- Email: [email protected]
[2] Preparation of myoglobins
[2]
PREPARATION OF MYOGLOBINS
29
1 hr against 1 liter of the same buffer. The Tris is the "ultra-pure" grade (Mann Laboratories, Inc.). The dialysis tubing is boiled with 10-4 M EDTA and is then washed exhaustively with deionized water before use. All steps of the preparation are done at 4°. The lysate is removed from the dialysis bag and centrifuged at 10,000 g for 30 min. The solution is removed from the center two-thirds of the tube and diluted with an equal volume of 0.05 M Tris, pH 8.0; the centrifugation process is repeated. About 40 ml of solution (approximately 2.0 g of hemoglobin) are applied to a 4 × 60 cm column of DEAE-Sephadex A-50 thoroughly equilibrated with 0.05 M Tris, pH 7.6 - 0.03. Elution is done with this buffer at no more than 160 ml/hr. The eluent is directed to a fraction collector by a three-way valve until the desired fraction emerges. This fraction is then directed through one channel of a two-channel peristaltic pump and is mixed with an equal volume of 0.05 M Tris, pH 8.4, coming through the other channel. The mixture than enters a short collection column of DEAE-Sephadex A-50. After collection of the chosen fraction (Ao), the column is removed and eluted with 0.2 M NaCl in 0.1 M Tris, pH 7.4. The use of such a collection column should be generally useful for many hemoglobins, especially those of lower vertebrates, which may be particularly unstable. Acknowledgments Original work by the author described here was supported by grants from the National Institutes of Health and the National Science Foundationand Grant F-213from the Robert A. WelchFoundation.
[2] P r e p a r a t i o n
of Myoglobins
By JONATHAN B. WITTENBERG and BEATRICE A. WITTENBERG This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called tissue hemoglobins in the widest sense. All of these, except Ascaris and yeast hemoglobin, are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity. Strictly, only muscle hemoglobin should be called myoglobin; by extension the term is often applied to other tissue hemoglobins as well. Isolation of leghemoglobin, the tissue hemoglobin of plants, has been treated in this series. 1 Monomeric blood
1 M. J. Dilworth,this series, Vol, 69 [74]. METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
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HEMOGLOBINS AND MYOGLOBINS
[2]
hemoglobins from the insect Chironomus and the annelid Glycera are included here because they are often studied together with myoglobin. Myoglobin was first obtained in pure form and crystallized by Theorell in 1932. 2 Well formed crystals suitable for X-ray diffraction analysis were achieved in 1950. 3 Most X-ray diffraction studies were done using myoglobin purified by a modification of Theorell's procedure involving precipitation of miscellaneous proteins with basic lead acetate, precipitation of myoglobin with ammonium sulfate, and fractionation of the myoglobin by repeated precipitation from ammonium sulfate or strong sodium potassium phosphate buffer solutions. This procedure, set forth in detail by Kendrew and Parrish, 4 is applicable to tissues, such as whale muscle, that contain much myoglobin; a variant relying heavily on fractionation from phosphate buffers has been used to isolate very pure myoglobin from muscles of several mammals. 5 Since the advent of reliable cellulose and Sephadex materials, this procedure has been supplanted by chromatographic methods. In general, isolation proceeds by extraction of myoglobin from the tissue into dilute buffer, ammonium sulfate fractionation, and gel filtration. Purification is achieved by ion-exchange chromatography. Ferric myoglobin may be purified by chromatography on carboxymethyl (CM) cellulose, usually at slightly acid pH 6-8 or on diethylaminoethyl (DEAE) cellulose, a-lz A crucial advance was the demonstration by Yamazaki, Yokata, and Shikama 1° that native oxymyoglobin (which had never been ferric) could be isolated using columns of DEAE-cellulose at alkaline pH. This material could be crystallized and remained in the oxygenated state almost indefinitely. Their essential discovery was that oxymoglobin is stable toward air oxidation at alkaline pH, pH 7.5-10, with greatest stability near pH 9.13 As a result of their work, the historic concept that myoglobin is unstable in the oxygenated state is no longer tenable. The choice of preparative procedure depends on the use to which the 2 H. Theorell, Biochem. Z. 252, 1 (1932). 3 j. C. Kendrew, Proc. R. Soc. (London), Set. A 201, 62 (1950). 4 j. C. Kendrew and R. G. Parrish, Proc. R. Soc. (London), Ser. A 238, 305 (1956). 5 M. Z. Atassi, Biochim. Biophys. Acta 221, 612 (1970). s A. Akeson and H. Theorell, Arch. Biochem. Biophys. 91, 319 (1960). 7 K. D. Hardman, E. H. Eylar, D. K. Ray, L. J. Banaszak, and F. R. N. Gurd, J. Biol. Chem. 241,432 (1966). s K. D. Hapner, R. A. Bradshaw, C. R. Hartzell, and F. R. N. Gurd, J. Biol. Chem. 243, 683 (1968). 9 W. D. Brown, J. Biol. Chem. 236, 2238 (1961). 10 I. Yamazald, K. Yokota, and K. Shikama, J. Biol. Chem. 239, 4151 (1964). 11 T. E. Hugli and F. R. N. Gurd, J. Biol. Chem. 245, 1930 (1970). 12 T. Gotoh, T. Ochiai, and K. Shikama, J. Chromatogr. 60, 260 (1971). la K. Shikama and Y. Sugawara, Eur. J. Biochem. 91, 407 (1978).
[2]
PREPARATION OF MYOGLOBINS
31
purified myoglobin will be put. Both DEAE and CM ion-exchange columns yield myoglobin that is pure in the sense of being free from contaminating polypeptide chains. Better resolution of forms of myoglobin differing only in charge is achieved on CM-cellulose. Such columns, however, are usually operated at acid pH, and it is a matter of experience that oxymyoglobin exposed to mildly acidic conditions becomes ferric and, in the process, undergoes some minor but apparently irreversible change. When subsequently reduced and again oxygenated, it is never as stable as before but is converted to ferric myoglobin at an accelerated rate. For this reason isolation at alkaline pH by slight modification of the procedure of Yamazaki et al. a° is preferred as a general preparative method. It had the additional advantages of speed and simplicity. Procedures are presented for the preparation of ferric or oxymyoglobin on columns of DEAE-Sephadex, and for the isolation of pure apomyoglobin in a form appropriate for amino acid sequence determination. A procedure for isolation of ferric myoglobin on CM-Sephadex is given in this series) 4 Isolation and Purification of Vertebrate Myoglobins Extraction and Fractionation with A m m o n i u m Sulfate. Fresh or frozen and thawed muscle or heart tissue (1 kg) is dissected free of gross fat, connective tissue, and larger blood vessels. It is chopped coarsely and homogenized in 2 - 3 volumes of oxygenated 0.01 M Tris-HCl buffer, pH 8, containing 1 mM EDTA. Any homogenizer (e.g., Waring blender) is satisfactory, but excessively fine homogenization is to be avoided because very fine (submitochondrial) particles may be difficult to sediment. The particulates are removed by centrifugation at about 10,000 g for 10 min, fat is decanted, and the hazy, red, slightly acid (about pH 6.5) supernatant is equilibrated with air or oxygen and restored to pH 8 by dropwise addition of ammonia. Solid ammonium sulfate is added to 65% saturation while maintaining the pH between 7.5 and 8.0 with ammonia. The voluminous precipitate, which contains more than half of the blood hemoglobin present, is discarded. Additional ammonium sulfate is added to 90% saturation, the pH is restored to 8, and the brick red precipitated myoglobin is collected by centrifugation. Occasionally precipitation is incomplete; in this event ammonium sulfate is added in excess of 100% saturation. The myoglobin and crystalline ammonium sulfate are then collected by filtration on glass wool or glass fiber filter paper. Gel Filtration. The precipitated myoglobin is dissolved in a minimum volume of 0.02 M Tris-HCl, pH 8.0, containing 1 mM EDTA, and fractionated on a column (5 × 50 cm) of Sephadex G-100 or preferably Se14 M. Rothgeb and F. R. N. Gurd, this series, Vol. 52 [50].
