[methods in enzymology] hemoglobins volume 76 || [5] preparation and properties of apohemoglobin and...

72 HEMOGLOBIN DERIVATIVES [5]

TABLE III g VALUES OF THE ELECTRON

PARAMAGNETIC RESONANCE SPECTRA OF HUMAN Hb HEMICHROME~

Derivative gl g2 g3

H type 2.80 2.26 1.67 C type 3.15 2.25 1.25 B type 2.95 2.26 1.47 P type 2.41 2.25 1.93

a Data are from Peisach and Blumberg. 25

1-5] P r e p a r a t i o n a n d P r o p e r t i e s o f A p o h e m o g l o b i n a n d R e c o n s t i t u t e d H e m o g l o b i n s

By FRANCA ASCOLI, MARIA ROSARIA R o s s I FANELLI ,*

and ERALDO ANTONINI

The specific, noncovalent interactions occurring between the heme group and globin, the apoprotein moiety of hemoglobins, are responsible for the assembly and the functional and structural properties of these proteins.

The study of hemoglobins and myoglobins containing prosthetic groups that differ from natural protoheme [iron (II) protoporphyrin IX] in the substituents of the porphyrin ring and/or in the central metal atom has been helpful in the elucidation of these interactions. Since the product obtained by recombination of the natural heme with human apohemoglobin is identical with human hemoglobin in its physicochemical and structural properties, 1-4 many studies have been devoted to the isolation of native globin from the hemoglobin under study and its reconstitution with modified hemes.

In this chapter, the procedures for the isolation of globin in the native state and for its reconstitution with metalloporphyrins are described in de- taft.

* Deceased March 6, 1981. Friends and colleagues express their sorrow. 1 A. Rossi Fanelli and E. Antonini, Arch. Biochem. Biophys. 80, 299 (1959). 2 A. Rossi Fanelli, E. Antonini and A. Caputo, Biochim. Biophys. Acta 35, 93 (1959). 3 E. Antonini and Q. H. Gibson, Biochem. J. 76, 534 (1960). 4 E. Antonini, M. Brunori, A. Caputo, E. Chiancone, A. Rossi Fanelli, and J. Wyman, Bio-

chim. Biophys. Acta 79, 284 (1964). Copyright © 1981 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 76 All rights of reproduction in any form reserved. ISBN 0-12-181976-0

[5] APOHEMOGLOBIN AND RECONSTITUTED HEMOGLOBINS 73

Preparat ion of Globin

Globins are isolated from the corresponding hemoproteins by removal of the heme moiety at acid pH, under controlled experimental conditions. The quality of the globin samples obtained can be checked by titration experiments with hemin (ferric protoheme) and by studying the properties of the reconstituted hemoproteins.

There are two known methods for the preparation of globin from the holoprotein; both are based on the decreased affinity of the heme for globin at acid pH values. The method described by Rossi Fanelli et al. 5,6 em- ploys acid-acetone at low temperature to split the heme group from the globin, which precipitates and is subsequently redissolved in water. A second method, first described by Teale 7 and recently applied to the isolation of globins from a number of hemoproteins, is based on heme extraction in methyl ethyl ketone at acid pH; in this case the apoprotein remains dissolved in the aqueous layer. Further purification of the globin is ac- complished similarly in both methods.

Oxy-, carbonyl-, or methemoglobin can be used as starting materials for the isolation of globin. Their preparation is described in this volume [4]. In the case of unstable globins, only the use of the ferric derivatives has given satisfactory results.

A c i d - A c e t o n e M e t h o d

The method requires the use of acid-acetone (prepared by adding 2.5 ml of 2 M HC1 to 1 liter of analytical grade acetone) and of carefully desalted hemoglobin solutions. The optimum protein concentration for complete removal of the heme group is - 1 mM on a heme basis, which corresponds to about 1.6% for monomeric or tetrameric hemoglobins.

