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[36] Affinity chromatography of heme-binding proteins: synthesis and characterization of hematin- and hematoporphyrin-agarose
324
H E M E PORPHYRINS A N D D E R I V A T I V E S
[36]
[36] A f f i n i t y C h r o m a t o g r a p h y o f H e m e - B i n d i n g P r o t e i n s : Synthesis and Characterization of Hematin- and Hematoporphyrin-Agarose
By
KENNETH
W.
OLSEN
Principle In recent years, the technique of affinity chromatography has revolutionized the purification of proteins. Several general affinity columns using immobilized coenzymes, such as nucleotides j 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 may prove to be successful for purifying a variety of proteins, including hemopexin, 3 ligandin, 4 and tryptophan 1,2dioxygenase (pyrrolase), 5 cytochromes, enzymes involved in heme metabolism, and heme-regulated factors in protein synthesis. 6 This chapter discusses a method for making a hematin-agarose resin that has been used to successfully purify hemopexin. 7,8 The method can be used to synthesize any porphyrin-agarose column packing. The reactions are summarized in Fig. 1. First, aminohexyl-agarose is made using the cyanogen bromide method, 9 and then the porphyrin is attached with a carbodiimide reaction. For the hematin-agarose, it is critical that the second reaction be done in dimethylformamide so that the hematin is soluble. Synthetic Procedures The synthesis of porphyrin-agarose resin is a two-step procedure. In the first phase the agarose is activated by cyanogen bromide, 9 and then 1 R. Barker, K. W. Olsen, J. H. Shaper, and R. L. Hill, J. Biol. Chem. 247, 7135 (1972). 2 C. R. Lowe, M. J. Harvey, D. B. Craven, and P. D. G. Dean, Biochem. J. 133, 499 (1973). 3 U. Miiller-Eberhard and W. T. Morgan, Ann. N . Y . Acad. Sci. 244, 624 (1975). 4 E. Tipping, B. Ketterer, and P. Koskelo, Biochem. J. 169, 509 (1978). 5 H. Marver, D. P. Tschudy, M. G. Perbroth, and A. Collins, Science 153, 501 (1966). 6 S. Ochoa and C. de Haro, Annu. Rev. Biochem. 48, 549 (1979). 7 K. W. Olsen, Anal. Biochem. 109, 250 (1980). 8 R. Majuri, Biochim. Biophys. Acta 719, 53 (1982). 9 p. Axen, J. Por~ith, and S. Ernback, Nature (London) 214, 1302 (1967).
METHODS IN ENZYMOLOGY, VOL. 123
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
325
A
~
OH OH
+
H2N__(CH2 )6__NH 2
(i) C N B r a c t i v a t i o n (2) a m i n o c o u p l i n g O --C--NH--(CH2) OH ,
--NH 6 2
Heme activated by Carbodiimide in DMF
~
O II
O ii
O--C--NH--(CH2)6---NH--C--Heme OH
B
CH
CH 3
CH:CH
C
_C
2
CH
I __O__C__NH__(CH2 )6__NH__~__CH2CH2" C ~ C
CH=CH2
~H~CH3 ~H2 FIG. 1. (A) Synthesis of hematin-agarose. (B) Structure of hematin-agarose.
coupled to 1,6-diaminohexane. l° In a typical synthesis, well-washed Sepharose 4B (150 ml) was activated by cyanogen bromide (37.5 g) at pH 10.5 and 25 °. After thoroughly washing with ice water (1500 ml) the activated resin was reacted with an equal volume of 1,6-diaminohexane solution (34.8 g) at pH 10 and 4 ° overnight. ~j The aminohexyl-agarose was washed with water (2 liters) 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 " a r m " by a carbodiimide reaction. The 10 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970). 11 M. Wilcheck, F E B S Lett. 33, 70 (1973).