32
HEMOGLOBINS AND MYOGLOBINS
[2]
phadex G-100 (superfine) (Pharmacia) equilibrated with the same buffer. About 50 ml of myoglobin solution are applied to the column and developed at a flow rate of 60 ml/hr. Hemoglobin emerges first, followed by myoglobin. This removes high molecular weight substances that would interfere in subsequent ion-exchange chromatography, small amounts of protein that otherwise would overlap the myoglobin peaks in ion-exchange chromatography, and residual blood hemoglobin. The myoglobin-containing fractions are pooled and concentrated to 50-100 ml in an Amicon ultrafiltration apparatus under 40 psi pressure of air (concentration under nitrogen may lead to partial deoxygenation with consequent conversion to ferric myoglobin), over a PM-10 membrane. Chromatography on DEAE-Sephadex. The procedure presented is essentially that of Yamazaki et al. 10 except that DEAE-Sephadex has been found to be superior to the DEAE-cellulose originally used. Purification of myoglobin and simultaneous separation of oxy from ferric myoglobin is achieved on a column (5 × 50 cm) of DEAE-Sephadex A-50 (Pharmacia) equilibrated with 0.02 M Tris-HCl buffer, pH 8.4, containing 1 mM EDTA. A solution of myoglobin that has been partially purified on a column of Sephadex G-100 is applied to the column in the same buffer (a buffer 0.1 pH unit more alkaline than the column-equilibrating buffer may be used to assure a narrow band at loading). Usually about 100/~mol (1.8 g) of myoglobin in no more than 100 ml are placed on the column, but up to 250/zmol (4.5 g) may be used. The column is developed at 4° with the column-equilibrating buffer, at a flow rate of I00 ml/hr. Cytochrome c is not adsorbed and emerges first. A symmetrical peak of ferric myoglobin emerges next, followed closely by oxymyoglobin. Hemoglobin (if it has not previously been removed) remains at the top of the column. If the myoglobin applied has been chromatographed previously on a column of Sephadex G-100, no detectable protein other than myoglobin is eluted in the region of the oxymyoglobin peak. A criterion of purity is the molar extinction coefficient at 280 nm; 30.6, 36.6, and 37.5 x 103 M -1 cm -~, for sperm whale aquoferric myoglobin, oxymyoglobin and carbon monoxymyoglobin, respectively. ~4 The corresponding extinctions at the Soret maxima are: 164 (409nm), 128 (418nm), and 187 × 103 (423 nm) M -~ cm -1. In practice the ratio of absorbance at the Soret maximum to that at the ultraviolet maximum is monitored. For sperm whale myoglobin, these are 5.36, 3.50, and 5.00, respectively) 4 Myoglobins from different sources may require slightly different conditions, and very slight changes in pH will affect the separation of oxymyoglobin from ferric myoglobin. In general myoglobin is more strongly adsorbed at more alkaline pH. 2-Amino-2-methyl-l,3-propanediol (AMPD) has been suggested as a buffer because at 25 ° the pK (pK 8,80) is farther into the alka-
[2]
PREPARATION OF MYOGLOBINS
33
line range than that o f Tris (pK 8.07). '1 Indeed columns operated with 0.03 M A M P D buffer, p H 8.6, give very satisfactory purification of myoglobin and separation of oxy from ferric myoglobin. AMPD suffers from the disadvantage of high cost. It must be recrystallized from ethanol before use, and it is not suitable for freezing or long-term storage o f some myoglobin solutions. Shikama and his colleagues have advocated returning to the use of DEAE-cellulose with a gradient o f decreasing pH. lz,la C h r o m a t o g r a p h y o n C M - C e l l u l o s e . M i c r o h e t e r o g e n e i t y . As shown by E d m u n d s o n and Hirs, 15 otherwise homogeneous preparations of ferric myoglobin from sperm whale, seal, or other sources may be separated into 5 - 1 2 fractions by chromatography on C M - c e l l u l o s e Y '15-17 by electrophoresis, 17 or by isoelectric fractionation. These fractions have identical amino acid compositions, T M identical kinetics in their reactions with ligands, TM and the same conformation as determined by X-ray diffraction. They differ only in charge. 1~ T h e y are not artifacts o f the isolation procedure or of proteolysis occurring in the muscle homogenate because very pure myoglobin prepared by rapid isolation and purified by a z i n c - e t h anol procedure 7 shows a pattern of microheterogeneity identical to that of samples prepared by classic salt fractionation. A likely explanation is that the differences may lie in variation of the amide content of glutamic and aspartic residues. A procedure for isolating these fractions on a preparative scale is given by Rothgeb and Gurd in this series TM (see also H a p n e r et al. 8) Isolation of A p o m y o g l o b i n R o m e r o - H e r r e r a et a1.,'9,2° whose objective is the determination of amino acid sequence, have developed a simple procedure in which myoglobin is isolated as ferric myoglobin cyanide; the heme group is removed, and the resulting apomyoglobin is brought to purity by chromatography on CM-cellulose. Skeletal muscle, 500 g, is minced and homogenized in a blender with 1.5 volumes of distilled water containing 2 m M K C N . The homogenate is centrifuged for 30 min at 25,000 g, the supernatant is recovered, and ammonium sulfate is added to 55% saturation. The material is then stirred for t~ A. B. Edmundson and C. H. W. Hirs, J. Mol. Biol. 5, 663 (1962). ~6N. M. Rumen, Acta Chem. Scand. 13, 1542 (1959). ~7M. Z. Atassi, Nature (London) 202, 496 (1964). ~8L. J. Parkhurst and J. LaGow, Biochemistry 14, 1200(1975). ,9 A. E. Romero-Herrera, H. Lehmann, K. A. Joysey, and A. E. Friday, Philos. Trans. R. Soc. London, Ser. B 283, 61 (1978). 20 H. Dene, M. Goodman, and A. E. Romero-Herrera, Proc. R. Soc. London, Ser. B 207, 111 (1980).
34
HEMOGLOBINS AND MYOGLOBINS
[2]
1 hr at 4 ° and centrifuged for 45 min at 43,000 g. The supernatant is dialyzed against distilled water containing 2 mM KCN for 48 hr and then concentrated in an Amicon ultrafiltration unit, using a PM-10 membrane under 40 psi nitrogen pressure, to a final volume of approximately 60 ml. Aliquots of 20 ml are applied to a column of Ultrogel AcA-54 (LKB) (2.6 cm x 180 cm) equilibrated with 50 mM Tris-HC1 buffer, pH 8.5, and 2 mM KCN. The gel filtration is performed at room temperature with a flow rate of 15 ml/hr. This procedure permits the separation of contaminating heavy molecular weight proteins including tetrameric hemoglobin. The eluted myogloblin is concentrated as above. After dialysis for 48 hr against distilled water, the heme group is removed by 1.5% HCI in acetone followed by three washes with cold acetone to eliminate the acid. 21 The precipitated apomyoglobin is dried under a stream of nitrogen. At this stage, 1.8 g of myoglobin has been recovered from 500 g of muscle. Further purification of the apomyoglobin is achieved by column chromatography on Whatman CM-23 microgranular CM-cellulose (2.6 cm x 12 cm) equilibrated with a solution of 8 M urea, 5 mM Na~HPO4, pH 6.5, 1 mM dithiothreitol (starting buffer). Six hundred milligrams of apomyoglobin are dissolved in 12 ml of this buffer and applied to the column. The column is developed with a linear gradient formed by 500 ml of starting buffer and 500 ml of elution buffer. The latter is prepared under the same conditions as the former but contains 40 mM Na~HPO4. Chromatography proceeds at room temperature with a flow rate of 100 ml/hr, and the effluent is monitored at 280 nm. Small quantities of contaminating proteins are eluted with the void volume. The apomyoglobin-containing fractions are pooled, the salts are removed by gel filtration using sephadex G-25, and the apomyoglobin is recovered by lyophilization. Crystallization S p e r m Whale Ferric Myoglobin. The best crystals 22 are obtained from solutions containing l ml of 6% solution of salt-free purified myoglobin and 2.5 ml of 100% solution of ammonium sulfate, pH 5.75, without added buffer. Crystallization is complete after about I day at room temperature. S p e r m Whale Deoxymyogiobin. Crystallization~3"~4was carded out in a nitrogen-filled glove box. A 50-fold molar excess of sodium dithionite was added to a salt-free 6% solution of ferric myoglobin; this was then mixed with a saturated solution of ammonium sulfate containing 0.01 M sodium EDTA and adjusted to pH 5.75 with 5 vol% of a 4 M solution of
21M. L. Anson and A. E. Mirsky,J. Gen. Physiol. 13, 469 (1930). 22T. Takano,J. Mol. Biol. 110, 537 (1977). 22C. L. Nobbs, H. C. Watson, and J. C. Kendrew,Nature (London) 209, 339 (1966). 24T. Takano,J. Mol. Biol. 110, 569 (1977).
[2]
PREPARATION OF MYOGLOBINS
35
K2HPO4 and NaH~PO4, pH 6.5. The best crystals grow in 72.4% saturated ammonium sulfate solutions at room temperature. Sperm Whale Oxymyoglobin. Deoxymyoglobin crystals were washed (to remove dithionite) and exposed to air immediately before mounting for X-ray analysis, z5 Oxymyoglobin crystals, apparently not suitable for Xray diffraction, have been obtained by adding 3-5 volumes of saturated ammonium sulfate, pH 5.7, to solutions of sperm whale oxymyoglobin, 3.4 mM protein, in water, apparently at 40.7 Seal Myoglobin. The terminal amino group of seal and horse myoglobin is glycine, whereas in sperm and finback whale it is valine. For this reason, and because the crystal form is different, it was of interest to determine the structure of seal myoglobin,z6 A saturated solution of ammonium sulfate was added to a salt-free solution of myoglobin, 5%, to the point of incipient turbidity. Best results were obtained in the range pH 5.6 to 7.3. 4,27 Tuna Myoglobin. This, the only fish myoglobin investigated intensively, was first crystallized by Rossi Fanelli and Antonini. z8 Partial amino acid sequence 29 and X-ray diffraction analysis to 6/k resolutiona° are available. Red muscle from yellowfin tuna, Thunnus albacares, was homogenized with an equal volume of water and fractionated with ammonium sulfate, 70-95% saturation; the myoglobin fraction was separated from hemoglobin and other proteins on a column of Sephadex G-75 in 0.05 M Tris-phosphate buffer, pH 8. Final purification was on a column of CMSephadex C-50 (2.5 × 34 cm) eluted with 0.015 M Tris-phosphate buffer, pH 7.1, 31 or on a column ofDEAE-Sephadex A-50 (2.4 × 85 cm, capacity up to 1.0 g of myoglobin) eluted with a linear gradient formed from 700 ml each of 0.025 M Tris-HCl, pH 8.7, and 0.05 M Tris-HCl, pH 7.2. 3°'a~Crystallization was from 70% saturated ammonium sulfate, pH 5.5-7.0, 1030 mg of protein per milliliter,z°'32 Other Species. The first crystalline myoglobins studied by X-ray diffraction were those of horse a and finback whale, a3 Kendrew et al. z4 crys25 S. E. V. Phillips, Nature (London) 273, 247 (1978). 26 H. Scouloudi and E. N. Baker, J. Mol. Biol. 126, 637 (1978). zr H. Scouloudi, Proc. Roy. Soc. London, Set. A. 258, 181 (1960). 2s A. Rossi Fanelli and E. Antonini, Arch. Biochem. Biophys, 58, 498 (1955). 29 R. H. Price, D. A. Watts, and W. D. Brown, Cornp. Biochem. Physiol. 62B, 481 (1979). z0 E. E. Lattman, C. E. Nockolds, R. H. Kretsinger, and W. E. Love, J. Mol. Biol. 60, 271 (1971). 3~ G. J. Fosmire and W. D. Brown, Comp. Biochem. Physiol. 5511, 293 (1976). 32 R. H. Kretsinger, J. Mol. Biol. 38, 141 (1968). aa j. C. Kendrew and P. J. Pauling, Proc. R. Soc. London, Ser. A 237, 255 (1956). a4 j, C. Kendrew, R, G. Panfish, J. R. Marrack, and E. S. Orlans, Nature (London) 174, 946 (1954).
