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A new method for affinity chromatography of heme-binding proteins: Synthesis and characterization of hematin- and hematoporphyrin-agarose
ANALYTICAL
BIOCHEMISTRY
109. 250-254
(1980)
A New Method for Affinity Chromatography of Heme-Binding Synthesis and Characterization of Hematinand Hematoporphyrin-Agarose W.
KENNETH Department
of Biochemistq,
The University Jackson. Mississippi Received
Proteins:
OLSEN
of
Mississippi 39216
Medical
Center.
June 3, 1980
Two new affinity columns, using hematin and hematoporphyrin as ligands, have been prepared. Both were made by first attaching 1,6-diaminohexane to Sepharose 4B by the cyanogen bromide procedure and then coupling the porphyrins to the free amino groups of this arm with carbodiimide. This second reaction was done in dimethylformamide to increase the solubility of the porphyrins at pH 4.7. This resin was then washed extensively with dimethylformamide to remove all of the unreacted porphyrin. The new affinity columns are able to bind apoglobin, albumin. and hemopexin, which demonstrates their ability to purify heme-binding proteins. The proteins could be removed by washing the column with a deforming buffer, such as 8 M urea or 0. I M sodium citrate buffer at pH 4. Neither lysozyme nor hemoglobin bound to these resins demonstrating that the absorbants are specific for apo heme-binding proteins.
In recent years, the technique of affinity chromatography has revolutionized the purification of proteins. Several general affinity columns using immobilized coenzymes, such as nucleotides (1) or NAD (2), have been developed. These resins are able to bind many different proteins and, therefore, have been used in a great number of purification schemes. The heme prosthetic group, however, has largely been ignored as a general ligand. A heme affinity column might prove successful for purifying a variety of proteins, including hemopexin (3). ligandin (4), trytophan pyrrolase (5). cytochromes. enzymes involved in heme metabolism, and heme-regulated factors in protein synthesis (6). Several attempts have been made to purify hemoproteins by affinity chromatography. This is an obvious approach, but it has been plagued with technical problems. As early as 1966, Heide (7) mentioned the possibility of isolating hemopexin with heme bound to Sephadex G-100, but no 0003-26971801 Copyright All rights
I80250-05$02.00/O
C 1980 by Academic I’rcra. Inc. of reproduction I” any form reerved.
250
experimental details were given. More recently, Conway and Muller-Eberhard (8) coupled hemin to aminoethyl-Sepharose, but the resulting resin had a relatively low capacity for both hemopexin and albumin. A better resin was made by using 2.4-disulfonic acid deuteroprophyrin instead of hemin (8). Rabbit hemopexin and this water-soluble porphyrin form an equimolar complex with an apparent dissociation constant of 1.8 x IO-” M (9). Although this resin requires a considerably more complex synthesis, the affinity column did bind hemopexin effectively. Apohemopexin could be eluted with 2.5 M guanidine -HCl (8). Unfortunately, the experimental details of this work have not yet been published. Recently, Suttnar ct (11. (10) have coupled hemin to Bio-Gel P-200 by a pnitrobenzoylazide spacer arm. This synthesis required seven reactions, and the resulting affinity column had low capacity and released some hemin when the hemopexin was eluted with glycine-HCl buffer (pH 2.4).
