromotive force of 50 agarose affinity matrix (prepared watts for 3 h. The resulting protein from bovine hemoglobin which was bands were stained with Coomassie chemically modified to remove the blue (5). Complex formation between heme group) was obtained from a the swine haptoglobin isolate and commercial source (Sigma Chemical, swine hemoglobin was induced in St. Louis, Missouri). The affinity vitro by mixing swine haptoglobin chromatography was performed in with a swine erythrocyte lysate. The three stages using 0.01 M Tris-HCl swine erythrocyte lysate was made containing 5 M NaCl (pH = 7.0) as the using 1 mL of a 10% packed cell sample loading buffer and 1.6 M volume of swine erythrocytes in guanidine-HCl (pH = 7.0) and 3.5 M 10 mL of distilled water. The guanidine-HCl (pH = 5.0) as the haptoglobin-hemoglobin complex elution buffers. Prior to chromato- was visualized using a commercially -
RESUME L'haptoglobine porcine a ete purifiee par chromatographie d'affiniti sur gel d'agarose-globine. Cette technique a permis, son isolement, en une seule etape sans contamination par les proteines seriques. La formation d'un complexe entre l'haptoglobine et l'hemoglobine a confirme le maintien de l'activite biologique au cours du processus de purification. Deux techniques dlimmunodiffusion revelent une reaction croisee contre un anticorps polyvalent anti-haptoglobine humaine. (Traduit par Dr NormandLarivi&re).
Department of Veterinary Biosciences (Eurell) and Department of Veterinary Clinical Medicine (Hall, Bane), College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801. Source of support: Illinois Department of Agriculture. Submitted December 18, 1989.
Can J Vet Res 1990; 54: 501-503
501
2
Cl a)
0
1
0
agarose gels following the procedures reported by Nilsson (6,7). A polyvalent human haptoglobin preparation (containing types 1-1, 2-1 and 2-2) and polyvalent antiserum against human haptoglobin (types 1-1, 2-1 and 2-2) were obtained from a commercial source (Sigma Chemical, St. Louis,
Missouri). RESULTS
Fraction Number Fig. 1. Densitometry scan (280 nm) of swine serum proteins eluted from globin-agarose affinity column. Peak #1 was recovered in sample loading buffer, peak #2 was recoverd in 1.6 M guanidine-HCI (pH = 7.0), and peak #3 was recovered in 3.5 M guanidine-HCI (pH = 5.0). Fractions were collected in 5 mL volumes.
available 0-dianisidine stain (Isolab, Akron, Ohio) which detects its peroxidase activity.
Immunoprecipitation analysis Immunoelectrophoresis and Ouchterlony analysis were performed in
Three peaks were obtained following affinity chromatography of the swine serum (Fig. 1). The peak eluted by 3.5 molar guanidine-HCl (peak 3) contained a single protein band analyzed using a 10% polyacrylamide gel (Fig. 2, band Bi). 0-dianisidine was used to confirm the complex formation of the affinity purified isolate with porcine hemoglobin (Fig. 2, band G3). Several components of the swine erythrocyte lysate were detected using the Coomassie blue stain (Fig. 2, E*). The hemoglobin component of the erythrocyte lysate was specifically detected using the 0dianisidine stain (Fig. 2, lane F). Immunoprecipitation analysis was used to further identify the affinity chromatography isolate (Peak 3) as haptoglobin (Fig. 3). Ouchterlony immunodiffusion using a polyvalent rabbit antihuman haptoglobin antiserum revealed a reaction of partial identity between swine haptoglobin and a polyvalent human haptoglobin preparation (Fig. 3, panel 1). The globin-affinity isolate of swine haptoglobin migrated as an alpha2 globulin and had an immunoelectrophoretic pattern similar to the polyvalent human haptoglobin preparation (Fig. 3, panel 2).
DISCUSSION Fig. 2. Native polyacrylamide gel electrophoresis of swine serum proteins. Lanes A through E were stained using Coomassie Blue. Lane A = whole swine serum, lane B = swine haptoglobin isolated by globin-affinity chromatography, lane C = whole swine serum + swine erythrocyte lysate, lane D = swine haptoglobin isolate + swine erythrocyte lysate, lane E = swine erythrocyte lysate (*) = protein components released by lysis of swine erythrocytes. Lanes F and G were stained using 0-dianisidine. Lane F = swine hemoglobin and lane G = swine haptoglobin + swine hemoglobin.
