- Email: [email protected]
[9] Preparation of hybrid hemoglobins with different prosthetic groups
[9]
PREPARATION
OF
HYBRID
HEMOGLOBINS
113
have also been made by fitting the oxygen binding equilibrium curves of valency hybrids. 2°'21'z9This analysis has not given a definite answer on the validity of the models, as it relies on the assumption that hybrids are rigorI I ously identical to the intermediates (do2fl~eoxyh and ~tgdeoxyjuOeI2t~lI /3II x , which are formed in the course of oxygen binding to deoxyHb. This fact is perhaps not strictly true as, for instance, kinetic evidence indicates that artificially prepared intermediates could exist in two different conformations in slow equilibrium. 9 Acknowledgments I wish to thank Drs. R. Banerjee and Y. Henry for their helpful comments on the manuscript. This work was supported by grants from the Centre National de ia Recherche Scientifique (E.R. 157), the Drlrgation Grnrrale/t la Recherche Scientifique et Technique, and the Institut National de la Santd et de la Recherche Mddicale. z9 A. P. Minton, Science 184, 577 (1974).
[9] P r e p a r a t i o n By
of Hybrid Hemoglobins Prosthetic Groups
with Different
MASAO IKEDA-SAITO, TOSHIRO INUBUSHI, and TAKASH1 Y O N E T A N I
An artificial hybrid hemoglobin, a(X)d3(Yh, carrying heme X in the o~ subunits and heme Y in the B subunits and its complementary form, a(Y)d3(X)2, can be prepared by mixing stoichiometric amounts of the isolated a(X) -sH and fl(y)-SH chains, or or(Y)-sH and fl(X) -su chains. As tabulated in the table, these kinds of hybrids afford the unique opportunity to investigate numerous properties of each subunit, a and fl, in a tetrameric hemoglobin molecule by a number of different spectroscopic methods, including optical, EPR, NMR, and Mfssbauer techniques, as well as by equilibrium and kinetic measurements of ligand binding, z-11 By use of these hybrid hemoglobins, the functional and structural properties of the a and/3 subunits in tetrameric hemoglobin are to be characterized in relation to the quaternary structure of hemoglobin. 1 M. Ikeda-Saito, H. Yamamoto, and T. Yonetani, J. Biol. Chem, 252, 8639 (1977). z M. Ikeda-Saito, T. Inubushi, G. G. McDonald, and T. Yonetani, J. Biol. Chem. 253, 7134 (1978). 3 K. Imai, M. Ikeda-Saito, H. Yamamoto, and T. Yonetani, J. Mol. Biol. 138, 635 (1980). 4 M. Ikeda-Saito and T. Yonetani, J. Mol.Biol. 138, 845 (1980). 5 L. J. Parkhurst, G. Geraci, and Q. H. Gibson, J. Biol. Chem. 245, 5131 (1970). METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
1 14
HEMOGLOBIN
CHAINS
~" . ~
~-~
o~ .~ .<
~
~.~
AND
~
~
-=--=
-
~= ~
HYBRID
[9]
MOLECULES
~,c= ~
"~
~
o ~
~,-,.~
~..,
~
~-~,
~
0
.=-~.~
o
~
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o ,..1 o o
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=.
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Z ~
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0
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[9]
P R E P A R A T I OOF N HYBRID HEMOGLOBINS
1 15
As a typical example, the m e t h o d o f preparation of F e - C o hybrid hemoglobins ~ ( C o ) # ( F e ) 2 , where the cz and/3 subunits carry cobaltous protoporphyrin I X and ferrous p r o t o p o r p h y r i n IX, respectively, and its c o m p l e m e n t a r y hybrid hemoglobin, a ( F e ) # ( C o ) 2 , will be described. Criteria for the evaluation of the quality o f the preparations will also be discussed. Since success in the preparation of the hybrid hemoglobins depends largely on the isolation of the a and/3 chains, a preparative method which has p r o v e d to be convenient for the isolation o f chains from iron hemoglobin (FeHb) and cobalt hemoglobin (CoHb) will be described in d e t a i l ) 2 This very same method can also be applied to prepare the isolated chains of m e s o F e H b , m e s o C o H b , and 57Fe-enriched F e H b . Therefore, by an appropriate combination of hemoglobin, one can easily prepare p r o t o - m e s o hybrid H b , o~(protoheme)~(mesoheme)2 and a(mesoheme)z/3(protoheme)2, and 56Fe-S7Fe hybrid hemoglobins a(S7Fe)a/3(S6Fe)2 and o~(S*Fe)z/3(57Fe)2 . Precautions The methods of preparation of artificial hemoglobins containing unnatural h e m e s or other metalloporphyrins can be found elsewhere in this series. 1z-15 The properties of these artificial hemoglobins have to be c h e c k e d before preparing the isolated chains. The oxygen equilibrium curve m e a s u r e m e n t is the m o s t effective method to evaluate the quality of the preparation. An automatic equilibrium curve recording system, such as the Imai apparatus, 16 which enables one to c o v e r a wide range of saturation, is quite suitable for this purpose. The criteria that have been used for F e H b and C o H b in this laboratory are briefly described. P r o t o C o H b , which is p r e p a r e d f r o m a p o H b and cobalt p r o t o p o r p h y r i n IX by the m e t h o d of Yonetani e t al., 17 has to exhibit the following oxygen
6 y. Sugita, S. Bannai, Y. Yoneyama, and T. Nakamura, J. Biol. Chem. 247, 6092 (1970). 7 T. Nakamura, Y. Sugita, and S. Bannai, J. Biol. Chem. 248, 4119 (1973). s H. Yamamoto and T. Yonetani, J. Biol. Chem. 249, 7964 (1974). 9 N. Makino and Y. Sugita, J. Biol. Chem. 253, 1174 (1978), 10T. Inubushi and T. Yonetani, unpublished findings. 11P.-W. Lau and T. Asakura, J. Biol. Chem. 254, 2595 (1979). 12 M. Ikeda-Saito, H. Yamamoto, K. Imai, F. J. Kayne, and T. Yonetani, J. Biol, Chem. 252, 620 (1977). 13 Section II of this volume. 14T. Asakura, this series, Vol. 52 p. 447. 15D. M. Scholler, M.-Y. R. Wang, and B. M. Hoffman, this series, Vol. 52, p. 487. 16K. Imai, H. Morimoto, M. Kotani, H. Watari, and M. Kuroda, Biochim. Biophys. Acta 200, 189 (1970). 17T. Yonetani, H. Yamamoto, and G. V. Woodrow, J. Biol. Chem. 249, 682 (1974).