AFFINITY
CHROMATOGRAPHY
OF
HEME-BINDING
PROTEINS
‘51
(34.8 g) at pH 10 and 4°C overnight (21). The aminohexyl agarose was washed with water (21) to remove excess 1.6-diaminohexane. In the second phase of the synthesis. the porphyrin is linked to the free amino group of the aminohexyl “arm” by a carbodiimide reaction. The aminohexyl agarose (100 ml) was washed several times with DMF. The hematoporphyrin or hematin EXPERIMENTAL PROCEDURES (0.S g) was dissolved in DMF (150 ml) by Materials. Hematin, hematoporphyrin, stirring overnight at room temperature. In Cohn fraction V of bovine serum. and this and all subsequent steps, the porphyrins undenatured globin were purchased from were protected from light as much as posICN Pharmaceuticals, Inc. Human hemosible. The hematin solution had to be filtered globin, chicken lysozyme, and l-ethyl-3-(3on a medium-sintered glass funnel to redimethylaminopropyl)carbodiimide were move insoluble particles. Each of the porobtained from Sigma Chemical Company. phyrin solutions were mixed with 100 ml Dimethylformamide (DMF)’ and 1,6of the aminohexyl agarose. A solution of Idiaminohexane were purchased from ethyl-3(3-dimethyl-aminopropyl)carbodiAldrich Chemical Company. Sepharose 4B imide (7.5 g) in 50% DMF (20 ml) was was obtained from Pharmacia Fine Chemiadded dropwise to the mixture, while the cals, dimethyl sulfoxide from J. T. Baker apparent pH was adjusted to 4.7 with I N Chemical Company, and cyanogen bromide HCI. The pH was readjusted to 4.7 every from Pierce Chemical Company. All re30 min. After 5 h, a second addition of agents were used without further purificacarbodiimide (7.5 g) was made. Several tion. more pH adjustments were made, and the An impure preparation of bovine hemoreaction was allowed to proceed overnight pexin was made from fresh blood obtained at room temperature. In the morning the pH from Jackson Packing Company. Following was adjusted to 4.7 several more times. the procedure introduced by Schultze et (11. After 18 h at pH 4.7 the reaction was (12), serum was prepared and made 0.2 M stopped by adding 1 N sodium hydroxide in perchloric acid. The precipitate was to bring the pH to 7.5. rapidly removed by centrifugation, and the The hematoporphyrin-agarose was washed solution was neutralized with 1 N NaOH. with 4 liters of distilled water to remove These operations were done at 4°C. the DMF and unreacted porphyrin. The Synthetic procedures. The synthesis of resulting affinity resin was pink, and further porphyrin-agarose resin is a two-step washing did not remove any additional procedure. In the first phase the agarose color. is activated by cyanogen bromide (13) and The hematin-agarose resin required more then coupled to 1,6-diaminohexane ( 14). extensive washing to remove the unreacted In a typical synthesis. well-washed Sephahematin. After adjusting the pH to 7.5, the rose 4B (150 ml) was activated by cyanogen resin was washed twice on a sintered glass bromide (37.5 g) at pH 10.5 and 25°C. After funnel with DMF (200 ml). The hematinthoroughly washing with ice water (1500 ml) agarose was suspended in DMF and shaken the activated resin was reacted with an equal in a waterbath at 30°C. The solvent was volume of 1,6-diaminohexane solution changed four times daily for the next 4 days ’ Abbreviation used: DMF, dimethylformamide. until two consecutive washes had no color. In the present study, a simple method for the synthesis of a hematin-agarose resin is presented. The new affinity material has a high capacity and is stable to the urea, guanidine-HCl and acidic solutions that are used to release proteins from the resin. A preliminary report of these studies was made earlier ( 1 I).
252
KENNETH
The resin was then washed extensively with water to remove the DMF. Both affinity resins were stored in dark bottles at 4°C under toluene when not in use. Resin stored in this manner appears to be stable for at least 6 months. Atrtd~ticrrt procedrrws. The amount of porphyrin attached to the resin was determined spectrophotometrically by modifying the method of Golovina et (I/. (15). A 50% (w/v) solution of polyethylene glycol 20,000 was used to suspend the affinity resins. The spectra were obtained with a Cary 2 19 double-beam spectrophotometer using an identical amount of suspended agarose as the reference. Standard curves were constructed by adding known amounts of hematin or hematoporphyrin to the same volume of suspended agarose. The concentration of the hematin in the stock solution was determined using an ,!$,!J of 170 in 80% dimethylsulfoxide (16). The concentration of hematoporphyrin was calculated from the amount used to make the stock solution. RESULTS The spectrophotometric analyses of the new affinity resins showed that 0.13 ,umol of hematin and 0.16 pmol of hematoporphyrin were attached per milliliter of agarose by the synthetic procedures described here. The usefulness of these new affinity resins was tested by chromatographing several apohemoproteins or heme-binding proteins. Undenatured globin (Fig. 1A) and bovine serum albumin (Fig. IB) can bind to hematin-agarose. Neither of these proteins was eluted by sodium phosphate buffer, but both proteins could be completely removed by any one of several deforming buffers. Similar results were obtained for the chromatography of albumin on hematoporphyrin-agarose (data not shown). The hematin-agarose resin can be used to further purify hemopexin from a crude preparation. as shown in Fig. 2. The material
W.