36
HEMOGLOBINS AND MYOGLOBINS
[2]
tallized, and examined crystallographically, myoglobins from 16 species including whales, porpoises, seals, tortoise, penguin, and carp. Horse oxymyoglobin crystallizes from 87% saturated ammonium sulfate solution at 4°. 1° Analytical Separation of Myoglobin from H e m o g l o b i n Three methods are available to separate myoglobin obtained from small samples of tissue from contaminating hemoglobin. In each case it is best to extract the tissue by the procedure of Schuder e t a1.,35 described here. A sample of muscle is minced coarsely with scissors, frozen, and ground to a fine powder in a procelain mortar cooled in Dry Ice or liquid nitrogen. Exactly 1 g of the frozen powder is weighed into a centrifuge tube and leached with 9.25 ml of 0.01 M potassium phosphate buffer, pH 7.0, containing 5 mM EDTA for 5 min at ice temperature, with occasional stirring. The sample is centrifuged at 20,000 g for 10 rain to obtain a crystal clear supernatant; 10.0 ml of this solution contains the myoglobin from 1.0 g of muscle. (It is assumed that the tissue myoglobin becomes distributed in the total water of the extract including that contributed by the tissue and that from added buffer. The water content of muscles from different sources is very nearly the same.) Very little cytochrome c is extracted. Hemoglobin is subsequently separated from myoglobin in the extract by subunit exchange chromatography on columns of a-fl dimers of human hemoglobin A immobilized on Sepharose 4B,3~ or by absorption on p-mercuribenzene-coupled Sepharose CI-4B, 3e or by gel filtration on columns (2 × 40 cm) of Sephadex G-100 (superfine). Useful absorbance maxima, largely free from interference by cytochrome c, arc: ferrous myoglobin, 434 nm, E = 114 x 103; and carbon monoxide myoglobin, 579nm, • = 12.2 × 10a cm -1 M - '. 35 Photometric errors may be minimized by taking the difference spectrum: carbon monoxide (dithionite) minus reduced (dithionite), taking A•, 422 minus 438 nm = 181 x 103 c m - ' M-'.
Conversion of Ferric to Oxymyoglobin Ferredoxin, in a coupled enzyme system, reduces ferric hemoglobin, myoglobin, or Icghemoglobin. 37 The reducing system (see Dilodo, this volume [4]) is added to the aerated myoglobin solution at room tempera3~S. Schuder, J. B. Wittenberg,B. Haseltine, and B. A. Wittenberg,Anal. Biochem. 92, 473 (1979). a6 D. J. Goss and L. J. Parkhurst, J. Biochem. Biophys. Methods 3, 315 (1980). 3r A. Hayashi, T. Suzuki, and M. Shin, Biochim. Biophys. Acta 310, 309 (1973).
[2]
PREPARATION OF MYOGLOBINS
37
ture. Reduction is complete within 20 min, and solutions of oxyhemoproteins so prepared are stable for about 1 week. Stock enzyme solutions or suspensions are used as purchased without further dilution. Glucose-6phosphate dehydrogenase and ferredoxin NADP reductase are apparently inactivated when stored with EDTA and should be stored in EDTA-free buffers .38 Myoglobin may also be reduced with dithionite. A column of Sephadex G-10 or G-25 or of BioGel P2-P6 (Bio-Rad) about 20 cm high is flushed with oxygen-free buffer, pH 7.5-9.0. A layer about 1 cm high, of an anaerobically prepared solution of dithionite, 10-100 mM, in the same buffer is carefully pipetted above the surface of the Sephadex bed, beneath a protecting column of anaerobic buffer, and is run into the column. A solution of ferric myoglobin is next applied to the column. It overtakes and passes the band of dithionite. Ferrous myoglobin so generated becomes oxygenated by residual oxygen in the column or as the effluent emerges into the air. The products of reaction of dithionite with oxygen are deleterious to proteins, and oxymyoglobin, so prepared, is less stable than that prepared by enzymic reduction. Storage Myoglobin may be stored at 4° in the form of a paste in saturated ammonium sulfate for periods of 2 years or longer without apparent alteration. 4 Solutions of ferric or oxymyoglobin in 0.05 M phosphate buffer, pH 7.5, may be stored for 5 years at liquid nitrogen temperature without apparent change. Solutions of myoglobin in 0.05 M Tris-HC1 buffer, pH 8, are stable to repeated freezing and thawing. We have no experience with prolonged storage in Tris buffer. We believe on limited experience that morpholinoethanesulfonic acid (MES) buffers are inappropriate for prolonged storage of myoglobin. Myoglobin in solution in AMPD buffer, pH 8.0-8.5, is destroyed by repeated freezing and thawing. I n v e r t e b r a t e Myoglobins Gastrophilus Myoglobin. Intracellular hemoglobins occur twice among insects: in the tracheal organs of a few aquatic notonectids (Hemiptera) 39 and in the tracheal organ of a larval stage of the bot fly Gastrophilus intestinalis, 4° which lives as a parasite in the stomach of the horse. This dimeric protein is noteworthy for the low value of the oxygen dissociation constant. 41
38F. C. Mills, M. L. Johnson, and G. K. Ackers, Biochemistry 15, 5350 (1976). 39G. Bergstrom,Insect Biochern. 7, 313 (1977). 40D. Keilin and Y. L. Wang, Biochem J. 40, 855 (1946). 41C. F. Phelps, E. Antonini, M. Brunori, and G. Kellet,Biochem. J. 129, 891 (1972).
38
HEMOGLOBINS AND MYOGLOBINS
[2]
Gastrophilus larvae are available from September to June, with local seasonal variations in abundance. Horse stomachs are opened at the knackery. Larvae found attached to the mucosa of the cardiac region of the stomach are returned to the laboratory at room temperature in moist filter paper or on slices of liver. The red tracheal organ, attached to the postabdominal spiracular plate, is dissected free and is carefully washed free of blood. The blood contains phenols and phenol oxidases, which otherwise would destroy the hemoglobin. The tracheal organs are ground with sand in 0.01 M phosphate buffer, pH 7.5, and centrifuged; the clear red supernatant solution is fractionated with ammonium sulfate. The fraction precipitating from 70-85% saturation is retained. This is dissolved in a minimum volume of phosphate buffer, pH 7.5, and further purified by passage over a column of Sephadex G-75. Chironomus Hemolymph Hemoglobin. The structure of monomeric hemoglobin (fraction III) from larvae of the fly Chironomus has been determined at 1.4/~ resolution for carbon monoxy, deoxy, aquoferric, cyanofen-ic, 42 and oxy 43 forms. These structures, together with the amino acid sequence, 44 are central to present discussions of oxygen binding to myoglobin.43-46 Hemolymph, separated from 20 g of frozen and thawed larvae, was fractionated on a column of Sephadex G-75 (5 x 43 cm) developed with a solution containing 0.1 M sodium acetate and 0.035 M magnesium chloride, pH 6.8-7.1.47 (Alternatively, larvae are homogenized in 1.5 volumes of 5 mM phosphate buffer, pH 8.6, containing 1.5 mM potassium cyanide, and the supernatant from the homogenate is fractionated with ammonium sulfate.) The hemoglobin monomer fractions from nine such columns were pooled to give 860 mg of hemoglobin. This was concentrated by precipitation with ammonium sulfate (to 85% saturation) dissolved in water, desalted, and further fractionated on a column of DEAE-cellulose (3 × 30 cm) at pH 8.6. A basic protein, fraction I, which isnot adsorbed, was eluted with 300 ml of a solution of 0.005 M potassium phosphate buffer, pH 8.6, containing 1.5 mM potassium cyanide. Monomer fractions III and IV were then separated by elution with a gradient formed between the starting buffer and a solution of 0.01 M potassium phosphate buffer, pH 8.0, containing 0.04 M potassium chloride and 1.5 mM potassium cyanide. A yield of 220 mg of monomer fraction III was obtained. 4~ W. Steigemann and E. Weber, J. Mol. Biol. 127, 309 (1979). 43 E. Weber, W. Steigemann, T. A. Jones, and R. Huber, J. Mol. Biol. 120, 327 (1978). 44 R. Huber, O. Epp, W. Steigemann, and H. Formanek, Eur. J. Biochem. 19, 42 (1971). 45 G. Steffens, G. Buse, and A. Wollmer, Eur. J. Biochem. 72, 201 (1977). 4e A. Wollmer, G. St¢ffens, and G. Buse, Eur. J. Biochem. 72, 207 (1977). 4r V. Braun, R. R. Crichton, and G. Braunitzer, Hoppe-Seyler's Z. Physiol. Chem. 349, 197
(1968).
[2]
PREPARATION OF MYOGLOBINS
39
After a final chromatographic purification on a column of CM-cellulose, crystals were obtained from 3.75 M ammonium sulfate, pH 7.0. 44 These were transferred to 3.75M phosphate buffer for X-ray diffraction studies. The cyanide derivative was formed by exposing the crystals of aquoferric hemoglobin to 0.01 M potassium cyanide, the deoxy derivative by exposing the crystals to 0.1 M dithionite in the same buffer, and the carbon monoxide derivative by subsequent exposure to gaseous carbon monoxide. Oxyhemoglobin crystals were obtained by exposing crystals of aquoferric hemoglobin to a mixture of hydrogen sulfide (a reducing agent) and oxygen in the proportion 1:6. 43 Glycera Monomeric Hemoglobin. Hemoglobin from the coelomic erythrocytes of the polychaete annelid Glycera dibranehiata is an interacting mixture of monomeric and oligomeric proteins. Monomeric hemoglobin, separated from the mixture, lacks the distal histidine characteristic of most hemoglobins and myoglobins and has in its place a leucine residue. 48 Optical spectra 4a'5° ligand binding affinities, 5° kinetics, 5~,51a amino acid sequence, 48 and X-ray crystallographic structure 52 are available. The oxygen combination and dissociation rate constants of monomer fraction I are the largest yet reported for a hemoglobin. 5~a This hemoglobin, together with Aplysia myoglobin is often used in studies of the role of the distal histidine residue. Glycera dibranchiata, blood worms, may be purchased from bait companies in New England (e.g., Maine Bait Company, Newcastle, Maine,). Worms are anesthetized in 10% ethanol in 3% sodium chloride, slit, and allowed to bleed in to 3% NaCI. Hemoglobin-containing cells of the coelomic fluid are collected and washed three times with 3% NaCI by low speed centrifugation (about 400 g). The washed cells are lysed by the addition of two volumes of distilled water, and cell residues are subsequently removed by centrifugation at 20,000 g for 20 rain. One-tenth volume of 1 M Tris-HC1 buffer, pH 8.42, is added, and a portion, 10-20 ml, of the clear supernatent is fractionated on a column of Sephadex G-75 (2.4 × 40 cm), equilibrated with 0.1 M Tris buffer, pH 8.42. 49 Two peaks are resolved. The slower moving component is the desired monomer. The monomer fraction from 100 worms in 35 ml of 0.01 M potassium phosphate buffer, pH 6.8, is applied to a 5 × 50 cm CM-Sephadex C-50 column previously equilibrated with 0.01 M potassium phosphate buffer, 48 T. Imamura, T. O. Baldwin, and A. Riggs, J. Biol. Chem. 247, 2785 (1972). 49 S. N. Vinogradov, C. A. Machlik, and L. L. Chao, J. Biol. Chem. 245, 6533 (1970). 5o B. Seamonds, R. E. Forster, and P. George, J. Biol. Chem. 246, 5391 (1971). 51 B. Seamonds, J. A. McCray, L. J. Parkhurst, and P. D. Smith, J. Biol. Chem. 251, 2579 (1976). 51a L. J. Parkhurst, P. Sima, and D. J. Goss, Biochemistry 19, 2688 (1980). 5a E. Padlan and W. E. Love, J. Biol. Chem. 249, 4067 (1974).