116
HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[9]
equilibrium propertieslS: nmax, maximum slope of the Hill plots, about 2.1; Pro, median oxygen pressure, about 70 Tort, in 0.1 M phosphate buffer, pH 7.4 at 15°. The Met(Co 3+) content should be less than 4% as measured by spectrophotometry.17 The light-absorption spectra in the visible region should be measured at several degrees of saturation during the deoxygenation and reoxygenation cycle. The spectral changes have to be completely reversible upon deoxygenation and reoxygenation with sharp isosbestic points at 526, 541, and 564 nm. A preparation of CoHb that meets these criteria is ready for chain separation and for other experiments. Since FeHb's with unnatural hemes are usually prepared in the Met(Fe 3+) form, the metal ion has to be reduced to the ferrous state for study of their functional properties. This can be carried out easily by use of the enzymic MetHb-reducing system described by Hayashi e t al. 10 in place of sodium dithionite or sodium borohydride. It is highly recommended that one should practice the reconstitution of Hb from apoHb and iron protoporphyrin IX before attempting the prepartion of Hb containing unnatural iron porphyrins. Reconstituted FeHb from apoHb and protoherain, reduced by the enzymic reducing system, should meet the following criteria. The oxygen equilibrium curves have to be the same as those of native FeHb over a wide range of saturation (1-99.5%), e.g., they should be characterized by the following values in 0.1 M phosphate buffer, pH 7.4 a t 25°: nmax, a b o u t 3.0, Pro, 7 T o r t . T h e M e t ( F e 3 + ) H b c o n t e n t s h o u l d
be less than 3% as determined by the method of Kilmartin e t al. ~° Although the preparation of hemoglobin chains and their treatment the regeneration of free -SH groups is described in this volume [7], procedure will be given that is currently used in this laboratory for preparation of the hybrids with different heroes. All manipulations of moglobin or its chains are done at 4 °.
for the the he-
Preparation of - p M B Chains This is a modification of the method proposed by Geraci et al. 21 F c H b or C o H b (2-3 m M as hcmc and about 20 ml) in the oxy form is allowed to react overnight with p-hydroxymercuribenzoate (pMB) (10-fold cxccss per H b tctramer) at p H 6. I in the prcsence of 0.25 M NaCl. The next 18 K. Imai, T. Yonetani, and M. Ikeda-Saito, J. Mol. Biol. 109, 83 (1979). 19 A. Hayashi, T. Suzuki, and M. Shin, Biochim. Biophys. Acta 310, 309 (1973). Also see this volume [27]. 20 j. V. Kilmartin, K. Imai, R. T. Jones, A. R. Faruqui, J. Fogg, and J. M. Baldwin, Biochim. Biophys. Acta 534, 15 (1978). ~1 G. Geraci, L. J. Parkhurst, and Q. H. Gibson, J. Biol. Chem. 244, 4664 (1969).