OLSEN
0.2 -
10
20
30
40
Elutlon
50
Volume
a
70
FIG. 1. Chromatography hematin-agarose. (A)
of globin and Undenatured globin
applied
of
to a column
80
(ml)
hematin-agarose
albumin on ( 10 mg) was (IO
ml)
in
0.05 M sodium phosphate buffer. ph 7.6. The column was washed with this buffer (50 ml), and then the globin was eluted with 6 M urea at the point indicated. (B) Bovine to a column
Cohn fraction of hematin-agarobe
phosphate
buffer.
pH
V 7.6.
(IO my) was t IO ml) in 0.05
After
the
applied sodium
contaminating
proteins were washed out of the column phate buffer, the albumin was eluted with
with phos6 M urea.
that came straight through the column contained no hemopexin, as judged by the spectral assay procedure of Drabkin (17). The protein eluted by the acidic buffer consisted almost exclusively of albumin and hemopexin (data not shown). as measured by cellulose acetate electrophoresis at pH 8.6 by the method of Kohn (IS). A third protein, which is present in very small amounts in the acidic eluate. is probably the histidine-rich glycoprotein (19). The spectrum of the eluate showed that no hematin was bound to these proteins. Elution by 8 M urea did not elute any additional protein from this column. To demonstrate that the columns are specific for heme-binding proteins, lysozyme and hemoglobin were applied to hematin-agarose. Both of these proteins
AFFINITY
CHROMATOGRAPHY
r
OF HEME-BINDING
1
PROTEINS
use, the binding capacity of the remained essentially constant.
253 resin
DISCUSSION
.6 -
20
60
40
Elution
Volume
80
(ml)
FIG. 2. Chromatography of bovine hemopexin on hematin-agarose. Partially purified hemopexin (1 ml) was applied to a hematin-agarose column (10 ml) in 0.05 M sodium phosphate buffer. pH 7.6. Proteins which were not absorbed by the affinity resin were washed out of the column with the same buffer. The heme-binding proteins were then eluted with 0. I M sodium citrate buffer. pH 4.0. The progress of this buffer through the column could be followed easily due to a reversible color change of the affinity resin from dark green to red-brown.