326
HEME PORPHYRINS AND DERIVATIVES
[36]
aminohexyl-agarose (100 ml) was washed four times with dimethylformamide (DMF). The hematoporphyrin or hematin (0.5 g) was dissolved in DMF (150 ml) by stirring overnight at room temperature. In this and all subsequent steps, the porphyrins were protected from light as much as possible. The hematin solution had to be filtered on a medium-sintered glass funnel to remove insoluble particles. Each of the porphyrin solutions were mixed with 100 ml of the aminohexyl-agarose. A solution of 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (7.5 g) in 50% DMF (20 ml) was added dropwise to the mixture, while the pH was adjusted to 4.7 with 1 N HCI. The pH was readjusted to 4.7 every 30 min. After 5 hr, a second addition of carbodiimide (7.5 g) was made. Several more pH adjustments were made at 30 min intervals, and the reaction was allowed to proceed overnight at room temperature. In the morning the pH was adjusted to 4.7 several more times. After 18 hr at pH 4.7, the reaction was stopped by adding 1 N sodium hydroxide to bring the pH to 7.5. The hematoporphyrin-agarose was washed with 4 liters of distilled water to remove the DMF and unreacted porphyrin. The resulting affinity resin was pink, and further washing did not remove any additional color. The hematin-agarose resin required more extensive washing to remove the unreacted hematin. After adjusting the pH to 7.5, the resin was washed twice on a sintered glass funnel with DMF (200 ml). The hematinagarose was suspended in DMF and shaken in a waterbath at 30°. The solvent was changed four times daily for the next 4 days until two consecutive washes had no color. The resin was then washed extensively with water to remove the DMF. Unreacted primary amino groups of the gel can be blocked by an additional carbodiimide reaction in acetate buffer. 8 Both affinity resins were stored in dark bottles at 4° under toluene when not in use. Resin stored in this manner appears to be stable for at least 6 months. Analytical Procedures The amount of porphyrin attached to the resin was determined spectrophotometrically by modifying the method of Golovina et al.J2 A 50% (w/v) solution of polyethylene glycol 20,000 was used to suspend the affinity resins. The spectra were obtained with a Cary 219 double-team 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. ~z T. O. Golovina, T. V. Cherednikova, A. T. Mevkh, and N. K. Nagradova, Anal. Biochem. 83, 778 (1977).
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
327
The concentration of the hematin in the stock solution was determined using an E40m~of 170 in 80% dimethyl sulfoxide. 13 The concentration of hematoporphyrin was calculated from the amount used to make the stock solution. The spectrophotometric analyses of the new affinity resins showed that 0.13 /xmol of hematin and 0.16/xmol of hematoporphyrin were attached per milliliter of agarose by the synthetic procedures described here. An alternative method of determining the amount of hematin bound involves hydrolysis in base followed by measuring the released hematin as a pyridine hemichrome.14 Applications The usefulness of these affinity resins was tested by chromatographing several apohemoproteins or heme-binding proteins. Undenatured globin (Fig. 2A) and bovine serum albumin (Fig. 2B) can bind to hematinagarose. 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. The hematin-agarose resin can be used to further purify hemopexin from a crude preparation, as shown in Fig. 3. This starting material was prepared from serum by the perchloric acid precipitation method15; however, if the rivanol precipitation method is used, the albumin can be removed at this step. 8 The material that came straight through the column contained no hemopexin, as judged by the spectral assay procedure of Drabkin. 16The protein eluted by the acidic buffer consisted almost exclusively of albumin and hemopexin, as measured by cellulose acetate electrophoresis at pH 8.6 by the method of Kohn.17 A third protein, which is present in very small amounts in the acidic eluate, is probably the histidine-rich glycoprotein. 18 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 ~3 W. T. Morgan, H. H. Liem, R. P. Sutor, and U. Mtiller-Eberhard, Biochim. Biophys. Acta 444, 435 (1976). ~4 K. T s u t s u i and G. C. Mueller, Anal. Biochem. 121, 244 (1982). 15 H. E. Schultze, K. Heide, and H. Haupt, Clin. Chim. Acta 7, 854 (1962). ~6 D. L. Drabkin, Proc. Natl. Acad. Sci. U.S.A. 68, 609 (1971). 17 j. K o h n , Clin. Chim. Acta 3, 450 (1958). 18 W. T. Morgan, Biochim. Biophys. Acta 533, 319 (1978).