[9]
PREPARATION OF H Y B R I D H E M O G L O B I N S
117
morning, the pMB-Hb solution is centrifuged at 10,000 rpm for 10 min to remove any precipitate and is then applied to a 4.5 cm x 30 cm Sephadex G-25 (fine) column equilibrated with 10 mM phosphate buffer, pH 8.0. The eluate is loaded on a well packed 4.5 cm x 15 cm DEAE-ceUulose column (Whatman DE-52) equilibrated with 10 mM phosphate buffer, pH 8.0. As the column is washed with the same buffer, a broad dilute band of Od-pMB chain comes off, while the/3 -pMB chain sticks at the top of the column. The a -~B chain is collected and is ready for removal of mercury by the method described later. After the a -pMBchain has been eluted, the column is washed with 30 mM phosphate buffer, pH 8.3, to elute the undissociated Hb tetramer. Usually the amount of undissociated Hb tetramer is very small and a small moving band is recognized in the column by careful visual observation. Automatic systems, such as fraction collector and a spectrophotometric monitor, are not necessary. While the column is washed with this buffer, the band of the fl-pMB chain diffuses slightly. After the undissociated tetramer is washed off from the column, the/~-pMB chain is eluted with 0.1 M phosphate buffer, pH 7.0. Removal of pMB The a-pMB or /3-pMB chains are incubated with 20 mM dithiothreitol (Sigma) in the presence of 5/zM catalase (Sigma C-100) for 2 hr. The mixture of t~-pMB, dithiothreitol, and catalase is passed through a 10 cm x 15 cm Sephadex G-25 (fine) column equilibrated with 10 mM phosphate buffer, pH 6.6. The colored fraction containing the a -sn chain is loaded on a 2.5 cm x 3 cm column of CM-cellulose (Whatman, CM-52) equilibrated with l0 mM phosphate buffer, pH 6.6. The a -sH chain sticks at the top of the column. The column is washed with the same buffer, then the a -sH chains is eluted with 50 mM phosphate buffer, pH 7.4. The mixture of fl-pMB, dithiothreitol, and catalase is passed through a l0 cm x 15 cm Sephadex G-25 column (fine) equilibrated with 7 mM Tris buffer, pH 8.6. The colored fraction containing the/3 -sn chain is loaded on a 2.5 cm x 7 cm column of DEAE-cellulose (Whatman, DE52) equilibrated with 7 mM Tris buffer, pH 8.6. The/3 -sH chain sticks at the top of the column. The column is washed with the same buffer, then the/3 -sn chain is eluted with 50 mM phosphate buffer, pH 7.4. An approximately equal amount of the a -sn and/3 -sn chain is obtained, and the yield is typically about 65%. MetHb is not formed during this procedure. Although the isolated chains of FeHb or CoHb can be stored at 0° for a couple of days, the recombination of the partner subunits into tetramers should be done at the earliest convenience. After the buffer is changed from phosphate to Tris buffer, such as 0.05 M, pH 7.4, the FeHb or CoHb chains (either proto- or meso-) can be stored at 77°K for a
l 18
H E M O G L O B I N CHAINS A N D HYBRID M O L E C U L E S
[9]
considerably longer period of time without MetHb formation. In this case, the solution should be divided into appropriate vials to avoid repeated freezing and thawing with each use. The presence of catalase during the regeneration of the - SH groups is essential to obtain good results, especially for the isolation of CoHb chains. When regeneration of the - SH groups of the isolated chains of CoHb is performed with dithiothreitol in the absence of catalase, a green compound is gradually formed and turbidity develops even under a nitrogen atmosphere. The ct chain is found to be more liable than the/3 chain during the dithiotreitol treatment, and the yield is less than 20%. The isolated chains of FeHb are more stable than those of CoHb during the regeneration of the - SH groups, but a small amount of oxidized material is formed in both oLand/3 chains during the dithiotreitol incubation without catalase. Oxidation of the chains can be prevented by use of the carbon monoxide form of FeHb as reported by Kilmartin e t a l . 2z This method, however, cannot be applied to CoHb, since CoHb does not combine with carbon monoxide. 17 The use of the carbon monoxide form greatly increases the yield of the preparation of the isolated chains or hybrids of relatively unstable hemoglobins such as deuteroFeHb. The method described above does not require any special apparatus or reagent, and the isolated chains are obtained in a day after the reaction of pMB with Hb is completed. Recombination of the Subunit Chains The ot-sH and j~-SH chains are incubated with dithiothreitol (2 mg/ml) for 1 hr in the presence of 10 p.M catalase before recombination. The t~-sH chains are mixed with a 1.2-fold excess of/3 -sH chains. After about 2 hr, the mixture is passed through a 2.5 cm x 20 cm column of Sephadex G25 (fine) equilibrated with 10 mM phosphate buffer, pH 6.5. The mixture is then applied to a 2.5 cm x 5 cm CM-cellulose column (Whatman, CM52) equilibrated with l0 mM phosphate buffer, pH 6.5. The excess/3 -s~ chain is washed off by the same buffer. The hybrid Hb tetramer is eluted with 50 mM phosphate buffer, pH 7.0 in about 2 mM per metal. The excess fl-SH chain can be removed by the following method as well. The mixture of a -sH and fl-sn chains is passed through a 2.5 cm x 20 cm column of Sephadex G-25 (fine) equilibrated with l0 mM phosphate buffer, pH 6.9, then passed through a 2.5 cm x 5 cm DEAE-cellulose column (Whatman DE-52) equilibrated with the same buffer. The excess /3-sn chain is removed. ~2 j. V. Kilmartin, J. A. Hewitt, and J. F. Wooton, J. Mol. Biol. 93, 203 (1975).
[9]
P R E P A R A T I OOF N HYBRID HEMOGLOBINS
i 3
i
I 19
I - 99.9
-
2
99
~TI>_ I
90
0
~ 20
50_ °
-I
I0
-2
I -
0
I
I I
2
log pO 2 FIG. l. The Hill plots for oxygen equilibrium of FeHb reconstituted from the isolated a -sH and/3 -sH chains, R-FeHb, (O-G-O) and native FeHb (O-11-O) in 0.1 M phosphate buffer, pH 7.4 at 15°. Hemoglobin concentrations were 60/xM as heme.