failed to interact with the resin. Elution of these columns with 8 M urea gave no evidence that these proteins had been absorbed, although a very small amount of protein which lacked heme was eluted in the case of the hemoglobin. The capacity of the new affinity resins has been measured by frontal analysis by saturating them with bovine serum albumin. The results depend on the history of the resin being used. Porphyrin-agarose that has never been used absorbs more albumin than resin which has already been saturated with protein in previous experiments. However, the extra absorbed protein cannot be removed from the affinity column, even by 6 M guanidine hydrochloride. The unused hematin-agarose absorbed 22 mgiml. while the used resin absorbed 16 mg/ml. From hematoporphyrin-agarose, the unused resin bound 19 mgiml, but the used resin, only 13 mg/ml. After the first
The synthesis of the porphyrin affinity resins is a two-step procedure in which aminohexylagarose is first synthesized by the cyanogen bromide technique (13) and then hematin is coupled to the “arm” by the carbodiimide reaction. The porphyrin in the affinity resin can be attached by either or both of its propionic acid groups. The novel and essential aspect of this reaction is the use of pure DMF. Aqueous DMF in concentrations up to 50%~ (v/v) has been used in similar reactions involving hydrophobic ligands, such as estradiol (14) or phenylalanine (20). Although pure dioxane has been used as a solvent for the carbodiimide reaction (2 l), higher concentrations of DMF have not been used, apparently for fear of damaging the agarose. The results presented here demonstrate that this is not a problem. For the synthesis of hematin-agarose the use of the pure solvent is a necessity. If it is replaced by 50% tv/v)DMF, the resulting resin is not an effective affinity column due to the low level of substitution and to the presence of considerable amounts of noncovalently attached heme in the resin. Both of these problems are caused by the low solubility of hematin in even partially aqueous solvents under acidic conditions. These results may explain the difficulties observed with other heme resin syntheses. Thus. the use of DMF as the solvent in the coupling reaction is essential to the success of this synthesis. The synthesis of hematin-agarose presented here has several advantages over the possible methods of making similar affinity resins. The only other synthesis that has been reported in detail is the hemin-Bio Gel P-200 resin made by Suttnar et nl. ( IO). The method reported here requires only three reactions. rather than the seven re-
254
KENNETH
actions which were required to attach hemin to a polyacrylamide matrix. In addition. the extensive washing with DMF eliminates the problem of noncovalently attached porphyrin. Hemopexin released from hematin-agarose was entirely free of heme, while 20% of the hemopexin from the hemin-Bio Gel P-200 column had bound heme. Finally, the protein binding capacity of the hematin-agarose appears to be at least 10 times that of the hemin-polyacrylamide. The most serious problem with hematin-agarose is the irreversible binding of protein when the column is first used. This situation is easily avoided by washing the resin with serum albumin before using it. The mechanism of the irreversible binding is not known. The hematin-agarose affinity resin should be useful in the preparation of many hemoproteins. The protein must be in the apo form to bind to the resin and be recoverable from the deforming buffer used to elute it. Elution by dilute hematin solutions was not effective (data not shown) because the hematin was noncovalently bound to the top portion of the affinity column and could only be removed by DMF. Hematinagarose has already been used to purify hemopexin (22). which exists without heme in the serum. Many other applications will be possible in the future. ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association-Mississippi Affiliate and the Biomedical Research Support Program. NIH (Grant 5 507 RR05386). The author would like to thank Mr. David Johnson and Mr. Danny Carey for their technical assistance, Dr. Philip Tucker for his critical comments, and Mrs. Romie Brown for her secretarial assistance.
W. OLSEN
REFERENCES Barker. R.. Olsen. K. W., Shaper, .I. H.. and Hill. R. L. t 1972) J. Biol. Chem. 247, 7135-7147. 2. Lowe. C. R.. Harvey. M. J.. Craven. D. B.. and Dean. P. D. G. (1973) Eioc~hem. J. 133, 499506. 3. Muller-Eberhard.
4. 5. 6. 7.
U., and Morgan, W. T. (1975) Anr~. N. Y. Acud. Sci. 244, 624-650. Tipping, E., Ketterer. B., and Koskelo. P. t 1978) Biochrm. .I. 169, 5099516. Marver, H.. Tschudy, D. P.. Perbroth. M. G., and Collins. A. (1966) Scic,rrc,ta 153. 501-503. Ochoa. S.. and de Haro. C. (1979) Annu. Rev. Biochrm. 48. 549-580. Heide, K. ( 1966) Protides Bid. Fluids 14, 593600.