40
HEMOGLOBINS AND MYOGLOBINS
[2]
pH 6.8, saturated with carbon monoxide; the column is developed at 4°. The flow rate is 45 ml/hr. After approximately 8 hr, four bands are observed on the column. Hemoglobins I and II (bottom and second band) are separated by - 10 cm; hemoglobins III and IV (top of column) are separated by - 3 cm. The column gel is carefully forced out of the column by gentle air pressure, and the bands are cut out. The proteins are eluted from the gel by repouring the gel into smaller columns and washing with 0.2 M potassium phosphate, pH 7.5. This also serves to concentrate the proteins. The recovery of total monomers is about 0.3-0.4/zmol of hemoglobin per worm. Fractions I and II show very rapid kinetics. Fraction II probably corresponds to that hemoglobin for which the sequence is rep o r t e d . 51a
Crystals of Glycera carbon monoxyhemoglobin were grown at 4° from 2.4 to 2.8 M solutions of ammonium sulfate, 0.06 M potassium phosphate, pH 6.8. 52 Aplysia Myoglobin. Myoglobin from the buccal muscles 53 and nerves 54 of the gastropod mollusc Aplysia limacina (Mediterranean) or A. californica (Pacific Coast) lacks a histidine residue distal to the heme. 55 It is unfolded reversibly by solvent change or high temperature.56.5r Optical spectra, 58 EPR spectra, 59 ligand affinity,5s'6° kinetics, 61 amino acid sequence, 55 and X-ray diffraction structure 62 are available. Buccal masses [approximately 2 g per animal; 150 ftmol (A. californica) or 500/zmol (A. limacina) of myoglobin per kilogram wet weight] are homogenized in 20 volumes of 0.05 M Tris-HC1 buffer, pH 8.0, containing I mM EDTA. The homogenate is centrifuged at 20,000 g for 10 min and fractionated by the addition of solid ammonium sulfate between 50 and 90% saturation, keeping the pH near 8 by the dropwise addition of ammonia. The dark red precipitate is collected by centrifugation, taken up in 1-2 volumes of buffer, and again fractionated with ammonium 53 A. Rossi Fanelli and E. Antonini, Biokhimiya 22, 336 (1957). 54 B. A. Wittenberg, R. W. Briehl, and J. B. Wittenberg, Biochem. J. 96, 363 (1965). 55 L. Tentori, G. Vivaldi, S. Carta, M. Marinucci, A. Massa, E. Antonini, and M. Brunori, Int. J. Pept. Protein Res. 5, 187 (1973). so M. Brunori, E. Antonini, P. Fasella, J. Wyman, and A. Rossi Fanelli, J. Mol. Biol. 34, 497
(1968). 5~ M. Brunori, G. M. Giacometti, E. Antonini, and J. Wyman, J. Mol. Biol. 63, 139 (1972). 5s A. Rossi Fanelli, E. Antonini, and D. Povoledo, in "I. U. P. A. C. Symposium on Protein Structure" (A. Neuberger, ed.), p. 144 (1958). 59 G. Rotilio, L. Calabrese, G. M. Giacometti, and M. Brunori, Biochim. Biophys. Acta 236, 234 (1971). a0 G. M. Giacometti, A. Da Ros, E. Antonini, and M. Brunori, Biochemistry 14, 1584 (1975). e~ B. A. Wittenberg, M. Brunori, E. Antonini, J. B. Wittenberg, and J. Wyman, Arch. Biochem. Biophys. 111, 576 (1965). as T. L. Blundell, M. Brunori, B. Curti, M. Bolognesi, A. Coda, M. Fumagalli, and L. Ungaretti, J. Mol. Biol. 97, 665 (1975).
[2]
PREPARATION OF MYOGLOBINS
41
sulfate. The precipitate is dissolved in a minimum volume of the same buffer (or 0.05 M potassium phosphate buffer, pH 7.5, containing 1 mM EDTA) and separated from high molecular weight substances by chromatography on a column of Sephadex G-75 (superfine) or G-100 (superfine) (Pharmacia). At this stage the protein is approaching homogeneity and is usually more than 90% oxymyoglobin. Crystals suitable for X-ray diffraction 62 were obtained by dialyzing a solution of the ferric protein, about 15 mg/ml, in a solution of 3.5 M ammonium sulfate, 0.05 M phosphate buffer, pH 7.2, against a solution of 3.8 M ammonium sulfate in the same buffer. Identical crystals were obtained by vapor diffusion by leaving the same protein solution in equilibrium with 4 M ammonium sulfate, pH 7.2. Other Annelid and Molluscan Myoglobins. Monomeric and dimeric myoglobins, some of which show cooperative oxygen binding, have been isolated from many annelids 63-6~ and molluscs. 65"66In general, apart from ligand affinity, little is known about these proteins. Purification is essentially by the procedure described for Aplysia myoglobin. The amino acid sequence of a dimeric myoglobin from the readily available gastropod mollusc Busycon caniculatum has been reported. 67 Ascaris Hemoglobin. Body walls of the nematode Ascaris lumbricoides, an intestinal parasite of pigs, contain two electrophoretically and chromatographically distinct oxygen-binding hemoproteins that differ in their affinity for ligands. 6s'69 The more abundant of these has been purified r°-Tz and is of interest because of the extraordinarily high oxygen affinity resulting from very slow dissociation of oxygen. 7a It differs from myoglobin in that the molecular weight is about 3 5 , 0 0 0 , 71 and in optical spectra 71 and ligand binding kinetics. TM Ascaris are chilled at the slaughterhouse, slit, and washed free of adhering perienteric fluid. (Ascaris is a potent allergen. Use of surgical mask 63 C. P. Mangum, in "Adaptation to Environment" (R. C. NeweU, ed.), p. 191. Butterworth, London, 1976. R. E. Weber, in "Physiology of Annelids" (P. J. Mill, ed.), p. 369. Academic Press, New York, 1978. 65 R. C. Terwilliger, Am. Zool. 20, 53 (1980). 66 K. R. n . Read, in "Physiology of Mollusca" (K. M. Wilbur and C. M. Yonge, eds.), Vol. 2, p. 209. Academic Press, New York, 1966. 67 A. S. Bonner and R. A. Laursen, FEBS Lett. 73, 201 (1977). K. Hamada, T. Okazaki, R. Shukuya, and K. Kaziro, J. Biochem. (Tokyo) 52, 290 (1962). 6~ K. Hamada, T. Okazaki, R. Shukuya and K. Kaziro, J. Biochem. (Tokyo) 53, 479 (1963). 7o H. E. Davenport, Proc. R. Soc. London, Set. B 136, 255 (1949). 7, T. Okazaki, B. A. Wittenberg, R. W. Briehl, and J. B. Wittenberg, Biochim. Biophys. Acta 140, 258 (1967). 72 j. B. Wittenberg, F. J. Bergersen, C. A. Appleby, and G. L. Turner, J. Biol. Chem. 249, 4057 (1974). 73 Q. H. Gibson and M. H. Smith, Proc. R. Soc. London, Ser. B 163, 206 (1965).
42
HEMOGLOBINS AND MYOGLOBINS
[2]
is advised.) Body walls (266 g) were homogenized in two volumes of 20 mM phosphate buffer, pH 7.4, containing 1 mM EDTA, and the supernatant solution was separated by centrifugation. The pale red supernatant, pH 6.7, was adjusted to pH 7.2 and fractionated by the addition of solid ammonium sulfate. The fraction precipitating between 56 and 80% saturation was retained. The precipitate was dissolved in a minimum volume of 10 mM phosphate buffer, pH 7.0, and was separated from high molecular weight substances by chromatography on a column of Sephadex G-75 (5 × 52 cm) in the same buffer. The yield at this stage was about 30/xmol of hemoglobin per kilogram of body walls. Final purification was by chromatography on a column of Whatman DE-52 microgranular DEAE-cellulose (2.5 x 20 cm), which had been equilibrated with 5 mM phosphate buffer pH 7.0. Elution was with a gradient of buffer concentration from 5 to 20 mM. The product is minimally 90% oxyhemoglobin. Trematode Hemoglobins. Monomeric hemoglobins have been purified from Fasciolopsis buski, the intestinal fluke of man and pigs, ~4 and from Dicrocoelium dendriticum, a fluke that infests the hepatic ducts of sheep. 75 The oxygen affinity of the latter hemoglobin is notably great. Paramecium Myoglobin. Myoglobin from the ciliate protozoan Paramecium aurelia 76-7a has been purified by fractionation with ammonium sulfate, 50-65% saturation, followed b y chromatography on Sephadex G75 and, finally, by chromatography on Sephadex G-50. 7s Five components apparently differing only in their isoelectric points were separated electrophoretically. Yeast Hemoglobin. Intracellular hemoglobin is abundant only in particular strains of yeast. It has been isolated from Candida mycoderma. 79 The molecule has two prosthetic groups, FAD and protoheme, apparently both attached to a single polypeptide chain, molecular weight 50,000. Except for the contribution by the flavin, the optical spectrum is that of a typical hemoglobin. The oxygen affinity is extraordinarily great, apparently as a result of very rapid combination with oxygen. Isolation involves mechanical disruption of the cells, ammonium sulfate fractionation between 45 and 60% saturation at pH 7, and, finally, chromatography on DEAE-cellulose in 20 mM acetate buffer pH 6.2. 74 G. D. Cain, J. Parasitol. 55, 311 (1969). 75 p. E. Tuchschmid, P. A. Kunz, and K. J. Wilson, Eur. J. Biochem. 88, 387 (1978). 76 D. Keilin and J. F. Ryley, Nature (London172, 451 (1953). r7 M. H. Smith, P. George, and J. R. Preer, Arch. Biochem. Biophys. 99, 313 (1962). 7a E. Steers and R. H. Davis, Comp. Biochem. Physiol. 6211, 393 (1979). 79 R. Oshino, J. Asakura, K. Takio, N. Oshino, and B. Chance, Eur. J. Biochem. 39, 581 (1973).