The preparation can be stored at 77°K as described in the preceding section. E v a l u a t i o n of t h e P r e p a r a t i o n s The purity o f the isolated chains can be easily checked by electrophoresis and pMB titrations as well as by oxygen equilibrium measurements. F o r the electrophoresis e x p e r i m e n t s any commercially available electrophoresis apparatus, such as the G e l m a n Separatic cellulose acetate gel electrophoresis, can be used. The r e m o v a l o f m e r c u r y can be checked b y the s p e c t r o p h o t o m e t r i c titration o f the preparation with PMB as described b y Boyer. 23 Especially for the/3 chains, the r e m o v a l o f p M B f r o m the/3 chain is checked by oxygen equilibrium m e a s u r e m e n t s . In fact the oxygen affinity of the/3-SH chain is usually a couple of times higher than that o f the/3-pMB chain. The /3-SH chain of F e H b exhibits a / ' 5 o value of 23 p. D. Boyer, J.
A m . Chem. Soc.
76, 4331 0954).
120
HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[9]
about 0.3 Torr whereas the fl--pMB chain of FeI-Ib has a Ps0 value of about 5 Torr at 24 ° in 0.1 M phosphate buffer, pH 7.0. The most straightforward way to evaluate the quality of the FeHb reconstituted from a -sa and fl-sa chains is to compare oxygen equilibrium properties with those of native FeHb under various anion and pH conditions. Figure I illustrates the Hill plots of the oxygen equilbrium for FeHb reconstituted by the method described here and native FeHb in 0.1 M phosphate buffer, pH 7.4 at 15°. The oxygen equilibrium curves were measured by the Imai automatic recording system TMinterfaced to a PDP 11/40 computer for the real-time data acquisition and manipulation. 24 The two curves agree very well over a wide range of saturation, showing that the FeHb reconstituted by this method has exactly the same oxygen equilibrium characteristics as native FeHb. Similar agreements in the Hill plots for oxygen equilibrium have been obtained between pH 6.5 and 7.9 in 0.1 M phosphate buffer, as well as in the presence of DPG, IHP, and/or 0.1 M NaCl in 0.05 M bis-Tris buffer, pH 7.4. Thus, the homotropic and heterotropic effects in Hb are not altered by the separation and recombination of the a and/3 chains by the procedure described above. As already mentioned, before proceeding to the preparation of hybrid hemoglobins it is most important to practice the separation and recombination of the isolated chains using native FeHb until one is skilled enough to produce reconstituted FeHb with the same functional properties as those of native FeHb. Alternative
Methods
Waterman and Yonetani 25 prepared apoHb isolated chains, oL(apo) -SH and/3(apo) -sH. By combination of them with manganese protoporphyrin IX, a(Mn a+) and /3(Mna+) were prepared. These manganese isolated chains were used to prepare manganese-iron hybrid hemoglobins a(Mn3+)~fl(Fe2+) z and a(Fe2+)2fl(Mn3+)2. SemiHb, a(apo)d3(Fe2+)2 were also prepared by the use of a(apo) -sH chain26; the complementary hybrid, a(Fe2+)~(apo)~, has to be obtained by the method of Winterhalter and Deranleau. 2~ Details on the preparation of semihemoglobins are given by Cassoly in this volume [10]. Isolated chains containing fluorescent porphyrins were also prepared by the use of apoHb isolated chains. 2s Zinc-iron hybrid hemoglobins, 24 K. Imai and T. Yonetani, Biochim. Biophys. Acta 490, 164 (1977). 2~ M. R. Waterman and T. Yonetani, J. Biol. Chem. 245, 5847 (1970). s6 M. R. Waterman, R. Gondko, and T. Yonetani, Arch. Biochem. Biophys. 145, 448 (1971). 27 K. H. Winterhaltcr and D. A. Deranlcau, Biochemistry 6, 3136 (1967). 2s j. j. Leonard, T. Yonctani, and J. B. Callis, Biochemistry 13, 1460 (1974).
[10]
PREPARATION O F S E M I H E M O G L O B I N S
121
a(Zn)~8(Fe)z and a(Fe)~(Zn)z, and porphyrin-zinc hybrid hemoglobins, a(des-Fe)d3(Zn)2 and a(Zn)di(des-Fe)2, were prepared and their fluorescent properties were studied. Acknowledgments The authors thank to Drs. K. Imai and H. Yamamotofor suggestionsand discussions. This workhas been supportedby ResearchGrant HL-14508fromthe NationalHeart, Lung, and BloodInstituteand ResearchGrants PCM79-22841 and OIP 75-10059fromthe National Science Foundation.