8. Conway, T. P., and Muller-Eberhard, U. t 1973) Fed. Pro<.. 32, 469. 9. Conway. T. P.. and Muller-Eberhard. U. (1976) Arch. Birxhrm. Eiophys. 172, 558-564. IO. Suttnar. J.. Hrkal. 2.. and Vodrazka, Z. t 1977) J. Chromcuogr. 131, 453-457. Il. Olsen, K. W. t 1977) Fed. Pro<.. 36, 756. 12. Schultze. H. E., Heide. K.. and Haupt, H. ( 1962) C/in. Chim. A~,Iu 7, 854-868. 13. Axen, P.. Porath, J.. and Emback. S. (1967)N~rture (London~ 214, l302- 1304. 14. Cuatrecasar, P. t 1970) .I. Biol. Chrm. 245, 30.593065.
15. Golovina, T. 0.. Cherednikova. T. V., Mevkh. A. T., and Nagradova. N. K. (1977) Anal. Biochrm. 83, 77X-781. 16. Morgan, W. T.. Liem, H. H.. Sutor, R. P., and Muller-Eberhard, U. (1976) Biochim. Bit>ph.v.c. Acre! 444, 435-445. D. L. (1971) Proc. Nor. Acad. Sc,i. USA 17. Drabkin, 68,609-613.
18. Kohn, J. t 1958) C‘lin. Chim. Actu 19. Morgan. W. T. t 1978) Bioc,him. 533,
3. 450-454. Biophys.
Acrcc
319-333.
20. Schiller. P. W., and Schechter. A. N. (1974) Methods in Enzymology (Jakoby, W. B., ed.), Vol. 34. pp. 513-516, Academic Press, New York. 21. Wilcheck, M. (1973) FEES Lrrr. 33, 70-72. K. W., Carey, D., Thomson, N., and 32. Olsen, Hutchinson, M. E. (1978) Fed. Proc. 37, 1514.
BIOCHEMISTRY
109. 250-254
(1980)
A New Method for Affinity Chromatography of Heme-Binding Synthesis and Characterization of Hematinand Hematoporphyrin-Agarose W.
KENNETH Department
of Biochemistq,
The University Jackson. Mississippi Received
Proteins:
OLSEN
of
Mississippi 39216
Medical
Center.
June 3, 1980
Two new affinity columns, using hematin and hematoporphyrin as ligands, have been prepared. Both were made by first attaching 1,6-diaminohexane to Sepharose 4B by the cyanogen bromide procedure and then coupling the porphyrins to the free amino groups of this arm with carbodiimide. This second reaction was done in dimethylformamide to increase the solubility of the porphyrins at pH 4.7. This resin was then washed extensively with dimethylformamide to remove all of the unreacted porphyrin. The new affinity columns are able to bind apoglobin, albumin. and hemopexin, which demonstrates their ability to purify heme-binding proteins. The proteins could be removed by washing the column with a deforming buffer, such as 8 M urea or 0. I M sodium citrate buffer at pH 4. Neither lysozyme nor hemoglobin bound to these resins demonstrating that the absorbants are specific for apo heme-binding proteins.
In recent years, the technique of affinity chromatography has revolutionized the purification of proteins. Several general affinity columns using immobilized coenzymes, such as nucleotides (1) or NAD (2), have been developed. These resins are able to bind many different proteins and, therefore, have been used in a great number of purification schemes. The heme prosthetic group, however, has largely been ignored as a general ligand. A heme affinity column might prove successful for purifying a variety of proteins, including hemopexin (3). ligandin (4), trytophan pyrrolase (5). cytochromes. enzymes involved in heme metabolism, and heme-regulated factors in protein synthesis (6). Several attempts have been made to purify hemoproteins by affinity chromatography. This is an obvious approach, but it has been plagued with technical problems. As early as 1966, Heide (7) mentioned the possibility of isolating hemopexin with heme bound to Sephadex G-100, but no 0003-26971801 Copyright All rights
I80250-05$02.00/O
C 1980 by Academic I’rcra. Inc. of reproduction I” any form reerved.