PREPARATION OF MYOGLOBINS
29
1 hr against 1 liter of the same buffer. The Tris is the "ultra-pure" grade (Mann Laboratories, Inc.). The dialysis tubing is boiled with 10-4 M EDTA and is then washed exhaustively with deionized water before use. All steps of the preparation are done at 4°. The lysate is removed from the dialysis bag and centrifuged at 10,000 g for 30 min. The solution is removed from the center two-thirds of the tube and diluted with an equal volume of 0.05 M Tris, pH 8.0; the centrifugation process is repeated. About 40 ml of solution (approximately 2.0 g of hemoglobin) are applied to a 4 × 60 cm column of DEAE-Sephadex A-50 thoroughly equilibrated with 0.05 M Tris, pH 7.6 - 0.03. Elution is done with this buffer at no more than 160 ml/hr. The eluent is directed to a fraction collector by a three-way valve until the desired fraction emerges. This fraction is then directed through one channel of a two-channel peristaltic pump and is mixed with an equal volume of 0.05 M Tris, pH 8.4, coming through the other channel. The mixture than enters a short collection column of DEAE-Sephadex A-50. After collection of the chosen fraction (Ao), the column is removed and eluted with 0.2 M NaCl in 0.1 M Tris, pH 7.4. The use of such a collection column should be generally useful for many hemoglobins, especially those of lower vertebrates, which may be particularly unstable. Acknowledgments Original work by the author described here was supported by grants from the National Institutes of Health and the National Science Foundationand Grant F-213from the Robert A. WelchFoundation.
[2] P r e p a r a t i o n
of Myoglobins
By JONATHAN B. WITTENBERG and BEATRICE A. WITTENBERG This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called tissue hemoglobins in the widest sense. All of these, except Ascaris and yeast hemoglobin, are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity. Strictly, only muscle hemoglobin should be called myoglobin; by extension the term is often applied to other tissue hemoglobins as well. Isolation of leghemoglobin, the tissue hemoglobin of plants, has been treated in this series. 1 Monomeric blood
1 M. J. Dilworth,this series, Vol, 69 [74]. METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
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HEMOGLOBINS AND MYOGLOBINS
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hemoglobins from the insect Chironomus and the annelid Glycera are included here because they are often studied together with myoglobin. Myoglobin was first obtained in pure form and crystallized by Theorell in 1932. 2 Well formed crystals suitable for X-ray diffraction analysis were achieved in 1950. 3 Most X-ray diffraction studies were done using myoglobin purified by a modification of Theorell's procedure involving precipitation of miscellaneous proteins with basic lead acetate, precipitation of myoglobin with ammonium sulfate, and fractionation of the myoglobin by repeated precipitation from ammonium sulfate or strong sodium potassium phosphate buffer solutions. This procedure, set forth in detail by Kendrew and Parrish, 4 is applicable to tissues, such as whale muscle, that contain much myoglobin; a variant relying heavily on fractionation from phosphate buffers has been used to isolate very pure myoglobin from muscles of several mammals. 5 Since the advent of reliable cellulose and Sephadex materials, this procedure has been supplanted by chromatographic methods. In general, isolation proceeds by extraction of myoglobin from the tissue into dilute buffer, ammonium sulfate fractionation, and gel filtration. Purification is achieved by ion-exchange chromatography. Ferric myoglobin may be purified by chromatography on carboxymethyl (CM) cellulose, usually at slightly acid pH 6-8 or on diethylaminoethyl (DEAE) cellulose, a-lz A crucial advance was the demonstration by Yamazaki, Yokata, and Shikama 1° that native oxymyoglobin (which had never been ferric) could be isolated using columns of DEAE-cellulose at alkaline pH. This material could be crystallized and remained in the oxygenated state almost indefinitely. Their essential discovery was that oxymoglobin is stable toward air oxidation at alkaline pH, pH 7.5-10, with greatest stability near pH 9.13 As a result of their work, the historic concept that myoglobin is unstable in the oxygenated state is no longer tenable. The choice of preparative procedure depends on the use to which the 2 H. Theorell, Biochem. Z. 252, 1 (1932). 3 j. C. Kendrew, Proc. R. Soc. (London), Set. A 201, 62 (1950). 4 j. C. Kendrew and R. G. Parrish, Proc. R. Soc. (London), Ser. A 238, 305 (1956). 5 M. Z. Atassi, Biochim. Biophys. Acta 221, 612 (1970). s A. Akeson and H. Theorell, Arch. Biochem. Biophys. 91, 319 (1960). 7 K. D. Hardman, E. H. Eylar, D. K. Ray, L. J. Banaszak, and F. R. N. Gurd, J. Biol. Chem. 241,432 (1966). s K. D. Hapner, R. A. Bradshaw, C. R. Hartzell, and F. R. N. Gurd, J. Biol. Chem. 243, 683 (1968). 9 W. D. Brown, J. Biol. Chem. 236, 2238 (1961). 10 I. Yamazald, K. Yokota, and K. Shikama, J. Biol. Chem. 239, 4151 (1964). 11 T. E. Hugli and F. R. N. Gurd, J. Biol. Chem. 245, 1930 (1970). 12 T. Gotoh, T. Ochiai, and K. Shikama, J. Chromatogr. 60, 260 (1971). la K. Shikama and Y. Sugawara, Eur. J. Biochem. 91, 407 (1978).
[2]
PREPARATION OF MYOGLOBINS
31
purified myoglobin will be put. Both DEAE and CM ion-exchange columns yield myoglobin that is pure in the sense of being free from contaminating polypeptide chains. Better resolution of forms of myoglobin differing only in charge is achieved on CM-cellulose. Such columns, however, are usually operated at acid pH, and it is a matter of experience that oxymyoglobin exposed to mildly acidic conditions becomes ferric and, in the process, undergoes some minor but apparently irreversible change. When subsequently reduced and again oxygenated, it is never as stable as before but is converted to ferric myoglobin at an accelerated rate. For this reason isolation at alkaline pH by slight modification of the procedure of Yamazaki et al. a° is preferred as a general preparative method. It had the additional advantages of speed and simplicity. Procedures are presented for the preparation of ferric or oxymyoglobin on columns of DEAE-Sephadex, and for the isolation of pure apomyoglobin in a form appropriate for amino acid sequence determination. A procedure for isolation of ferric myoglobin on CM-Sephadex is given in this series) 4 Isolation and Purification of Vertebrate Myoglobins Extraction and Fractionation with A m m o n i u m Sulfate. Fresh or frozen and thawed muscle or heart tissue (1 kg) is dissected free of gross fat, connective tissue, and larger blood vessels. It is chopped coarsely and homogenized in 2 - 3 volumes of oxygenated 0.01 M Tris-HCl buffer, pH 8, containing 1 mM EDTA. Any homogenizer (e.g., Waring blender) is satisfactory, but excessively fine homogenization is to be avoided because very fine (submitochondrial) particles may be difficult to sediment. The particulates are removed by centrifugation at about 10,000 g for 10 min, fat is decanted, and the hazy, red, slightly acid (about pH 6.5) supernatant is equilibrated with air or oxygen and restored to pH 8 by dropwise addition of ammonia. Solid ammonium sulfate is added to 65% saturation while maintaining the pH between 7.5 and 8.0 with ammonia. The voluminous precipitate, which contains more than half of the blood hemoglobin present, is discarded. Additional ammonium sulfate is added to 90% saturation, the pH is restored to 8, and the brick red precipitated myoglobin is collected by centrifugation. Occasionally precipitation is incomplete; in this event ammonium sulfate is added in excess of 100% saturation. The myoglobin and crystalline ammonium sulfate are then collected by filtration on glass wool or glass fiber filter paper. Gel Filtration. The precipitated myoglobin is dissolved in a minimum volume of 0.02 M Tris-HCl, pH 8.0, containing 1 mM EDTA, and fractionated on a column (5 × 50 cm) of Sephadex G-100 or preferably Se14 M. Rothgeb and F. R. N. Gurd, this series, Vol. 52 [50].