A 5-ml aliquot of the hemoglobin solution, cooled at 4 °, is added drop- wise and under vigorous stirring into a 250-ml glass centrifuge bottle containing 200 ml of acid-acetone cooled at - 20°; the addition is completed in about 2 min. At the end of the addition, the acid-acetone is slightly reddish, and globin precipitates as a white material. The suspension is centrifuged at 5000 rpm in a centrifuge precooled at - 2 0 °. The heme-containing su- pernatant is carefully removed by suction, and the precipitate, if colored, is resuspended in 50 ml of acid-acetone cooled at - 2 0 ° and separated again by centrifugation at the same temperature. The final colorless precipitate of globin is resuspended in - 4 ml of cold distilled water and dialyzed at 4 ° against 500 ml of 0.1% sodium bicarbonate and then against 500 ml of 0.05 M phosphate buffer at pH 7.4. The solution is then centri-

5 A. Rossi FaneUi, and E. Antonini, Arch. Biochem. Biophys. 77, 478 (1958). s A. Rossi Fanelli, E. Antonini and A. Caputo, Biochirn Biophys. Acta 30, 608 (1958). 7 F. W. J. Teale, Biochim. Biophys. Acta 35, 543 (1959).


fuged at 6000 rpm, 4 °, to remove any precipitate of denatured globin and may be used directly for titration or reconstitution experiments. Alterna- tively, the solution may be dialyzed exhaustively against water, centrifuged as above, and freeze-dried. In some cases the precipitated globin obtained after treatment with acid-acetone is insoluble in water and can be dissolved only by raising the pH to 9.5-10 with small aliquots of 0.1 M NaOH; the solution is then neutralized by addition of diluted HCI or by two dialysis steps against 1.6 mM sodium bicarbonate and 0.01 M phosphate buffer at pH 7.0. With this procedure, native globin was prepared from Aplysia myoglobin a and Chironomus thummi thummi hemoglobin. 9

The yield is usually 90% for mammalian tetrameric or monomeric hemoglobins. The acid-acetone method has also been used for the isolation of globins from hemoglobin chains 1° and from high molecular weight hemoproteins, erythrocruorins, and chlorocruorins. H'~z

Methyl Ethyl Ketone Method

This method was introduced by Teale 7 for the isolation of human apohemoglobin and reexamined by Yonetani. 13 A small-scale experiment should be carried out first to determine the minimal acidity required for the complete splitting of the heme group and its extraction in the organic phase; for most hemoproteins this corresponds to pH values between 2.4 and 2.8.

To this end, 0.2-0.3-ml aliquots of a cold ferric hemoglobin solution ( - 1 mM on a heme basis) are placed in small test tubes and brought to different pH values, ranging between 2.3 and 3.0, by addition of small amounts of cold 0.1 M HC1. Cold methyl ethyl ketone (0.2-0.3 ml) is im- mediately added to each tube, and, after vigorous shaking, the solutions are left to stand in the cold for 1 min; this period of time usually allows good separation of the two phases. The highest pH value at which complete extraction of the ferric heme occurs is chosen for the large-scale experiment, which is performed as follows at 4 °. An aliquot of the cold ferric hemoglobin solution in water or dilute buffer, at the same concentration as in the small-scale experiment, is added to an equal volume of cold methyl ethyl ketone in a separatory funnel; the pH is adjusted to the ap- propriate value by rapid addition of the required amount of cold 0.1 M

a A. Rossi Faneili and E. Antonini, Biokhimiya (Moscow) 22, 336 (1957). 9 G. Amiconi, E. Antonini, M. Brunori, H. Formaneck and R. Huber, Eur. J. Biochem. 31,

52 (1972). 10 y . K. Yip, M. Waks and S. Beychok, J. Biol. Chem. 247, 7237 (1972). H E. Antonini, A. Rossi Fanelli and A. Caputo, Arch. Biochem. Biophys. 97, 343 (1962). ~2 E. Chiancone, M. R. Rossi Fanelli, F. Ascoli, P. Vecchini, and E. Antonini, Biochim.

Biophys. Acta 535, 150 (1978). ta T. Yonetani, J. Biol. Chem. 242, 5008 (1967).