250
experimental details were given. More recently, Conway and Muller-Eberhard (8) coupled hemin to aminoethyl-Sepharose, but the resulting resin had a relatively low capacity for both hemopexin and albumin. A better resin was made by using 2.4-disulfonic acid deuteroprophyrin instead of hemin (8). Rabbit hemopexin and this water-soluble porphyrin form an equimolar complex with an apparent dissociation constant of 1.8 x IO-” M (9). Although this resin requires a considerably more complex synthesis, the affinity column did bind hemopexin effectively. Apohemopexin could be eluted with 2.5 M guanidine -HCl (8). Unfortunately, the experimental details of this work have not yet been published. Recently, Suttnar ct (11. (10) have coupled hemin to Bio-Gel P-200 by a pnitrobenzoylazide spacer arm. This synthesis required seven reactions, and the resulting affinity column had low capacity and released some hemin when the hemopexin was eluted with glycine-HCl buffer (pH 2.4).
AFFINITY
CHROMATOGRAPHY
OF
HEME-BINDING
PROTEINS
‘51
(34.8 g) at pH 10 and 4°C overnight (21). The aminohexyl agarose was washed with water (21) to remove excess 1.6-diaminohexane. In the second phase of the synthesis. the porphyrin is linked to the free amino group of the aminohexyl “arm” by a carbodiimide reaction. The aminohexyl agarose (100 ml) was washed several times with DMF. The hematoporphyrin or hematin EXPERIMENTAL PROCEDURES (0.S g) was dissolved in DMF (150 ml) by Materials. Hematin, hematoporphyrin, stirring overnight at room temperature. In Cohn fraction V of bovine serum. and this and all subsequent steps, the porphyrins undenatured globin were purchased from were protected from light as much as posICN Pharmaceuticals, Inc. Human hemosible. The hematin solution had to be filtered globin, chicken lysozyme, and l-ethyl-3-(3on a medium-sintered glass funnel to redimethylaminopropyl)carbodiimide were move insoluble particles. Each of the porobtained from Sigma Chemical Company. phyrin solutions were mixed with 100 ml Dimethylformamide (DMF)’ and 1,6of the aminohexyl agarose. A solution of Idiaminohexane were purchased from ethyl-3(3-dimethyl-aminopropyl)carbodiAldrich Chemical Company. Sepharose 4B imide (7.5 g) in 50% DMF (20 ml) was was obtained from Pharmacia Fine Chemiadded dropwise to the mixture, while the cals, dimethyl sulfoxide from J. T. Baker apparent pH was adjusted to 4.7 with I N Chemical Company, and cyanogen bromide HCI. The pH was readjusted to 4.7 every from Pierce Chemical Company. All re30 min. After 5 h, a second addition of agents were used without further purificacarbodiimide (7.5 g) was made. Several tion. more pH adjustments were made, and the An impure preparation of bovine hemoreaction was allowed to proceed overnight pexin was made from fresh blood obtained at room temperature. In the morning the pH from Jackson Packing Company. Following was adjusted to 4.7 several more times. the procedure introduced by Schultze et (11. After 18 h at pH 4.7 the reaction was (12), serum was prepared and made 0.2 M stopped by adding 1 N sodium hydroxide in perchloric acid. The precipitate was to bring the pH to 7.5. rapidly removed by centrifugation, and the The hematoporphyrin-agarose was washed solution was neutralized with 1 N NaOH. with 4 liters of distilled water to remove These operations were done at 4°C. the DMF and unreacted porphyrin. The Synthetic procedures. The synthesis of resulting affinity resin was pink, and further porphyrin-agarose resin is a two-step washing did not remove any additional procedure. In the first phase the agarose color. is activated by cyanogen bromide (13) and The hematin-agarose resin required more then coupled to 1,6-diaminohexane ( 14). extensive washing to remove the unreacted In a typical synthesis. well-washed Sephahematin. After adjusting the pH to 7.5, the rose 4B (150 ml) was activated by cyanogen resin was washed twice on a sintered glass bromide (37.5 g) at pH 10.5 and 25°C. After funnel with DMF (200 ml). The hematinthoroughly washing with ice water (1500 ml) agarose was suspended in DMF and shaken the activated resin was reacted with an equal in a waterbath at 30°C. The solvent was volume of 1,6-diaminohexane solution changed four times daily for the next 4 days ’ Abbreviation used: DMF, dimethylformamide. until two consecutive washes had no color. In the present study, a simple method for the synthesis of a hematin-agarose resin is presented. The new affinity material has a high capacity and is stable to the urea, guanidine-HCl and acidic solutions that are used to release proteins from the resin. A preliminary report of these studies was made earlier ( 1 I).