328
HEMEPORPHYRINSAND DERIVATIVES A
•
"
'
U'~EAr(
'
[36]
'
0.8
0.6
~ 0.4 C) (("3 DO 0.2 O
L
B
I
U
I
I
I
I
UREA
'~ 0.4 0.6 i
~
~
t
0.2 I0
20
50
40
50
60
70
80
Elution Volume (ml)
FIG. 2. Chromatography of globin and albumin on hematin-agarose. (A) Undenatured globin (10 mg) was applied to a column of hematin-agarose (10 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 Cohn fraction V (10 mg) was applied to a column of hematin-agarose (10 ml)in 0.05 sodium phosphate buffer, pH 7.6. After the contaminating proteins were washed out of the column with the same phosphate buffer, the albumin was eluted with 6 M urea.
these proteins 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 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 mg/ml, while the used resin absorbed 16 mg/ml. From hematoporphyrin-agarose, the unused resin bound 19 mg/ml, but the used resin, only 13 mg/ml. After the first use, the binding capacity of the resin remained essentially constant.
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
329
i
1.5
°i 4
"•'1.2 0 O0 ~--~--"0.9 0
"~0.6
0.3~ 20
i 40
60
80
Elution Volume (ml) FIG. 3. 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 0.1 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.
Although most of the interest in hematin-agarose affinity chromatography has concerned the purification of hemopexin, 7,8,14.19-21this matrix has also been used to isolate the RNA-containing fraction of the enzyme system that converts glutamic acid into &aminolevulinic acid in plants.22, 23 Alternative Methods
Several attempts have been made to purify hemoproteins by affinity chromatography. This is an obvious approach, but it has been plagued 19j. Suttnar, Z. Hrkal, and Z. Vodrazka, J. Chromatogr. 131, 453 (1977). 2o j. Suttnar, Z. Hrkal, Z. Vodrazka, and J. Rejnkova, J. Chromatogr. 169, 500 (1979). 2~ p. Strop, J. Borvak, V. Kasicka, Z. Prusik, and L. Moravek, J. Chromatogr. 214, 317 (1981). 22 D.-D. Huang, W.-Y. Wang, S. P. Gough, and C. G. Kannangara, Science 225, 1482 (1984). 23 W.-Y. Wang, D.-D. Huang, D. Stachon, S. P. Gough, and C. G. Kannangara, Plant Physiol. 74, 569 (1984).
330
HEME PORPHYRINS AND DERIVATIVES
[36]
with technical problems. As early as 1966, Heide 24mentioned the possibility of isolating hemopexin with heme bound to Sephadex G-100, but no experimental details were given. More recently, Conway and MtillerEberhard 25 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 deuteroporphyrin instead of hemin. 25 Rabbit hemopexin and this water-soluble porphyrin form an equimolar complex with an apparent dissociation constant of 1.8 × 10-6 M. 26 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-HCI. 8 Unfortunately, the experimental details of this work have not yet been published. Suttnar e t al.19 have coupled hemin to BioGel P-200 by a p-nitrobenzoylazide 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-HCI buffer (pH 2.4). This synthetic method can also be used to make a heme-agarose matrix, which allowed a much higher recovery of hemopexin from human serum than did the hemin-BioGel P-200 matrix. 2° Two other heme-agarose gels have been developed that can be used to isolate hemopexin. Strop et al. 2J have attached heme to agarose via an 8amino-2-hydroxy-4-thiooctyl linkage. This method requires six synthetic steps but the resulting gel appears to have properties similar to the matrix described here. Tsutsui and Mueller 14 coupled hemin to aminoethylagarose with 1,1-carbonyldiimidazole. The details of this method are given in this volume. 27 This synthetic procedure is as easy to accomplish as the one reported here. Since the hemin-agarose linkage is the same chemical type as in the present method, the lack of serum albumin binding by the Tsutsui and Mueller matrix must be due to the shorter spacer employed. 14 Comments The synthesis of the porphyrin affinity resins described here is a twostep procedure in which aminohexylagarose is first synthesized by the cyanogen bromide technique 9 and then hematin is coupled to the " a r m " by the carbodiimide reaction. The porphyrin in the affinity resin can be z4 K. Heide, Protides Biol. Fluids 14, 593 (1966). 25 T. P. Conway and U. Miiller-Eberhard, Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 469 (1973). 26 T. P. Conway and U. Miiller-Eberhard, Arch. Biochem. Biophys. 172, 558 (1976). 27 K. Tsutsui, this volume [37].