HCI under vigorous stirring. After standing 1 min in the cold, the aqueous phase, which increases slightly in volume owing to the partial solubility of methyl ethyl ketone in water, is separated from the organic layer. Any residual heme in the aqueous phase can be removed by extraction with approximately the same volume of cold methyl ethyl ketone; after ex- haustive dialysis against various changes of cold distilled water, the pale- yellow solution of globin is finally freed from any precipitated material by centrifugation. The apoprotein solution is then lyophilized or dialyzed against the desired buffer in the cold. In some cases ~4 further purification from any denatured material can be achieved by passage through a Sepha- dex G-75 column. Yields are comparable to those obtained with the acid- acetone method. The procedure involving methyl ethyl ketone is particu- larly suitable for the isolation of globins, which are per se quite unstable, like the apohemoglobins from trout, 14,15 since the protein remains always in solution and irreversible denaturation favored by precipitation is avoided.

Physicochemical Properties of Globin

Lyophilized globin (from human hemoglobin or myoglobin) is a white powder that can be kept for months in the refrigerator without any loss of its physicochemical properties; it is very soluble in water and in dilute neutral buffers. Its aqueous solution is not very stable even in the cold and tends to become turbid with time.

The absorption spectrum of globin is a typical protein spectrum with a band centered at 280 nm. The EmM a t 280 nm is 12.7 for human apohemoglobin 1°'1~ and 15.9 for human apomyoglobin17; it is higher for globins with higher tryptophan content. ~2'~4 The presence of an absorption band in the Soret region indicates incomplete removal of heme ( - 1% residual heme gives a A405 :A280 ratio of - 0.1).

Human apohemoglobin at neutral pH is substantially dissociated into dimers; this is indicated by a sedimentation coefficient of 2.8, which corresponds to a molecular weight of 40,000. TM The sedimentation constant at neutral pH of apomyoglobin from various sources is similar to that of myoglobin. ~° Globins obtained from high molecular weight erythrocruorins have a molecular weight of about 45,000.12

14 G. Falcioni, E. Fioretti, B. Giardina, I. Ariani, F. Ascoli and M. Brunori, Biochemistry 17, 1229 (1978).

15 E. Fioretti, F. Ascoli and M. Brunori, Biochem. Biophys. Res. Commun. 6, 1169 (1976). 16 Q. H. Gibson and E. Antonini, J. Biol. Chem. 238, 1384 (1963). 17 S. C. Harrison and E. R. Blout, J. Biol, Chem. 240, 299 (1964). 18 A. Rossi Fanelli, E. Antonini and A. Caputo, J. Biol. Chem. 234, 2906 (1959). is N. Rumen and E. Appella, Arch. Biochem. Biophys. 97, 128 (1962).

7 6 HEMOGLOBIN DERIVATIVES [5]

Optical rotatory dispersion and circular dichroism measurements of globins from various sources 1r,2° indicate that these molecules have

50% of the residues in a-helical conformation; globins from trout, which are fairly unstable, have an a-helical content of about 40%14; these values are significantly lower than those calculated for the corresponding hemoproteins, which range between 70 and 75%.

Titration of Globin with Hemin

The quality of globin samples can be checked by determining the yield in reconstituted holoprotein obtained upon titration with hemin. A typical experiment may be performed as follows: Hemin ( - 6 mg) is dissolved in a minimal volume of 0.1 N NaOH and diluted with water to 5 ml to a final concentration of - 1.9 mM (¢mM = 50 at 390 nm in borate 2%21); this solution is prepared just before use. A known volume (approximately 2.5 ml) of globin solution ( -20 / zM on a momomer basis) in phosphate buffer 0.2 M, pH 7.0 is placed in the sample cell of the spectrophotometer; the reference cell contains the same volume of buffer. Both cells are thermo- statted at 15 °. Aliquots of the hemin solution (5/.d) are added to both cells, and the absorption is read at 400 nm after each addition; the optical density increase in the sample cell is proportional to the amount of reconstituted ferric hemoprotein. In human hemoglobin the final absorption is attained rapidly; in other hemoproteins the recombination reaction is complex, in that it has a fast phase with a half-time of a few seconds and a slow phase with a half-time of the order of minutes. The titration is complete when the absorption remains constant upon further addition of hemin. Sharp end points are usually obtained for globin isolated from monomeric and tetrameric hemoglobins; the ratio between the molar concentration of the hemin and the globin at the end point gives the fraction of native globin present in the sample.