[10] P r e p a r a t i o n o f G l o b i n - H e m o g l o b i n Hybrids: Artificially Prepared and Naturally Occurring Semihemoglobins B y R O B E R T CASSOLY
Semihemoglobins are derivatives of human hemoglobin (Hb) that carry half the normal number of hemes, i.e., heme on only one kind of chain, the complementary subunit being heme free. These compounds are called semihemoglobins a, 1-4/3, 5 or y,6 according to the nature of the heme-carrying chain. They can be considered as symmetrical globin-Hb hybrids and represent a good model for the study of the properties of individual chains within a tetramer with limited interference from the other type of subunit. Semihemoglobins are also used to prepare Hb hybrids carrying different prosthetic groups in a straightforward way. The interest in these compounds increased after the discovery of a naturally occurring semihemoglobin a (Hb Gun Hill) whose 13 chain is unable to bind heme owing to deletion of five amino acids. 7 Several pathological semihemoglobins have since been characterized. In this chapter, a general method for preparing semihemoglobins is described. The implications of these hybrids in the study of Hb properties
1 R. Banerjee and R. Cassoly, Biochim. Biophys. Acta 133, 545 (1967). 2 R. Cassoly, E. Bucci, M. Iwatsubo, and R. Banerjee, Biochim. Biophys. Acta 133, 557 (1967). a K. H. Winterhaiter and D. A. Deranleau, Biochemistry, 6, 3136-3143 (1967). 4 K. H. Winterhalter, C. Ioppolo, and E. Antonini, Biochemistry 10, 3790 (1971). R. Cassoly, Biochim. Biophys. Acta 168, 370 (1968). 6 R. Gondko, M. J. Obreska, and M. R. Waterman, Biochem. Biophys. Res. Commun. 56, 444 (1974). 7 T. B. Bradley, Jr., R. C. Wohl, and R. F. Rieder, Science 157, 1581 (1967).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181976-0
PREPARATION
OF
HYBRID
HEMOGLOBINS
113
have also been made by fitting the oxygen binding equilibrium curves of valency hybrids. 2°'21'z9This analysis has not given a definite answer on the validity of the models, as it relies on the assumption that hybrids are rigorI I ously identical to the intermediates (do2fl~eoxyh and ~tgdeoxyjuOeI2t~lI /3II x , which are formed in the course of oxygen binding to deoxyHb. This fact is perhaps not strictly true as, for instance, kinetic evidence indicates that artificially prepared intermediates could exist in two different conformations in slow equilibrium. 9 Acknowledgments I wish to thank Drs. R. Banerjee and Y. Henry for their helpful comments on the manuscript. This work was supported by grants from the Centre National de ia Recherche Scientifique (E.R. 157), the Drlrgation Grnrrale/t la Recherche Scientifique et Technique, and the Institut National de la Santd et de la Recherche Mddicale. z9 A. P. Minton, Science 184, 577 (1974).
[9] P r e p a r a t i o n By
of Hybrid Hemoglobins Prosthetic Groups
with Different
MASAO IKEDA-SAITO, TOSHIRO INUBUSHI, and TAKASH1 Y O N E T A N I
An artificial hybrid hemoglobin, a(X)d3(Yh, carrying heme X in the o~ subunits and heme Y in the B subunits and its complementary form, a(Y)d3(X)2, can be prepared by mixing stoichiometric amounts of the isolated a(X) -sH and fl(y)-SH chains, or or(Y)-sH and fl(X) -su chains. As tabulated in the table, these kinds of hybrids afford the unique opportunity to investigate numerous properties of each subunit, a and fl, in a tetrameric hemoglobin molecule by a number of different spectroscopic methods, including optical, EPR, NMR, and Mfssbauer techniques, as well as by equilibrium and kinetic measurements of ligand binding, z-11 By use of these hybrid hemoglobins, the functional and structural properties of the a and/3 subunits in tetrameric hemoglobin are to be characterized in relation to the quaternary structure of hemoglobin. 1 M. Ikeda-Saito, H. Yamamoto, and T. Yonetani, J. Biol. Chem, 252, 8639 (1977). z M. Ikeda-Saito, T. Inubushi, G. G. McDonald, and T. Yonetani, J. Biol. Chem. 253, 7134 (1978). 3 K. Imai, M. Ikeda-Saito, H. Yamamoto, and T. Yonetani, J. Mol. Biol. 138, 635 (1980). 4 M. Ikeda-Saito and T. Yonetani, J. Mol.Biol. 138, 845 (1980). 5 L. J. Parkhurst, G. Geraci, and Q. H. Gibson, J. Biol. Chem. 245, 5131 (1970). METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
1 14
HEMOGLOBIN
CHAINS
~" . ~
~-~
o~ .~ .<
~
~.~
AND
~
~
-=--=
-
~= ~
HYBRID
[9]
MOLECULES
~,c= ~
"~
~
o ~
~,-,.~
~..,
~
~-~,
~
0
.=-~.~
o
~
o N
om
©
z < .1 < z < ;>,
o ,..1 o o
•~ ""
=.