252
KENNETH
The resin was then washed extensively with water to remove the DMF. Both affinity resins were stored in dark bottles at 4°C under toluene when not in use. Resin stored in this manner appears to be stable for at least 6 months. Atrtd~ticrrt procedrrws. The amount of porphyrin attached to the resin was determined spectrophotometrically by modifying the method of Golovina et (I/. (15). A 50% (w/v) solution of polyethylene glycol 20,000 was used to suspend the affinity resins. The spectra were obtained with a Cary 2 19 double-beam spectrophotometer using an identical amount of suspended agarose as the reference. Standard curves were constructed by adding known amounts of hematin or hematoporphyrin to the same volume of suspended agarose. The concentration of the hematin in the stock solution was determined using an ,!$,!J of 170 in 80% dimethylsulfoxide (16). The concentration of hematoporphyrin was calculated from the amount used to make the stock solution. RESULTS The spectrophotometric analyses of the new affinity resins showed that 0.13 ,umol of hematin and 0.16 pmol of hematoporphyrin were attached per milliliter of agarose by the synthetic procedures described here. The usefulness of these new affinity resins was tested by chromatographing several apohemoproteins or heme-binding proteins. Undenatured globin (Fig. 1A) and bovine serum albumin (Fig. IB) can bind to hematin-agarose. Neither of these proteins was eluted by sodium phosphate buffer, but both proteins could be completely removed by any one of several deforming buffers. Similar results were obtained for the chromatography of albumin on hematoporphyrin-agarose (data not shown). The hematin-agarose resin can be used to further purify hemopexin from a crude preparation. as shown in Fig. 2. The material
W.
OLSEN
0.2 -
10
20
30
40
Elutlon
50
Volume
a
70
FIG. 1. Chromatography hematin-agarose. (A)
of globin and Undenatured globin
applied
of
to a column
80
(ml)
hematin-agarose
albumin on ( 10 mg) was (IO
ml)
in
0.05 M sodium phosphate buffer. ph 7.6. The column was washed with this buffer (50 ml), and then the globin was eluted with 6 M urea at the point indicated. (B) Bovine to a column
Cohn fraction of hematin-agarobe
phosphate
buffer.
pH
V 7.6.
(IO my) was t IO ml) in 0.05
After
the
applied sodium
contaminating
proteins were washed out of the column phate buffer, the albumin was eluted with
with phos6 M urea.
that came straight through the column contained no hemopexin, as judged by the spectral assay procedure of Drabkin (17). The protein eluted by the acidic buffer consisted almost exclusively of albumin and hemopexin (data not shown). as measured by cellulose acetate electrophoresis at pH 8.6 by the method of Kohn (IS). A third protein, which is present in very small amounts in the acidic eluate. is probably the histidine-rich glycoprotein (19). The spectrum of the eluate showed that no hematin was bound to these proteins. Elution by 8 M urea did not elute any additional protein from this column. To demonstrate that the columns are specific for heme-binding proteins, lysozyme and hemoglobin were applied to hematin-agarose. Both of these proteins
AFFINITY
CHROMATOGRAPHY
r
OF HEME-BINDING
1
PROTEINS
use, the binding capacity of the remained essentially constant.