[37]
SYNTHESIS OF HEMIN-AGAROSE
331
attached by either or both of its propionic acid groups. The novel and essential aspect of this reaction is the use of pure DMF as a solvent for hematin. Aqueous DMF in concentrations up to 50% (v/v) has been used in similar reactions involving hydrophobic ligands, such as estradiol. 1° Although pure dioxane has been used as a solvent for the carbodiimide reaction, 1~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% (v/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. Thus, the use of DMF as the solvent in the coupling reaction is essential to the success of this synthesis.
[37] A f f i n i t y C h r o m a t o g r a p h y o f H e m e - B i n d i n g P r o t e i n s : Synthesis of Hemin-Agarose
By KEN TSUTSUI Heme (ferroprotoporphyrin IX) plays an important role as a prosthetic group of many proteins such as oxygen-carrier proteins, proteins of the electron transport system, mixed function oxidases, and peroxidases. In addition to being directly involved in biological reactions as a cofactor, heme also appears to serve as a regulatory molecule in such processes as the initiation of protein synthesis,l ATP/ubiquitin-dependent protein degradation, 2 inhibition of DNA polymerase, 3 transcriptional regulation of a cytochrome c g e n e : and enhancement of cell differentiation) Specific binding of heme to some of the proteins involved in these functions has been demonstrated. Hemopexin, 6 serum albumin, 6 histidine-rich glycoprotein, 7 and HBP.938 are serum proteins that have been shown to bind I S. Ochoa and C. de Haro, Annu. Rev. Biochem. 48, 549 (1979). 2 A. L. Haas and I. A. Rose, Proc. Natl. Acad. Sci. U,S.A. 78, 6845 (1981). 3 j. j. Byrnes, K. M. Downey, L. Esserman, and A. G. So, Biochemistry 14, 796 (1975). 4 L. Guarente and T. Mason, Cell 32, 1279 (1983). 5 j._j. Chen and I. M. London, Cell 26, 117 (1981). 6 U. Mfiller-Eberhard and W. T. Morgan, Ann. N.Y. Acad. Sci. 244, 624 (1975). 7 W. T. Morgan, Biochim. Biophys. Acta 535, 319 (1978). 8 K. Tsutsui and G. C. Mueller, J. Biol. Chem. 257, 3925 (1982).
METHODS 1N ENZYMOLOGY.VOL. 123
Copyright © 1986by Academic Press, Inc. All rights of reproductionin any form reserved.
H E M E PORPHYRINS A N D D E R I V A T I V E S
[36]
[36] A f f i n i t y C h r o m a t o g r a p h y o f H e m e - B i n d i n g P r o t e i n s : Synthesis and Characterization of Hematin- and Hematoporphyrin-Agarose
By
KENNETH
W.
OLSEN
Principle In recent years, the technique of affinity chromatography has revolutionized the purification of proteins. Several general affinity columns using immobilized coenzymes, such as nucleotides j 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 may prove to be successful for purifying a variety of proteins, including hemopexin, 3 ligandin, 4 and tryptophan 1,2dioxygenase (pyrrolase), 5 cytochromes, enzymes involved in heme metabolism, and heme-regulated factors in protein synthesis. 6 This chapter discusses a method for making a hematin-agarose resin that has been used to successfully purify hemopexin. 7,8 The method can be used to synthesize any porphyrin-agarose column packing. The reactions are summarized in Fig. 1. First, aminohexyl-agarose is made using the cyanogen bromide method, 9 and then the porphyrin is attached with a carbodiimide reaction. For the hematin-agarose, it is critical that the second reaction be done in dimethylformamide so that the hematin is soluble. Synthetic Procedures The synthesis of porphyrin-agarose resin is a two-step procedure. In the first phase the agarose is activated by cyanogen bromide, 9 and then 1 R. Barker, K. W. Olsen, J. H. Shaper, and R. L. Hill, J. Biol. Chem. 247, 7135 (1972). 2 C. R. Lowe, M. J. Harvey, D. B. Craven, and P. D. G. Dean, Biochem. J. 133, 499 (1973). 3 U. Miiller-Eberhard and W. T. Morgan, Ann. N . Y . Acad. Sci. 244, 624 (1975). 4 E. Tipping, B. Ketterer, and P. Koskelo, Biochem. J. 169, 509 (1978). 5 H. Marver, D. P. Tschudy, M. G. Perbroth, and A. Collins, Science 153, 501 (1966). 6 S. Ochoa and C. de Haro, Annu. Rev. Biochem. 48, 549 (1979). 7 K. W. Olsen, Anal. Biochem. 109, 250 (1980). 8 R. Majuri, Biochim. Biophys. Acta 719, 53 (1982). 9 p. Axen, J. Por~ith, and S. Ernback, Nature (London) 214, 1302 (1967).