m
<
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~
~
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Z ~
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0
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0
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[9]
P R E P A R A T I OOF N HYBRID HEMOGLOBINS
1 15
As a typical example, the m e t h o d o f preparation of F e - C o hybrid hemoglobins ~ ( C o ) # ( F e ) 2 , where the cz and/3 subunits carry cobaltous protoporphyrin I X and ferrous p r o t o p o r p h y r i n IX, respectively, and its c o m p l e m e n t a r y hybrid hemoglobin, a ( F e ) # ( C o ) 2 , will be described. Criteria for the evaluation of the quality o f the preparations will also be discussed. Since success in the preparation of the hybrid hemoglobins depends largely on the isolation of the a and/3 chains, a preparative method which has p r o v e d to be convenient for the isolation o f chains from iron hemoglobin (FeHb) and cobalt hemoglobin (CoHb) will be described in d e t a i l ) 2 This very same method can also be applied to prepare the isolated chains of m e s o F e H b , m e s o C o H b , and 57Fe-enriched F e H b . Therefore, by an appropriate combination of hemoglobin, one can easily prepare p r o t o - m e s o hybrid H b , o~(protoheme)~(mesoheme)2 and a(mesoheme)z/3(protoheme)2, and 56Fe-S7Fe hybrid hemoglobins a(S7Fe)a/3(S6Fe)2 and o~(S*Fe)z/3(57Fe)2 . Precautions The methods of preparation of artificial hemoglobins containing unnatural h e m e s or other metalloporphyrins can be found elsewhere in this series. 1z-15 The properties of these artificial hemoglobins have to be c h e c k e d before preparing the isolated chains. The oxygen equilibrium curve m e a s u r e m e n t is the m o s t effective method to evaluate the quality of the preparation. An automatic equilibrium curve recording system, such as the Imai apparatus, 16 which enables one to c o v e r a wide range of saturation, is quite suitable for this purpose. The criteria that have been used for F e H b and C o H b in this laboratory are briefly described. P r o t o C o H b , which is p r e p a r e d f r o m a p o H b and cobalt p r o t o p o r p h y r i n IX by the m e t h o d of Yonetani e t al., 17 has to exhibit the following oxygen
6 y. Sugita, S. Bannai, Y. Yoneyama, and T. Nakamura, J. Biol. Chem. 247, 6092 (1970). 7 T. Nakamura, Y. Sugita, and S. Bannai, J. Biol. Chem. 248, 4119 (1973). s H. Yamamoto and T. Yonetani, J. Biol. Chem. 249, 7964 (1974). 9 N. Makino and Y. Sugita, J. Biol. Chem. 253, 1174 (1978), 10T. Inubushi and T. Yonetani, unpublished findings. 11P.-W. Lau and T. Asakura, J. Biol. Chem. 254, 2595 (1979). 12 M. Ikeda-Saito, H. Yamamoto, K. Imai, F. J. Kayne, and T. Yonetani, J. Biol, Chem. 252, 620 (1977). 13 Section II of this volume. 14T. Asakura, this series, Vol. 52 p. 447. 15D. M. Scholler, M.-Y. R. Wang, and B. M. Hoffman, this series, Vol. 52, p. 487. 16K. Imai, H. Morimoto, M. Kotani, H. Watari, and M. Kuroda, Biochim. Biophys. Acta 200, 189 (1970). 17T. Yonetani, H. Yamamoto, and G. V. Woodrow, J. Biol. Chem. 249, 682 (1974).
116
HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[9]
equilibrium propertieslS: nmax, maximum slope of the Hill plots, about 2.1; Pro, median oxygen pressure, about 70 Tort, in 0.1 M phosphate buffer, pH 7.4 at 15°. The Met(Co 3+) content should be less than 4% as measured by spectrophotometry.17 The light-absorption spectra in the visible region should be measured at several degrees of saturation during the deoxygenation and reoxygenation cycle. The spectral changes have to be completely reversible upon deoxygenation and reoxygenation with sharp isosbestic points at 526, 541, and 564 nm. A preparation of CoHb that meets these criteria is ready for chain separation and for other experiments. Since FeHb's with unnatural hemes are usually prepared in the Met(Fe 3+) form, the metal ion has to be reduced to the ferrous state for study of their functional properties. This can be carried out easily by use of the enzymic MetHb-reducing system described by Hayashi e t al. 10 in place of sodium dithionite or sodium borohydride. It is highly recommended that one should practice the reconstitution of Hb from apoHb and iron protoporphyrin IX before attempting the prepartion of Hb containing unnatural iron porphyrins. Reconstituted FeHb from apoHb and protoherain, reduced by the enzymic reducing system, should meet the following criteria. The oxygen equilibrium curves have to be the same as those of native FeHb over a wide range of saturation (1-99.5%), e.g., they should be characterized by the following values in 0.1 M phosphate buffer, pH 7.4 a t 25°: nmax, a b o u t 3.0, Pro, 7 T o r t . T h e M e t ( F e 3 + ) H b c o n t e n t s h o u l d
be less than 3% as determined by the method of Kilmartin e t al. ~° Although the preparation of hemoglobin chains and their treatment the regeneration of free -SH groups is described in this volume [7], procedure will be given that is currently used in this laboratory for preparation of the hybrids with different heroes. All manipulations of moglobin or its chains are done at 4 °.
for the the he-
Preparation of - p M B Chains This is a modification of the method proposed by Geraci et al. 21 F c H b or C o H b (2-3 m M as hcmc and about 20 ml) in the oxy form is allowed to react overnight with p-hydroxymercuribenzoate (pMB) (10-fold cxccss per H b tctramer) at p H 6. I in the prcsence of 0.25 M NaCl. The next 18 K. Imai, T. Yonetani, and M. Ikeda-Saito, J. Mol. Biol. 109, 83 (1979). 19 A. Hayashi, T. Suzuki, and M. Shin, Biochim. Biophys. Acta 310, 309 (1973). Also see this volume [27]. 20 j. V. Kilmartin, K. Imai, R. T. Jones, A. R. Faruqui, J. Fogg, and J. M. Baldwin, Biochim. Biophys. Acta 534, 15 (1978). ~1 G. Geraci, L. J. Parkhurst, and Q. H. Gibson, J. Biol. Chem. 244, 4664 (1969).