32
HEMOGLOBINS AND MYOGLOBINS
[2]
phadex G-100 (superfine) (Pharmacia) equilibrated with the same buffer. About 50 ml of myoglobin solution are applied to the column and developed at a flow rate of 60 ml/hr. Hemoglobin emerges first, followed by myoglobin. This removes high molecular weight substances that would interfere in subsequent ion-exchange chromatography, small amounts of protein that otherwise would overlap the myoglobin peaks in ion-exchange chromatography, and residual blood hemoglobin. The myoglobin-containing fractions are pooled and concentrated to 50-100 ml in an Amicon ultrafiltration apparatus under 40 psi pressure of air (concentration under nitrogen may lead to partial deoxygenation with consequent conversion to ferric myoglobin), over a PM-10 membrane. Chromatography on DEAE-Sephadex. The procedure presented is essentially that of Yamazaki et al. 10 except that DEAE-Sephadex has been found to be superior to the DEAE-cellulose originally used. Purification of myoglobin and simultaneous separation of oxy from ferric myoglobin is achieved on a column (5 × 50 cm) of DEAE-Sephadex A-50 (Pharmacia) equilibrated with 0.02 M Tris-HCl buffer, pH 8.4, containing 1 mM EDTA. A solution of myoglobin that has been partially purified on a column of Sephadex G-100 is applied to the column in the same buffer (a buffer 0.1 pH unit more alkaline than the column-equilibrating buffer may be used to assure a narrow band at loading). Usually about 100/~mol (1.8 g) of myoglobin in no more than 100 ml are placed on the column, but up to 250/zmol (4.5 g) may be used. The column is developed at 4° with the column-equilibrating buffer, at a flow rate of I00 ml/hr. Cytochrome c is not adsorbed and emerges first. A symmetrical peak of ferric myoglobin emerges next, followed closely by oxymyoglobin. Hemoglobin (if it has not previously been removed) remains at the top of the column. If the myoglobin applied has been chromatographed previously on a column of Sephadex G-100, no detectable protein other than myoglobin is eluted in the region of the oxymyoglobin peak. A criterion of purity is the molar extinction coefficient at 280 nm; 30.6, 36.6, and 37.5 x 103 M -1 cm -~, for sperm whale aquoferric myoglobin, oxymyoglobin and carbon monoxymyoglobin, respectively. ~4 The corresponding extinctions at the Soret maxima are: 164 (409nm), 128 (418nm), and 187 × 103 (423 nm) M -~ cm -1. In practice the ratio of absorbance at the Soret maximum to that at the ultraviolet maximum is monitored. For sperm whale myoglobin, these are 5.36, 3.50, and 5.00, respectively) 4 Myoglobins from different sources may require slightly different conditions, and very slight changes in pH will affect the separation of oxymyoglobin from ferric myoglobin. In general myoglobin is more strongly adsorbed at more alkaline pH. 2-Amino-2-methyl-l,3-propanediol (AMPD) has been suggested as a buffer because at 25 ° the pK (pK 8,80) is farther into the alka-
[2]
PREPARATION OF MYOGLOBINS
33
line range than that o f Tris (pK 8.07). '1 Indeed columns operated with 0.03 M A M P D buffer, p H 8.6, give very satisfactory purification of myoglobin and separation of oxy from ferric myoglobin. AMPD suffers from the disadvantage of high cost. It must be recrystallized from ethanol before use, and it is not suitable for freezing or long-term storage o f some myoglobin solutions. Shikama and his colleagues have advocated returning to the use of DEAE-cellulose with a gradient o f decreasing pH. lz,la C h r o m a t o g r a p h y o n C M - C e l l u l o s e . M i c r o h e t e r o g e n e i t y . As shown by E d m u n d s o n and Hirs, 15 otherwise homogeneous preparations of ferric myoglobin from sperm whale, seal, or other sources may be separated into 5 - 1 2 fractions by chromatography on C M - c e l l u l o s e Y '15-17 by electrophoresis, 17 or by isoelectric fractionation. These fractions have identical amino acid compositions, T M identical kinetics in their reactions with ligands, TM and the same conformation as determined by X-ray diffraction. They differ only in charge. 1~ T h e y are not artifacts o f the isolation procedure or of proteolysis occurring in the muscle homogenate because very pure myoglobin prepared by rapid isolation and purified by a z i n c - e t h anol procedure 7 shows a pattern of microheterogeneity identical to that of samples prepared by classic salt fractionation. A likely explanation is that the differences may lie in variation of the amide content of glutamic and aspartic residues. A procedure for isolating these fractions on a preparative scale is given by Rothgeb and Gurd in this series TM (see also H a p n e r et al. 8) Isolation of A p o m y o g l o b i n R o m e r o - H e r r e r a et a1.,'9,2° whose objective is the determination of amino acid sequence, have developed a simple procedure in which myoglobin is isolated as ferric myoglobin cyanide; the heme group is removed, and the resulting apomyoglobin is brought to purity by chromatography on CM-cellulose. Skeletal muscle, 500 g, is minced and homogenized in a blender with 1.5 volumes of distilled water containing 2 m M K C N . The homogenate is centrifuged for 30 min at 25,000 g, the supernatant is recovered, and ammonium sulfate is added to 55% saturation. The material is then stirred for t~ A. B. Edmundson and C. H. W. Hirs, J. Mol. Biol. 5, 663 (1962). ~6N. M. Rumen, Acta Chem. Scand. 13, 1542 (1959). ~7M. Z. Atassi, Nature (London) 202, 496 (1964). ~8L. J. Parkhurst and J. LaGow, Biochemistry 14, 1200(1975). ,9 A. E. Romero-Herrera, H. Lehmann, K. A. Joysey, and A. E. Friday, Philos. Trans. R. Soc. London, Ser. B 283, 61 (1978). 20 H. Dene, M. Goodman, and A. E. Romero-Herrera, Proc. R. Soc. London, Ser. B 207, 111 (1980).
34
HEMOGLOBINS AND MYOGLOBINS
[2]
1 hr at 4 ° and centrifuged for 45 min at 43,000 g. The supernatant is dialyzed against distilled water containing 2 mM KCN for 48 hr and then concentrated in an Amicon ultrafiltration unit, using a PM-10 membrane under 40 psi nitrogen pressure, to a final volume of approximately 60 ml. Aliquots of 20 ml are applied to a column of Ultrogel AcA-54 (LKB) (2.6 cm x 180 cm) equilibrated with 50 mM Tris-HC1 buffer, pH 8.5, and 2 mM KCN. The gel filtration is performed at room temperature with a flow rate of 15 ml/hr. This procedure permits the separation of contaminating heavy molecular weight proteins including tetrameric hemoglobin. The eluted myogloblin is concentrated as above. After dialysis for 48 hr against distilled water, the heme group is removed by 1.5% HCI in acetone followed by three washes with cold acetone to eliminate the acid. 21 The precipitated apomyoglobin is dried under a stream of nitrogen. At this stage, 1.8 g of myoglobin has been recovered from 500 g of muscle. Further purification of the apomyoglobin is achieved by column chromatography on Whatman CM-23 microgranular CM-cellulose (2.6 cm x 12 cm) equilibrated with a solution of 8 M urea, 5 mM Na~HPO4, pH 6.5, 1 mM dithiothreitol (starting buffer). Six hundred milligrams of apomyoglobin are dissolved in 12 ml of this buffer and applied to the column. The column is developed with a linear gradient formed by 500 ml of starting buffer and 500 ml of elution buffer. The latter is prepared under the same conditions as the former but contains 40 mM Na~HPO4. Chromatography proceeds at room temperature with a flow rate of 100 ml/hr, and the effluent is monitored at 280 nm. Small quantities of contaminating proteins are eluted with the void volume. The apomyoglobin-containing fractions are pooled, the salts are removed by gel filtration using sephadex G-25, and the apomyoglobin is recovered by lyophilization. Crystallization S p e r m Whale Ferric Myoglobin. The best crystals 22 are obtained from solutions containing l ml of 6% solution of salt-free purified myoglobin and 2.5 ml of 100% solution of ammonium sulfate, pH 5.75, without added buffer. Crystallization is complete after about I day at room temperature. S p e r m Whale Deoxymyogiobin. Crystallization~3"~4was carded out in a nitrogen-filled glove box. A 50-fold molar excess of sodium dithionite was added to a salt-free 6% solution of ferric myoglobin; this was then mixed with a saturated solution of ammonium sulfate containing 0.01 M sodium EDTA and adjusted to pH 5.75 with 5 vol% of a 4 M solution of
21M. L. Anson and A. E. Mirsky,J. Gen. Physiol. 13, 469 (1930). 22T. Takano,J. Mol. Biol. 110, 537 (1977). 22C. L. Nobbs, H. C. Watson, and J. C. Kendrew,Nature (London) 209, 339 (1966). 24T. Takano,J. Mol. Biol. 110, 569 (1977).
[2]
PREPARATION OF MYOGLOBINS
35
K2HPO4 and NaH~PO4, pH 6.5. The best crystals grow in 72.4% saturated ammonium sulfate solutions at room temperature. Sperm Whale Oxymyoglobin. Deoxymyoglobin crystals were washed (to remove dithionite) and exposed to air immediately before mounting for X-ray analysis, z5 Oxymyoglobin crystals, apparently not suitable for Xray diffraction, have been obtained by adding 3-5 volumes of saturated ammonium sulfate, pH 5.7, to solutions of sperm whale oxymyoglobin, 3.4 mM protein, in water, apparently at 40.7 Seal Myoglobin. The terminal amino group of seal and horse myoglobin is glycine, whereas in sperm and finback whale it is valine. For this reason, and because the crystal form is different, it was of interest to determine the structure of seal myoglobin,z6 A saturated solution of ammonium sulfate was added to a salt-free solution of myoglobin, 5%, to the point of incipient turbidity. Best results were obtained in the range pH 5.6 to 7.3. 4,27 Tuna Myoglobin. This, the only fish myoglobin investigated intensively, was first crystallized by Rossi Fanelli and Antonini. z8 Partial amino acid sequence 29 and X-ray diffraction analysis to 6/k resolutiona° are available. Red muscle from yellowfin tuna, Thunnus albacares, was homogenized with an equal volume of water and fractionated with ammonium sulfate, 70-95% saturation; the myoglobin fraction was separated from hemoglobin and other proteins on a column of Sephadex G-75 in 0.05 M Tris-phosphate buffer, pH 8. Final purification was on a column of CMSephadex C-50 (2.5 × 34 cm) eluted with 0.015 M Tris-phosphate buffer, pH 7.1, 31 or on a column ofDEAE-Sephadex A-50 (2.4 × 85 cm, capacity up to 1.0 g of myoglobin) eluted with a linear gradient formed from 700 ml each of 0.025 M Tris-HCl, pH 8.7, and 0.05 M Tris-HCl, pH 7.2. 3°'a~Crystallization was from 70% saturated ammonium sulfate, pH 5.5-7.0, 1030 mg of protein per milliliter,z°'32 Other Species. The first crystalline myoglobins studied by X-ray diffraction were those of horse a and finback whale, a3 Kendrew et al. z4 crys25 S. E. V. Phillips, Nature (London) 273, 247 (1978). 26 H. Scouloudi and E. N. Baker, J. Mol. Biol. 126, 637 (1978). zr H. Scouloudi, Proc. Roy. Soc. London, Set. A. 258, 181 (1960). 2s A. Rossi Fanelli and E. Antonini, Arch. Biochem. Biophys, 58, 498 (1955). 29 R. H. Price, D. A. Watts, and W. D. Brown, Cornp. Biochem. Physiol. 62B, 481 (1979). z0 E. E. Lattman, C. E. Nockolds, R. H. Kretsinger, and W. E. Love, J. Mol. Biol. 60, 271 (1971). 3~ G. J. Fosmire and W. D. Brown, Comp. Biochem. Physiol. 5511, 293 (1976). 32 R. H. Kretsinger, J. Mol. Biol. 38, 141 (1968). aa j. C. Kendrew and P. J. Pauling, Proc. R. Soc. London, Ser. A 237, 255 (1956). a4 j, C. Kendrew, R, G. Panfish, J. R. Marrack, and E. S. Orlans, Nature (London) 174, 946 (1954).