502
Globin-agarose affinity chromatography is simple to perform and results in a swine haptoglobin isolate which maintains the ability to react with hemoglobin and lacks the contaminants of previously reported methods. The globin-agarose affinity matrix used in the present study was chemically modified to remove the heme group from the hemoglobinagarose ligand. This modification improves the ligand specificity of the
Fig. 3. Immunoprecipitation in gel techniques. Panel 1: Ouchterlony immunodiffusion-well A = polyvalent rabbit anti-human haptoglobin, well B = polyvalent human haptoglobin preparation, well C = swine haptoglobin. Panel 2: Immunoelectrophoresis-well A = polyvalent human haptoglobin preparation, well B = swine haptoglobin, trough C = polyvalent rabbit antihuman haptoglobin.
affinity matrix by utilizing a feature of the haptoglobin-hemoglobin interaction. The binding of haptoglobin to the hemoglobin molecule is solely dependent on the globin portion of the hemoglobin molecule, whereas hemopexin binds to the heme moiety of hemoglobin (8). Thus, the globinagarose matrix provides: (A) a simple method to separate haptoglobin from all contaminant plasma proteins and (B) a specific method to separate haptoglobin from hemopexin. Immunoelectrophoresis and Ouchterlony immunodiffusion techniques were used to demonstrate that swine haptoglobin cross-reacts with a polyvalent antiserum against human haptoglobin types 1-1, 2-1 and 2-2 (Fig. 3). This immunological crossreactivity is consistent with the biochemical profile of swine haptoglobin. Amino acid analysis, polypeptide mapping and swine haptoglobin/ human hemoglobin complex formation indicate a high degree of homology between swine haptoglobin and human haptoglobin type 1-1 (9). The reaction of partial identity in the Ouchterlony immunodiffusion (Fig. 3, panel 2) indicates that there are shared binding sites for the polyvalent
antiserum between the human haptoglobin preparation (Well B) and the swine haptoglobin isolate (Well C).
ACKNOWLEDGMENTS We are grateful to Mses. Janet Sinn and Lisa Graning for technical assistance.
REFERENCES 1. SHIM B, YOON C, OH S, LEE T, KANG Y. Studies on swine and canine serum haptoglobins. Biochim Biophys Acta 1971; 243: 126-136.
2. TERPSTRA FA, SMITH DB. The purification of porcine haptoglobin by affinity chromatography with concanavalin ASepharose. Can J Biochem 1976; 54: 999-1001. 3. GRUNDER AA. Distinction between heme and hemoglobin-binding proteins of swine and rabbits by electrophoreis. Vox Sang 1968; 14: 236-240. 4. JAVID J, LIANG JC. The hemoglobinhaptoglobin bond. I. Dissociation of the complex and recovery of the native haptoglobin in an affinity chromatography system. J Lab Clin Med 1973; 82: 991-1002. 5. HAMES BD. An introduction to polyacrylamide gel electrophoresis. In: Hames BD, Rickwood D, eds. Gel Electrophoresis of Proteins: A Practical Approach. Oxford, England: IRL Press, 1981: 1-86.
6. NILSSON LA. Immunoelectrophoresis. In: Nils HA, ed. Handbook of Immunoprecipitation in Gel Techniques. Oxford, England: Blackwell, 1983: 71-76. 7. NILSSON LA. Double diffusion in gel. In: Nils HA, ed. Handbook of Immunoprecipitation in Gel Techniques. Oxford, England: Blackwell, 1983: 57-68. 8. PUTNAM FW. Haptoglobin. In: Putnam FW, ed. The Plasma Proteins: Structure, Function, and Genetic Control. Vol. 2. New York: Academic Press, 1975: 2-46. 9. FRASER IH, SMITH DB. Studics on porcine haptoglobin and its complex with human hemoglobin. Can J Biochem 1971; 49: 141-147.
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