[9]
PREPARATION OF H Y B R I D H E M O G L O B I N S
117
morning, the pMB-Hb solution is centrifuged at 10,000 rpm for 10 min to remove any precipitate and is then applied to a 4.5 cm x 30 cm Sephadex G-25 (fine) column equilibrated with 10 mM phosphate buffer, pH 8.0. The eluate is loaded on a well packed 4.5 cm x 15 cm DEAE-ceUulose column (Whatman DE-52) equilibrated with 10 mM phosphate buffer, pH 8.0. As the column is washed with the same buffer, a broad dilute band of Od-pMB chain comes off, while the/3 -pMB chain sticks at the top of the column. The a -~B chain is collected and is ready for removal of mercury by the method described later. After the a -pMBchain has been eluted, the column is washed with 30 mM phosphate buffer, pH 8.3, to elute the undissociated Hb tetramer. Usually the amount of undissociated Hb tetramer is very small and a small moving band is recognized in the column by careful visual observation. Automatic systems, such as fraction collector and a spectrophotometric monitor, are not necessary. While the column is washed with this buffer, the band of the fl-pMB chain diffuses slightly. After the undissociated tetramer is washed off from the column, the/~-pMB chain is eluted with 0.1 M phosphate buffer, pH 7.0. Removal of pMB The a-pMB or /3-pMB chains are incubated with 20 mM dithiothreitol (Sigma) in the presence of 5/zM catalase (Sigma C-100) for 2 hr. The mixture of t~-pMB, dithiothreitol, and catalase is passed through a 10 cm x 15 cm Sephadex G-25 (fine) column equilibrated with 10 mM phosphate buffer, pH 6.6. The colored fraction containing the a -sn chain is loaded on a 2.5 cm x 3 cm column of CM-cellulose (Whatman, CM-52) equilibrated with l0 mM phosphate buffer, pH 6.6. The a -sH chain sticks at the top of the column. The column is washed with the same buffer, then the a -sH chains is eluted with 50 mM phosphate buffer, pH 7.4. The mixture of fl-pMB, dithiothreitol, and catalase is passed through a l0 cm x 15 cm Sephadex G-25 column (fine) equilibrated with 7 mM Tris buffer, pH 8.6. The colored fraction containing the/3 -sn chain is loaded on a 2.5 cm x 7 cm column of DEAE-cellulose (Whatman, DE52) equilibrated with 7 mM Tris buffer, pH 8.6. The/3 -sH chain sticks at the top of the column. The column is washed with the same buffer, then the/3 -sn chain is eluted with 50 mM phosphate buffer, pH 7.4. An approximately equal amount of the a -sn and/3 -sn chain is obtained, and the yield is typically about 65%. MetHb is not formed during this procedure. Although the isolated chains of FeHb or CoHb can be stored at 0° for a couple of days, the recombination of the partner subunits into tetramers should be done at the earliest convenience. After the buffer is changed from phosphate to Tris buffer, such as 0.05 M, pH 7.4, the FeHb or CoHb chains (either proto- or meso-) can be stored at 77°K for a
l 18
H E M O G L O B I N CHAINS A N D HYBRID M O L E C U L E S
[9]
considerably longer period of time without MetHb formation. In this case, the solution should be divided into appropriate vials to avoid repeated freezing and thawing with each use. The presence of catalase during the regeneration of the - SH groups is essential to obtain good results, especially for the isolation of CoHb chains. When regeneration of the - SH groups of the isolated chains of CoHb is performed with dithiothreitol in the absence of catalase, a green compound is gradually formed and turbidity develops even under a nitrogen atmosphere. The ct chain is found to be more liable than the/3 chain during the dithiotreitol treatment, and the yield is less than 20%. The isolated chains of FeHb are more stable than those of CoHb during the regeneration of the - SH groups, but a small amount of oxidized material is formed in both oLand/3 chains during the dithiotreitol incubation without catalase. Oxidation of the chains can be prevented by use of the carbon monoxide form of FeHb as reported by Kilmartin e t a l . 2z This method, however, cannot be applied to CoHb, since CoHb does not combine with carbon monoxide. 17 The use of the carbon monoxide form greatly increases the yield of the preparation of the isolated chains or hybrids of relatively unstable hemoglobins such as deuteroFeHb. The method described above does not require any special apparatus or reagent, and the isolated chains are obtained in a day after the reaction of pMB with Hb is completed. Recombination of the Subunit Chains The ot-sH and j~-SH chains are incubated with dithiothreitol (2 mg/ml) for 1 hr in the presence of 10 p.M catalase before recombination. The t~-sH chains are mixed with a 1.2-fold excess of/3 -sH chains. After about 2 hr, the mixture is passed through a 2.5 cm x 20 cm column of Sephadex G25 (fine) equilibrated with 10 mM phosphate buffer, pH 6.5. The mixture is then applied to a 2.5 cm x 5 cm CM-cellulose column (Whatman, CM52) equilibrated with l0 mM phosphate buffer, pH 6.5. The excess/3 -s~ chain is washed off by the same buffer. The hybrid Hb tetramer is eluted with 50 mM phosphate buffer, pH 7.0 in about 2 mM per metal. The excess fl-SH chain can be removed by the following method as well. The mixture of a -sH and fl-sn chains is passed through a 2.5 cm x 20 cm column of Sephadex G-25 (fine) equilibrated with l0 mM phosphate buffer, pH 6.9, then passed through a 2.5 cm x 5 cm DEAE-cellulose column (Whatman DE-52) equilibrated with the same buffer. The excess /3-sn chain is removed. ~2 j. V. Kilmartin, J. A. Hewitt, and J. F. Wooton, J. Mol. Biol. 93, 203 (1975).