253 resin
DISCUSSION
.6 -
20
60
40
Elution
Volume
80
(ml)
FIG. 2. Chromatography of bovine hemopexin on hematin-agarose. Partially purified hemopexin (1 ml) was applied to a hematin-agarose column (10 ml) in 0.05 M sodium phosphate buffer. pH 7.6. Proteins which were not absorbed by the affinity resin were washed out of the column with the same buffer. The heme-binding proteins were then eluted with 0. I M sodium citrate buffer. pH 4.0. The progress of this buffer through the column could be followed easily due to a reversible color change of the affinity resin from dark green to red-brown.
failed to interact with the resin. Elution of these columns with 8 M urea gave no evidence that these proteins had been absorbed, although a very small amount of protein which lacked heme was eluted in the case of the hemoglobin. The capacity of the new affinity resins has been measured by frontal analysis by saturating them with bovine serum albumin. The results depend on the history of the resin being used. Porphyrin-agarose that has never been used absorbs more albumin than resin which has already been saturated with protein in previous experiments. However, the extra absorbed protein cannot be removed from the affinity column, even by 6 M guanidine hydrochloride. The unused hematin-agarose absorbed 22 mgiml. while the used resin absorbed 16 mg/ml. From hematoporphyrin-agarose, the unused resin bound 19 mgiml, but the used resin, only 13 mg/ml. After the first
The synthesis of the porphyrin affinity resins is a two-step procedure in which aminohexylagarose is first synthesized by the cyanogen bromide technique (13) and then hematin is coupled to the “arm” by the carbodiimide reaction. The porphyrin in the affinity resin can be attached by either or both of its propionic acid groups. The novel and essential aspect of this reaction is the use of pure DMF. Aqueous DMF in concentrations up to 50%~ (v/v) has been used in similar reactions involving hydrophobic ligands, such as estradiol (14) or phenylalanine (20). Although pure dioxane has been used as a solvent for the carbodiimide reaction (2 l), higher concentrations of DMF have not been used, apparently for fear of damaging the agarose. The results presented here demonstrate that this is not a problem. For the synthesis of hematin-agarose the use of the pure solvent is a necessity. If it is replaced by 50% tv/v)DMF, the resulting resin is not an effective affinity column due to the low level of substitution and to the presence of considerable amounts of noncovalently attached heme in the resin. Both of these problems are caused by the low solubility of hematin in even partially aqueous solvents under acidic conditions. These results may explain the difficulties observed with other heme resin syntheses. Thus. the use of DMF as the solvent in the coupling reaction is essential to the success of this synthesis. The synthesis of hematin-agarose presented here has several advantages over the possible methods of making similar affinity resins. The only other synthesis that has been reported in detail is the hemin-Bio Gel P-200 resin made by Suttnar et nl. ( IO). The method reported here requires only three reactions. rather than the seven re-
254
KENNETH
actions which were required to attach hemin to a polyacrylamide matrix. In addition. the extensive washing with DMF eliminates the problem of noncovalently attached porphyrin. Hemopexin released from hematin-agarose was entirely free of heme, while 20% of the hemopexin from the hemin-Bio Gel P-200 column had bound heme. Finally, the protein binding capacity of the hematin-agarose appears to be at least 10 times that of the hemin-polyacrylamide. The most serious problem with hematin-agarose is the irreversible binding of protein when the column is first used. This situation is easily avoided by washing the resin with serum albumin before using it. The mechanism of the irreversible binding is not known. The hematin-agarose affinity resin should be useful in the preparation of many hemoproteins. The protein must be in the apo form to bind to the resin and be recoverable from the deforming buffer used to elute it. Elution by dilute hematin solutions was not effective (data not shown) because the hematin was noncovalently bound to the top portion of the affinity column and could only be removed by DMF. Hematinagarose has already been used to purify hemopexin (22). which exists without heme in the serum. Many other applications will be possible in the future. ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association-Mississippi Affiliate and the Biomedical Research Support Program. NIH (Grant 5 507 RR05386). The author would like to thank Mr. David Johnson and Mr. Danny Carey for their technical assistance, Dr. Philip Tucker for his critical comments, and Mrs. Romie Brown for her secretarial assistance.
W. OLSEN
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