METHODS IN ENZYMOLOGY, VOL. 123
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
325
A
~
OH OH
+
H2N__(CH2 )6__NH 2
(i) C N B r a c t i v a t i o n (2) a m i n o c o u p l i n g O --C--NH--(CH2) OH ,
--NH 6 2
Heme activated by Carbodiimide in DMF
~
O II
O ii
O--C--NH--(CH2)6---NH--C--Heme OH
B
CH
CH 3
CH:CH
C
_C
2
CH
I __O__C__NH__(CH2 )6__NH__~__CH2CH2" C ~ C
CH=CH2
~H~CH3 ~H2 FIG. 1. (A) Synthesis of hematin-agarose. (B) Structure of hematin-agarose.
coupled to 1,6-diaminohexane. l° In a typical synthesis, well-washed Sepharose 4B (150 ml) was activated by cyanogen bromide (37.5 g) at pH 10.5 and 25 °. After thoroughly washing with ice water (1500 ml) the activated resin was reacted with an equal volume of 1,6-diaminohexane solution (34.8 g) at pH 10 and 4 ° overnight. ~j The aminohexyl-agarose was washed with water (2 liters) 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 " a r m " by a carbodiimide reaction. The 10 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970). 11 M. Wilcheck, F E B S Lett. 33, 70 (1973).
326
HEME PORPHYRINS AND DERIVATIVES
[36]
aminohexyl-agarose (100 ml) was washed four times with dimethylformamide (DMF). The hematoporphyrin or hematin (0.5 g) was dissolved in DMF (150 ml) by stirring overnight at room temperature. In this and all subsequent steps, the porphyrins were protected from light as much as possible. The hematin solution had to be filtered on a medium-sintered glass funnel to remove insoluble particles. Each of the porphyrin solutions were mixed with 100 ml of the aminohexyl-agarose. A solution of 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (7.5 g) in 50% DMF (20 ml) was added dropwise to the mixture, while the pH was adjusted to 4.7 with 1 N HCI. The pH was readjusted to 4.7 every 30 min. After 5 hr, a second addition of carbodiimide (7.5 g) was made. Several more pH adjustments were made at 30 min intervals, and the reaction was allowed to proceed overnight at room temperature. In the morning the pH was adjusted to 4.7 several more times. After 18 hr at pH 4.7, the reaction was stopped by adding 1 N sodium hydroxide to bring the pH to 7.5. The hematoporphyrin-agarose was washed with 4 liters of distilled water to remove the DMF and unreacted porphyrin. The resulting affinity resin was pink, and further washing did not remove any additional color. The hematin-agarose resin required more extensive washing to remove the unreacted hematin. After adjusting the pH to 7.5, the resin was washed twice on a sintered glass funnel with DMF (200 ml). The hematinagarose was suspended in DMF and shaken in a waterbath at 30°. The solvent was changed four times daily for the next 4 days until two consecutive washes had no color. The resin was then washed extensively with water to remove the DMF. Unreacted primary amino groups of the gel can be blocked by an additional carbodiimide reaction in acetate buffer. 8 Both affinity resins were stored in dark bottles at 4° under toluene when not in use. Resin stored in this manner appears to be stable for at least 6 months. Analytical Procedures The amount of porphyrin attached to the resin was determined spectrophotometrically by modifying the method of Golovina et al.J2 A 50% (w/v) solution of polyethylene glycol 20,000 was used to suspend the affinity resins. The spectra were obtained with a Cary 219 double-team 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. ~z T. O. Golovina, T. V. Cherednikova, A. T. Mevkh, and N. K. Nagradova, Anal. Biochem. 83, 778 (1977).