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HEMOGLOBINS AND MYOGLOBINS
[2]
tallized, and examined crystallographically, myoglobins from 16 species including whales, porpoises, seals, tortoise, penguin, and carp. Horse oxymyoglobin crystallizes from 87% saturated ammonium sulfate solution at 4°. 1° Analytical Separation of Myoglobin from H e m o g l o b i n Three methods are available to separate myoglobin obtained from small samples of tissue from contaminating hemoglobin. In each case it is best to extract the tissue by the procedure of Schuder e t a1.,35 described here. A sample of muscle is minced coarsely with scissors, frozen, and ground to a fine powder in a procelain mortar cooled in Dry Ice or liquid nitrogen. Exactly 1 g of the frozen powder is weighed into a centrifuge tube and leached with 9.25 ml of 0.01 M potassium phosphate buffer, pH 7.0, containing 5 mM EDTA for 5 min at ice temperature, with occasional stirring. The sample is centrifuged at 20,000 g for 10 rain to obtain a crystal clear supernatant; 10.0 ml of this solution contains the myoglobin from 1.0 g of muscle. (It is assumed that the tissue myoglobin becomes distributed in the total water of the extract including that contributed by the tissue and that from added buffer. The water content of muscles from different sources is very nearly the same.) Very little cytochrome c is extracted. Hemoglobin is subsequently separated from myoglobin in the extract by subunit exchange chromatography on columns of a-fl dimers of human hemoglobin A immobilized on Sepharose 4B,3~ or by absorption on p-mercuribenzene-coupled Sepharose CI-4B, 3e or by gel filtration on columns (2 × 40 cm) of Sephadex G-100 (superfine). Useful absorbance maxima, largely free from interference by cytochrome c, arc: ferrous myoglobin, 434 nm, E = 114 x 103; and carbon monoxide myoglobin, 579nm, • = 12.2 × 10a cm -1 M - '. 35 Photometric errors may be minimized by taking the difference spectrum: carbon monoxide (dithionite) minus reduced (dithionite), taking A•, 422 minus 438 nm = 181 x 103 c m - ' M-'.
Conversion of Ferric to Oxymyoglobin Ferredoxin, in a coupled enzyme system, reduces ferric hemoglobin, myoglobin, or Icghemoglobin. 37 The reducing system (see Dilodo, this volume [4]) is added to the aerated myoglobin solution at room tempera3~S. Schuder, J. B. Wittenberg,B. Haseltine, and B. A. Wittenberg,Anal. Biochem. 92, 473 (1979). a6 D. J. Goss and L. J. Parkhurst, J. Biochem. Biophys. Methods 3, 315 (1980). 3r A. Hayashi, T. Suzuki, and M. Shin, Biochim. Biophys. Acta 310, 309 (1973).
[2]
PREPARATION OF MYOGLOBINS
37
ture. Reduction is complete within 20 min, and solutions of oxyhemoproteins so prepared are stable for about 1 week. Stock enzyme solutions or suspensions are used as purchased without further dilution. Glucose-6phosphate dehydrogenase and ferredoxin NADP reductase are apparently inactivated when stored with EDTA and should be stored in EDTA-free buffers .38 Myoglobin may also be reduced with dithionite. A column of Sephadex G-10 or G-25 or of BioGel P2-P6 (Bio-Rad) about 20 cm high is flushed with oxygen-free buffer, pH 7.5-9.0. A layer about 1 cm high, of an anaerobically prepared solution of dithionite, 10-100 mM, in the same buffer is carefully pipetted above the surface of the Sephadex bed, beneath a protecting column of anaerobic buffer, and is run into the column. A solution of ferric myoglobin is next applied to the column. It overtakes and passes the band of dithionite. Ferrous myoglobin so generated becomes oxygenated by residual oxygen in the column or as the effluent emerges into the air. The products of reaction of dithionite with oxygen are deleterious to proteins, and oxymyoglobin, so prepared, is less stable than that prepared by enzymic reduction. Storage Myoglobin may be stored at 4° in the form of a paste in saturated ammonium sulfate for periods of 2 years or longer without apparent alteration. 4 Solutions of ferric or oxymyoglobin in 0.05 M phosphate buffer, pH 7.5, may be stored for 5 years at liquid nitrogen temperature without apparent change. Solutions of myoglobin in 0.05 M Tris-HC1 buffer, pH 8, are stable to repeated freezing and thawing. We have no experience with prolonged storage in Tris buffer. We believe on limited experience that morpholinoethanesulfonic acid (MES) buffers are inappropriate for prolonged storage of myoglobin. Myoglobin in solution in AMPD buffer, pH 8.0-8.5, is destroyed by repeated freezing and thawing. I n v e r t e b r a t e Myoglobins Gastrophilus Myoglobin. Intracellular hemoglobins occur twice among insects: in the tracheal organs of a few aquatic notonectids (Hemiptera) 39 and in the tracheal organ of a larval stage of the bot fly Gastrophilus intestinalis, 4° which lives as a parasite in the stomach of the horse. This dimeric protein is noteworthy for the low value of the oxygen dissociation constant. 41
38F. C. Mills, M. L. Johnson, and G. K. Ackers, Biochemistry 15, 5350 (1976). 39G. Bergstrom,Insect Biochern. 7, 313 (1977). 40D. Keilin and Y. L. Wang, Biochem J. 40, 855 (1946). 41C. F. Phelps, E. Antonini, M. Brunori, and G. Kellet,Biochem. J. 129, 891 (1972).
38
HEMOGLOBINS AND MYOGLOBINS
[2]
Gastrophilus larvae are available from September to June, with local seasonal variations in abundance. Horse stomachs are opened at the knackery. Larvae found attached to the mucosa of the cardiac region of the stomach are returned to the laboratory at room temperature in moist filter paper or on slices of liver. The red tracheal organ, attached to the postabdominal spiracular plate, is dissected free and is carefully washed free of blood. The blood contains phenols and phenol oxidases, which otherwise would destroy the hemoglobin. The tracheal organs are ground with sand in 0.01 M phosphate buffer, pH 7.5, and centrifuged; the clear red supernatant solution is fractionated with ammonium sulfate. The fraction precipitating from 70-85% saturation is retained. This is dissolved in a minimum volume of phosphate buffer, pH 7.5, and further purified by passage over a column of Sephadex G-75. Chironomus Hemolymph Hemoglobin. The structure of monomeric hemoglobin (fraction III) from larvae of the fly Chironomus has been determined at 1.4/~ resolution for carbon monoxy, deoxy, aquoferric, cyanofen-ic, 42 and oxy 43 forms. These structures, together with the amino acid sequence, 44 are central to present discussions of oxygen binding to myoglobin.43-46 Hemolymph, separated from 20 g of frozen and thawed larvae, was fractionated on a column of Sephadex G-75 (5 x 43 cm) developed with a solution containing 0.1 M sodium acetate and 0.035 M magnesium chloride, pH 6.8-7.1.47 (Alternatively, larvae are homogenized in 1.5 volumes of 5 mM phosphate buffer, pH 8.6, containing 1.5 mM potassium cyanide, and the supernatant from the homogenate is fractionated with ammonium sulfate.) The hemoglobin monomer fractions from nine such columns were pooled to give 860 mg of hemoglobin. This was concentrated by precipitation with ammonium sulfate (to 85% saturation) dissolved in water, desalted, and further fractionated on a column of DEAE-cellulose (3 × 30 cm) at pH 8.6. A basic protein, fraction I, which isnot adsorbed, was eluted with 300 ml of a solution of 0.005 M potassium phosphate buffer, pH 8.6, containing 1.5 mM potassium cyanide. Monomer fractions III and IV were then separated by elution with a gradient formed between the starting buffer and a solution of 0.01 M potassium phosphate buffer, pH 8.0, containing 0.04 M potassium chloride and 1.5 mM potassium cyanide. A yield of 220 mg of monomer fraction III was obtained. 4~ W. Steigemann and E. Weber, J. Mol. Biol. 127, 309 (1979). 43 E. Weber, W. Steigemann, T. A. Jones, and R. Huber, J. Mol. Biol. 120, 327 (1978). 44 R. Huber, O. Epp, W. Steigemann, and H. Formanek, Eur. J. Biochem. 19, 42 (1971). 45 G. Steffens, G. Buse, and A. Wollmer, Eur. J. Biochem. 72, 201 (1977). 4e A. Wollmer, G. St¢ffens, and G. Buse, Eur. J. Biochem. 72, 207 (1977). 4r V. Braun, R. R. Crichton, and G. Braunitzer, Hoppe-Seyler's Z. Physiol. Chem. 349, 197
(1968).
[2]
PREPARATION OF MYOGLOBINS
39
After a final chromatographic purification on a column of CM-cellulose, crystals were obtained from 3.75 M ammonium sulfate, pH 7.0. 44 These were transferred to 3.75M phosphate buffer for X-ray diffraction studies. The cyanide derivative was formed by exposing the crystals of aquoferric hemoglobin to 0.01 M potassium cyanide, the deoxy derivative by exposing the crystals to 0.1 M dithionite in the same buffer, and the carbon monoxide derivative by subsequent exposure to gaseous carbon monoxide. Oxyhemoglobin crystals were obtained by exposing crystals of aquoferric hemoglobin to a mixture of hydrogen sulfide (a reducing agent) and oxygen in the proportion 1:6. 43 Glycera Monomeric Hemoglobin. Hemoglobin from the coelomic erythrocytes of the polychaete annelid Glycera dibranehiata is an interacting mixture of monomeric and oligomeric proteins. Monomeric hemoglobin, separated from the mixture, lacks the distal histidine characteristic of most hemoglobins and myoglobins and has in its place a leucine residue. 48 Optical spectra 4a'5° ligand binding affinities, 5° kinetics, 5~,51a amino acid sequence, 48 and X-ray crystallographic structure 52 are available. The oxygen combination and dissociation rate constants of monomer fraction I are the largest yet reported for a hemoglobin. 5~a This hemoglobin, together with Aplysia myoglobin is often used in studies of the role of the distal histidine residue. Glycera dibranchiata, blood worms, may be purchased from bait companies in New England (e.g., Maine Bait Company, Newcastle, Maine,). Worms are anesthetized in 10% ethanol in 3% sodium chloride, slit, and allowed to bleed in to 3% NaCI. Hemoglobin-containing cells of the coelomic fluid are collected and washed three times with 3% NaCI by low speed centrifugation (about 400 g). The washed cells are lysed by the addition of two volumes of distilled water, and cell residues are subsequently removed by centrifugation at 20,000 g for 20 rain. One-tenth volume of 1 M Tris-HC1 buffer, pH 8.42, is added, and a portion, 10-20 ml, of the clear supernatent is fractionated on a column of Sephadex G-75 (2.4 × 40 cm), equilibrated with 0.1 M Tris buffer, pH 8.42. 49 Two peaks are resolved. The slower moving component is the desired monomer. The monomer fraction from 100 worms in 35 ml of 0.01 M potassium phosphate buffer, pH 6.8, is applied to a 5 × 50 cm CM-Sephadex C-50 column previously equilibrated with 0.01 M potassium phosphate buffer, 48 T. Imamura, T. O. Baldwin, and A. Riggs, J. Biol. Chem. 247, 2785 (1972). 49 S. N. Vinogradov, C. A. Machlik, and L. L. Chao, J. Biol. Chem. 245, 6533 (1970). 5o B. Seamonds, R. E. Forster, and P. George, J. Biol. Chem. 246, 5391 (1971). 51 B. Seamonds, J. A. McCray, L. J. Parkhurst, and P. D. Smith, J. Biol. Chem. 251, 2579 (1976). 51a L. J. Parkhurst, P. Sima, and D. J. Goss, Biochemistry 19, 2688 (1980). 5a E. Padlan and W. E. Love, J. Biol. Chem. 249, 4067 (1974).