R e c o n s f i t u t i o n E x p e r i m e n t s

Hemes with different substituents in the porphyrin r i n g 4'14"15"22-~4

and/or metals different from iron, such as cobalt, 25-27 copper, 2s zinc, 29'3° and manganese, 31"32 have been used in the reconstitution experi-

20 S. Beychok, in "Polyaminoacids" (G. D. Fasman, ed.), pp. 318-321. Dekker, New York, 1967.

21 E. Antonini and M. Brunori, "Hemoglobin and Myoglobin in Their Reaction with Lig- ands." North-Holland Publ., Amsterdam, 1971.

22 D. W. Seybert, K. Moffat and Q. H. Gibson, Biochim. Biophys. Res. Commun. 63, 43 (1975).

~3 M. Sono and T. Asakura, J. Biol. Chem. 249, 7087 (1974).


ments. 33-35 A recent review has discussed the properties of metal-substi- tuted hemoglobins and myoglobins. 36

The preparation of a reconstituted hemoglobin involves the reaction of globin at pH around 7 with the metalloporphyrin and purification of the reconstitution product by ion-exchange chromatography; the reaction can be performed under anaerobic or aerobic conditions. Depending on the characteristics of the metalloporphyrin, reconstitution is carried out in aqueous solution or in aqueous pyridine solution. Hereafter, examples of the various procedures used are reported. The experimental conditions, such as pH, chromatographic column, and buffer, refer to the preparation and purification of reconstitution products from human apohemoglobin; modifications may be necessary in the case of other hemoproteins. It should be emphasized that careful purification of the reconstitution product is a necessary prerequisite to the study of its functional and structural properties. References to the preparation and properties of various reconstituted hemoproteins are given in the table. Special mention should be made of recent proton NMR studies on some reconstituted hemoglobins and myoglobins that have been used to determine the orientation of the porphyrin within the heme pocketY

Reconstitution in Aerobic Conditions

This method is applied in most cases, such as reconstitution experiments with proto-, meso-, and deuterohemes and with zinc, copper, and manganese porphyrins. All steps are performed at 4 °. A 10-ml aliquot of 0.3-0.5 mM globin solution (on a monomer basis) in 0. ! M phosphate buffer at pH around 6.5 is cooled at 4°; a 10% excess over the stoichio-

24 M. Sono and T. Asakura, J. Biol. Chem. 250, 5227 (1975). 25 B. M. Hoffman and D. H. Petering, Proc. Natl. Acad. Sci. U.S.A. 67, 637 (1970). 26 T. Yonetani, H. Yamamoto and G. V. Woodrow III, J. Biol. Chem. 249, 682 (1974). 27 E. Di Iorio, E. Fioretti, I. Ariani, F. Ascoli, G. Rotilio and M. Brunori, FEBS Lett. 105,

229 (1979). 28 T. L. Fabry, C. Simo and K. Javaherian, Biochirn. Biophys. Acta 1611, 118 (1968). 29 S. F. Andres and M. F. Atassi, Biochemistry 9, 2268 (1970). 3o j. I. Leonard, T. Yonetani and J. B. Collis, Biochemistry 13, 1461 (1974). 31 T. Yonetani and T. Asakura, J. Biol. Chem. 244, 4580 (1969). 3z T. Yonetani, H. R. Drott, J. S. Leigh, J. H. Reed, M. R. Waterman and T. Asakura, J.

Biol. Chem. 245, 2998 (1970). 33 T. Asakura, this series Vol. 52, p. 447. 34 D. M. Scholler, M. R. Wang and B. M. Hoffman, this series, Vol. 52, p. 487. 35 S. Sano, in "The Porphyrins" (D. Dolphin, ed.), Voi. VIIB, pp. 377-402. Academic

Press, New York, 1979. 36 B. M. Hoffman, in "The Porphyrins" (D. Dolphin, ed.), Vol. VIIB, pp. 403-444. Aca-

demic Press, New York, 1979. 37 G. La Mar, D. L. Budd, D. B. Viscio, K. M. Smith and K. C. Langry, Proc, Natl. Acad.