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
327
The concentration of the hematin in the stock solution was determined using an E40m~of 170 in 80% dimethyl sulfoxide. 13 The concentration of hematoporphyrin was calculated from the amount used to make the stock solution. The spectrophotometric analyses of the new affinity resins showed that 0.13 /xmol of hematin and 0.16/xmol of hematoporphyrin were attached per milliliter of agarose by the synthetic procedures described here. An alternative method of determining the amount of hematin bound involves hydrolysis in base followed by measuring the released hematin as a pyridine hemichrome.14 Applications The usefulness of these affinity resins was tested by chromatographing several apohemoproteins or heme-binding proteins. Undenatured globin (Fig. 2A) and bovine serum albumin (Fig. 2B) can bind to hematinagarose. 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. The hematin-agarose resin can be used to further purify hemopexin from a crude preparation, as shown in Fig. 3. This starting material was prepared from serum by the perchloric acid precipitation method15; however, if the rivanol precipitation method is used, the albumin can be removed at this step. 8 The material that came straight through the column contained no hemopexin, as judged by the spectral assay procedure of Drabkin. 16The protein eluted by the acidic buffer consisted almost exclusively of albumin and hemopexin, as measured by cellulose acetate electrophoresis at pH 8.6 by the method of Kohn.17 A third protein, which is present in very small amounts in the acidic eluate, is probably the histidine-rich glycoprotein. 18 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 ~3 W. T. Morgan, H. H. Liem, R. P. Sutor, and U. Mtiller-Eberhard, Biochim. Biophys. Acta 444, 435 (1976). ~4 K. T s u t s u i and G. C. Mueller, Anal. Biochem. 121, 244 (1982). 15 H. E. Schultze, K. Heide, and H. Haupt, Clin. Chim. Acta 7, 854 (1962). ~6 D. L. Drabkin, Proc. Natl. Acad. Sci. U.S.A. 68, 609 (1971). 17 j. K o h n , Clin. Chim. Acta 3, 450 (1958). 18 W. T. Morgan, Biochim. Biophys. Acta 533, 319 (1978).
328
HEMEPORPHYRINSAND DERIVATIVES A
•
"
'
U'~EAr(
'
[36]
'
0.8
0.6
~ 0.4 C) (("3 DO 0.2 O
L
B
I
U
I
I
I
I
UREA
'~ 0.4 0.6 i
~
~
t
0.2 I0
20
50
40
50
60
70
80
Elution Volume (ml)
FIG. 2. Chromatography of globin and albumin on hematin-agarose. (A) Undenatured globin (10 mg) was applied to a column of hematin-agarose (10 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 Cohn fraction V (10 mg) was applied to a column of hematin-agarose (10 ml)in 0.05 sodium phosphate buffer, pH 7.6. After the contaminating proteins were washed out of the column with the same phosphate buffer, the albumin was eluted with 6 M urea.
these proteins 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 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 mg/ml, while the used resin absorbed 16 mg/ml. From hematoporphyrin-agarose, the unused resin bound 19 mg/ml, but the used resin, only 13 mg/ml. After the first use, the binding capacity of the resin remained essentially constant.
[36]
HEMATIN- AND HEMATOPORPHYRIN-AGAROSE
329
i
1.5
°i 4
"•'1.2 0 O0 ~--~--"0.9 0
"~0.6
0.3~ 20
i 40
60
80
Elution Volume (ml) FIG. 3. 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 0.1 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.
Although most of the interest in hematin-agarose affinity chromatography has concerned the purification of hemopexin, 7,8,14.19-21this matrix has also been used to isolate the RNA-containing fraction of the enzyme system that converts glutamic acid into &aminolevulinic acid in plants.22, 23 Alternative Methods
Several attempts have been made to purify hemoproteins by affinity chromatography. This is an obvious approach, but it has been plagued 19j. Suttnar, Z. Hrkal, and Z. Vodrazka, J. Chromatogr. 131, 453 (1977). 2o j. Suttnar, Z. Hrkal, Z. Vodrazka, and J. Rejnkova, J. Chromatogr. 169, 500 (1979). 2~ p. Strop, J. Borvak, V. Kasicka, Z. Prusik, and L. Moravek, J. Chromatogr. 214, 317 (1981). 22 D.-D. Huang, W.-Y. Wang, S. P. Gough, and C. G. Kannangara, Science 225, 1482 (1984). 23 W.-Y. Wang, D.-D. Huang, D. Stachon, S. P. Gough, and C. G. Kannangara, Plant Physiol. 74, 569 (1984).