40
HEMOGLOBINS AND MYOGLOBINS
[2]
pH 6.8, saturated with carbon monoxide; the column is developed at 4°. The flow rate is 45 ml/hr. After approximately 8 hr, four bands are observed on the column. Hemoglobins I and II (bottom and second band) are separated by - 10 cm; hemoglobins III and IV (top of column) are separated by - 3 cm. The column gel is carefully forced out of the column by gentle air pressure, and the bands are cut out. The proteins are eluted from the gel by repouring the gel into smaller columns and washing with 0.2 M potassium phosphate, pH 7.5. This also serves to concentrate the proteins. The recovery of total monomers is about 0.3-0.4/zmol of hemoglobin per worm. Fractions I and II show very rapid kinetics. Fraction II probably corresponds to that hemoglobin for which the sequence is rep o r t e d . 51a
Crystals of Glycera carbon monoxyhemoglobin were grown at 4° from 2.4 to 2.8 M solutions of ammonium sulfate, 0.06 M potassium phosphate, pH 6.8. 52 Aplysia Myoglobin. Myoglobin from the buccal muscles 53 and nerves 54 of the gastropod mollusc Aplysia limacina (Mediterranean) or A. californica (Pacific Coast) lacks a histidine residue distal to the heme. 55 It is unfolded reversibly by solvent change or high temperature.56.5r Optical spectra, 58 EPR spectra, 59 ligand affinity,5s'6° kinetics, 61 amino acid sequence, 55 and X-ray diffraction structure 62 are available. Buccal masses [approximately 2 g per animal; 150 ftmol (A. californica) or 500/zmol (A. limacina) of myoglobin per kilogram wet weight] are homogenized in 20 volumes of 0.05 M Tris-HC1 buffer, pH 8.0, containing I mM EDTA. The homogenate is centrifuged at 20,000 g for 10 min and fractionated by the addition of solid ammonium sulfate between 50 and 90% saturation, keeping the pH near 8 by the dropwise addition of ammonia. The dark red precipitate is collected by centrifugation, taken up in 1-2 volumes of buffer, and again fractionated with ammonium 53 A. Rossi Fanelli and E. Antonini, Biokhimiya 22, 336 (1957). 54 B. A. Wittenberg, R. W. Briehl, and J. B. Wittenberg, Biochem. J. 96, 363 (1965). 55 L. Tentori, G. Vivaldi, S. Carta, M. Marinucci, A. Massa, E. Antonini, and M. Brunori, Int. J. Pept. Protein Res. 5, 187 (1973). so M. Brunori, E. Antonini, P. Fasella, J. Wyman, and A. Rossi Fanelli, J. Mol. Biol. 34, 497
(1968). 5~ M. Brunori, G. M. Giacometti, E. Antonini, and J. Wyman, J. Mol. Biol. 63, 139 (1972). 5s A. Rossi Fanelli, E. Antonini, and D. Povoledo, in "I. U. P. A. C. Symposium on Protein Structure" (A. Neuberger, ed.), p. 144 (1958). 59 G. Rotilio, L. Calabrese, G. M. Giacometti, and M. Brunori, Biochim. Biophys. Acta 236, 234 (1971). a0 G. M. Giacometti, A. Da Ros, E. Antonini, and M. Brunori, Biochemistry 14, 1584 (1975). e~ B. A. Wittenberg, M. Brunori, E. Antonini, J. B. Wittenberg, and J. Wyman, Arch. Biochem. Biophys. 111, 576 (1965). as T. L. Blundell, M. Brunori, B. Curti, M. Bolognesi, A. Coda, M. Fumagalli, and L. Ungaretti, J. Mol. Biol. 97, 665 (1975).
[2]
PREPARATION OF MYOGLOBINS
41
sulfate. The precipitate is dissolved in a minimum volume of the same buffer (or 0.05 M potassium phosphate buffer, pH 7.5, containing 1 mM EDTA) and separated from high molecular weight substances by chromatography on a column of Sephadex G-75 (superfine) or G-100 (superfine) (Pharmacia). At this stage the protein is approaching homogeneity and is usually more than 90% oxymyoglobin. Crystals suitable for X-ray diffraction 62 were obtained by dialyzing a solution of the ferric protein, about 15 mg/ml, in a solution of 3.5 M ammonium sulfate, 0.05 M phosphate buffer, pH 7.2, against a solution of 3.8 M ammonium sulfate in the same buffer. Identical crystals were obtained by vapor diffusion by leaving the same protein solution in equilibrium with 4 M ammonium sulfate, pH 7.2. Other Annelid and Molluscan Myoglobins. Monomeric and dimeric myoglobins, some of which show cooperative oxygen binding, have been isolated from many annelids 63-6~ and molluscs. 65"66In general, apart from ligand affinity, little is known about these proteins. Purification is essentially by the procedure described for Aplysia myoglobin. The amino acid sequence of a dimeric myoglobin from the readily available gastropod mollusc Busycon caniculatum has been reported. 67 Ascaris Hemoglobin. Body walls of the nematode Ascaris lumbricoides, an intestinal parasite of pigs, contain two electrophoretically and chromatographically distinct oxygen-binding hemoproteins that differ in their affinity for ligands. 6s'69 The more abundant of these has been purified r°-Tz and is of interest because of the extraordinarily high oxygen affinity resulting from very slow dissociation of oxygen. 7a It differs from myoglobin in that the molecular weight is about 3 5 , 0 0 0 , 71 and in optical spectra 71 and ligand binding kinetics. TM Ascaris are chilled at the slaughterhouse, slit, and washed free of adhering perienteric fluid. (Ascaris is a potent allergen. Use of surgical mask 63 C. P. Mangum, in "Adaptation to Environment" (R. C. NeweU, ed.), p. 191. Butterworth, London, 1976. R. E. Weber, in "Physiology of Annelids" (P. J. Mill, ed.), p. 369. Academic Press, New York, 1978. 65 R. C. Terwilliger, Am. Zool. 20, 53 (1980). 66 K. R. n . Read, in "Physiology of Mollusca" (K. M. Wilbur and C. M. Yonge, eds.), Vol. 2, p. 209. Academic Press, New York, 1966. 67 A. S. Bonner and R. A. Laursen, FEBS Lett. 73, 201 (1977). K. Hamada, T. Okazaki, R. Shukuya, and K. Kaziro, J. Biochem. (Tokyo) 52, 290 (1962). 6~ K. Hamada, T. Okazaki, R. Shukuya and K. Kaziro, J. Biochem. (Tokyo) 53, 479 (1963). 7o H. E. Davenport, Proc. R. Soc. London, Set. B 136, 255 (1949). 7, T. Okazaki, B. A. Wittenberg, R. W. Briehl, and J. B. Wittenberg, Biochim. Biophys. Acta 140, 258 (1967). 72 j. B. Wittenberg, F. J. Bergersen, C. A. Appleby, and G. L. Turner, J. Biol. Chem. 249, 4057 (1974). 73 Q. H. Gibson and M. H. Smith, Proc. R. Soc. London, Ser. B 163, 206 (1965).
42
HEMOGLOBINS AND MYOGLOBINS
[2]
is advised.) Body walls (266 g) were homogenized in two volumes of 20 mM phosphate buffer, pH 7.4, containing 1 mM EDTA, and the supernatant solution was separated by centrifugation. The pale red supernatant, pH 6.7, was adjusted to pH 7.2 and fractionated by the addition of solid ammonium sulfate. The fraction precipitating between 56 and 80% saturation was retained. The precipitate was dissolved in a minimum volume of 10 mM phosphate buffer, pH 7.0, and was separated from high molecular weight substances by chromatography on a column of Sephadex G-75 (5 × 52 cm) in the same buffer. The yield at this stage was about 30/xmol of hemoglobin per kilogram of body walls. Final purification was by chromatography on a column of Whatman DE-52 microgranular DEAE-cellulose (2.5 x 20 cm), which had been equilibrated with 5 mM phosphate buffer pH 7.0. Elution was with a gradient of buffer concentration from 5 to 20 mM. The product is minimally 90% oxyhemoglobin. Trematode Hemoglobins. Monomeric hemoglobins have been purified from Fasciolopsis buski, the intestinal fluke of man and pigs, ~4 and from Dicrocoelium dendriticum, a fluke that infests the hepatic ducts of sheep. 75 The oxygen affinity of the latter hemoglobin is notably great. Paramecium Myoglobin. Myoglobin from the ciliate protozoan Paramecium aurelia 76-7a has been purified by fractionation with ammonium sulfate, 50-65% saturation, followed b y chromatography on Sephadex G75 and, finally, by chromatography on Sephadex G-50. 7s Five components apparently differing only in their isoelectric points were separated electrophoretically. Yeast Hemoglobin. Intracellular hemoglobin is abundant only in particular strains of yeast. It has been isolated from Candida mycoderma. 79 The molecule has two prosthetic groups, FAD and protoheme, apparently both attached to a single polypeptide chain, molecular weight 50,000. Except for the contribution by the flavin, the optical spectrum is that of a typical hemoglobin. The oxygen affinity is extraordinarily great, apparently as a result of very rapid combination with oxygen. Isolation involves mechanical disruption of the cells, ammonium sulfate fractionation between 45 and 60% saturation at pH 7, and, finally, chromatography on DEAE-cellulose in 20 mM acetate buffer pH 6.2. 74 G. D. Cain, J. Parasitol. 55, 311 (1969). 75 p. E. Tuchschmid, P. A. Kunz, and K. J. Wilson, Eur. J. Biochem. 88, 387 (1978). 76 D. Keilin and J. F. Ryley, Nature (London172, 451 (1953). r7 M. H. Smith, P. George, and J. R. Preer, Arch. Biochem. Biophys. 99, 313 (1962). 7a E. Steers and R. H. Davis, Comp. Biochem. Physiol. 6211, 393 (1979). 79 R. Oshino, J. Asakura, K. Takio, N. Oshino, and B. Chance, Eur. J. Biochem. 39, 581 (1973).