Sci. U.S.A. 75, 5755 (1978).

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metric amount of the metalloporphyrin (for protoheme, 2.2-3.5 mg) in a minimal amount ( -0 .2 ml) of 0.01 M NaOH is added to the globin solution under gentle stirring. After a few minutes any precipitated, colored material (denatured reconstitution products) is removed by centrifugation at 6000 rpm for 10 min. The solution is freed from the unreacted heme by passage through a Sephadex G-25 column equilibrated in 0.01 M phosphate buffer at pH 6.3; the protein is then adsorbed on a CM-cellulose column equilibrated with the same buffer; after washing the column in 0.01 M phosphate buffer, pH 6.3, the reconstitution product is eluted with 30 mM phosphate buffer at pH 7.0 and dialyzed against the desired buffer.

The reconstituted protein obtained with this procedure contains the central atom in the porphyrin ring in its higher oxidation state. In the case of iron-containing porphyrins, ferric hemoglobins are formed and the study of the functional properties of the reconstituted products requires reduction to the ferrous derivatives. This is usually done by addition of a small amount of sodium dithionite to the ferric protein; the deoxygenated ferrous protein solution is then freed from dithionite by passage through a Sephadex G-25 column equilibrated with a buffer at neutral pH; the oxy- genated hemoprotein thus obtained can be used directly for the physicochemical and functional studies.

Reconstitution in Anaerobic Conditions

Anaerobic conditions are preferred in reconstitution experiments where formation of the reconstituted protein having the metal in its higher oxidation state is not desired. This is the case of earthworm erythro- cruorin, where direct reconstitution in the ferrous state favors the formation of high molecular weight molecules. 12 The same method is applied routinely in the reconstitution experiments with cobalt-containing por- phyrinsY ~ Cobalt (II) hemoglobins and myoglobins have received much attentions in recent years because they bind oxygen reversibly and are amenable to EPR studies. Their preparation involves the use of Co(II) porphyrins that are prepared in situ by reduction of the Co(III) porphyrins; the reaction is conducted under anaerobic conditions and does not require reduction of the cobaltic reconstituted protein.

The experiments are performed at 4 °. Aliquots of 5 ml of 0.3-0.5 mM globin solution (on a monomer basis) in 0.1 M phosphate buffer at pH 7.0 are placed in a modified tonometer with a side ampulla (Fig. 1) that contains a few grains of sodium dithionite. The ampulla is connected to a test tube containing 1.2 equivalents of the ferric heme dissolved in a minimal volume ( - 0 . 2 ml) of 0.01 M NaOH or of the cobaltic porphyrin dissolved in 0.5 ml of 50% aqueous pyridine. The solutions are deoxygenated by a gentle flow of cold nitrogen gas, which is passed over the surface of the


o N2

Metallo - Globin ~ porphyrin

Stirrer

FIG. 1. Scheme of the apparatus used in the reconstitution experiments under anaerobic conditions. The arrows indicate the direction of the nitrogen gas flow: d, deaeration; t, m, transfer of the metalloporphyrin solution into the ampulla containing dithionite and its mix- ing with the globin solution. For further details see text.

globin solution and bubbled into the porphyrin one. If desired, the globin solution may be gently stirred with a magnetic stirrer. When deoxygena- tion is completed, the heme solution is forced into the side ampulla and mixed with dithionite by reversing the gas flow. Subsequently the reduced metalloporphyrin is mixed with the globin solution and the mixture is freed from excess reagents by passage through a Sephadex G-25 column equilibrated with 10 mM phosphate buffer at pH around 6.0. The reconstituted protein is purified on a CM-cellulose column, elution is carried out with 30-100 mM phosphate buffer at pH 7.0. The fraction containing the reconstituted hemoprotein is collected and can be stored at 4 ° for a few days without loss of the physical and functional properties.

[methods in enzymology] hemoglobins volume 76 || [5] preparation and properties of apohemoglobin and...

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