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HEME PORPHYRINS AND DERIVATIVES
[36]
with technical problems. As early as 1966, Heide 24mentioned the possibility of isolating hemopexin with heme bound to Sephadex G-100, but no experimental details were given. More recently, Conway and MtillerEberhard 25 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 deuteroporphyrin instead of hemin. 25 Rabbit hemopexin and this water-soluble porphyrin form an equimolar complex with an apparent dissociation constant of 1.8 × 10-6 M. 26 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-HCI. 8 Unfortunately, the experimental details of this work have not yet been published. Suttnar e t al.19 have coupled hemin to BioGel P-200 by a p-nitrobenzoylazide 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-HCI buffer (pH 2.4). This synthetic method can also be used to make a heme-agarose matrix, which allowed a much higher recovery of hemopexin from human serum than did the hemin-BioGel P-200 matrix. 2° Two other heme-agarose gels have been developed that can be used to isolate hemopexin. Strop et al. 2J have attached heme to agarose via an 8amino-2-hydroxy-4-thiooctyl linkage. This method requires six synthetic steps but the resulting gel appears to have properties similar to the matrix described here. Tsutsui and Mueller 14 coupled hemin to aminoethylagarose with 1,1-carbonyldiimidazole. The details of this method are given in this volume. 27 This synthetic procedure is as easy to accomplish as the one reported here. Since the hemin-agarose linkage is the same chemical type as in the present method, the lack of serum albumin binding by the Tsutsui and Mueller matrix must be due to the shorter spacer employed. 14 Comments The synthesis of the porphyrin affinity resins described here is a twostep procedure in which aminohexylagarose is first synthesized by the cyanogen bromide technique 9 and then hematin is coupled to the " a r m " by the carbodiimide reaction. The porphyrin in the affinity resin can be z4 K. Heide, Protides Biol. Fluids 14, 593 (1966). 25 T. P. Conway and U. Miiller-Eberhard, Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 469 (1973). 26 T. P. Conway and U. Miiller-Eberhard, Arch. Biochem. Biophys. 172, 558 (1976). 27 K. Tsutsui, this volume [37].
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SYNTHESIS OF HEMIN-AGAROSE
331
attached by either or both of its propionic acid groups. The novel and essential aspect of this reaction is the use of pure DMF as a solvent for hematin. Aqueous DMF in concentrations up to 50% (v/v) has been used in similar reactions involving hydrophobic ligands, such as estradiol. 1° Although pure dioxane has been used as a solvent for the carbodiimide reaction, 1~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% (v/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. Thus, the use of DMF as the solvent in the coupling reaction is essential to the success of this synthesis.
[37] A f f i n i t y C h r o m a t o g r a p h y o f H e m e - B i n d i n g P r o t e i n s : Synthesis of Hemin-Agarose
By KEN TSUTSUI Heme (ferroprotoporphyrin IX) plays an important role as a prosthetic group of many proteins such as oxygen-carrier proteins, proteins of the electron transport system, mixed function oxidases, and peroxidases. In addition to being directly involved in biological reactions as a cofactor, heme also appears to serve as a regulatory molecule in such processes as the initiation of protein synthesis,l ATP/ubiquitin-dependent protein degradation, 2 inhibition of DNA polymerase, 3 transcriptional regulation of a cytochrome c g e n e : and enhancement of cell differentiation) Specific binding of heme to some of the proteins involved in these functions has been demonstrated. Hemopexin, 6 serum albumin, 6 histidine-rich glycoprotein, 7 and HBP.938 are serum proteins that have been shown to bind I S. Ochoa and C. de Haro, Annu. Rev. Biochem. 48, 549 (1979). 2 A. L. Haas and I. A. Rose, Proc. Natl. Acad. Sci. U,S.A. 78, 6845 (1981). 3 j. j. Byrnes, K. M. Downey, L. Esserman, and A. G. So, Biochemistry 14, 796 (1975). 4 L. Guarente and T. Mason, Cell 32, 1279 (1983). 5 j._j. Chen and I. M. London, Cell 26, 117 (1981). 6 U. Mfiller-Eberhard and W. T. Morgan, Ann. N.Y. Acad. Sci. 244, 624 (1975). 7 W. T. Morgan, Biochim. Biophys. Acta 535, 319 (1978). 8 K. Tsutsui and G. C. Mueller, J. Biol. Chem. 257, 3925 (1982).
METHODS 1N ENZYMOLOGY.VOL. 123
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