[9]
P R E P A R A T I OOF N HYBRID HEMOGLOBINS
i 3
i
I 19
I - 99.9
-
2
99
~TI>_ I
90
0
~ 20
50_ °
-I
I0
-2
I -
0
I
I I
2
log pO 2 FIG. l. The Hill plots for oxygen equilibrium of FeHb reconstituted from the isolated a -sH and/3 -sH chains, R-FeHb, (O-G-O) and native FeHb (O-11-O) in 0.1 M phosphate buffer, pH 7.4 at 15°. Hemoglobin concentrations were 60/xM as heme.
The preparation can be stored at 77°K as described in the preceding section. E v a l u a t i o n of t h e P r e p a r a t i o n s The purity o f the isolated chains can be easily checked by electrophoresis and pMB titrations as well as by oxygen equilibrium measurements. F o r the electrophoresis e x p e r i m e n t s any commercially available electrophoresis apparatus, such as the G e l m a n Separatic cellulose acetate gel electrophoresis, can be used. The r e m o v a l o f m e r c u r y can be checked b y the s p e c t r o p h o t o m e t r i c titration o f the preparation with PMB as described b y Boyer. 23 Especially for the/3 chains, the r e m o v a l o f p M B f r o m the/3 chain is checked by oxygen equilibrium m e a s u r e m e n t s . In fact the oxygen affinity of the/3-SH chain is usually a couple of times higher than that o f the/3-pMB chain. The /3-SH chain of F e H b exhibits a / ' 5 o value of 23 p. D. Boyer, J.
A m . Chem. Soc.
76, 4331 0954).
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HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[9]
about 0.3 Torr whereas the fl--pMB chain of FeI-Ib has a Ps0 value of about 5 Torr at 24 ° in 0.1 M phosphate buffer, pH 7.0. The most straightforward way to evaluate the quality of the FeHb reconstituted from a -sa and fl-sa chains is to compare oxygen equilibrium properties with those of native FeHb under various anion and pH conditions. Figure I illustrates the Hill plots of the oxygen equilbrium for FeHb reconstituted by the method described here and native FeHb in 0.1 M phosphate buffer, pH 7.4 at 15°. The oxygen equilibrium curves were measured by the Imai automatic recording system TMinterfaced to a PDP 11/40 computer for the real-time data acquisition and manipulation. 24 The two curves agree very well over a wide range of saturation, showing that the FeHb reconstituted by this method has exactly the same oxygen equilibrium characteristics as native FeHb. Similar agreements in the Hill plots for oxygen equilibrium have been obtained between pH 6.5 and 7.9 in 0.1 M phosphate buffer, as well as in the presence of DPG, IHP, and/or 0.1 M NaCl in 0.05 M bis-Tris buffer, pH 7.4. Thus, the homotropic and heterotropic effects in Hb are not altered by the separation and recombination of the a and/3 chains by the procedure described above. As already mentioned, before proceeding to the preparation of hybrid hemoglobins it is most important to practice the separation and recombination of the isolated chains using native FeHb until one is skilled enough to produce reconstituted FeHb with the same functional properties as those of native FeHb. Alternative
Methods
Waterman and Yonetani 25 prepared apoHb isolated chains, oL(apo) -SH and/3(apo) -sH. By combination of them with manganese protoporphyrin IX, a(Mn a+) and /3(Mna+) were prepared. These manganese isolated chains were used to prepare manganese-iron hybrid hemoglobins a(Mn3+)~fl(Fe2+) z and a(Fe2+)2fl(Mn3+)2. SemiHb, a(apo)d3(Fe2+)2 were also prepared by the use of a(apo) -sH chain26; the complementary hybrid, a(Fe2+)~(apo)~, has to be obtained by the method of Winterhalter and Deranleau. 2~ Details on the preparation of semihemoglobins are given by Cassoly in this volume [10]. Isolated chains containing fluorescent porphyrins were also prepared by the use of apoHb isolated chains. 2s Zinc-iron hybrid hemoglobins, 24 K. Imai and T. Yonetani, Biochim. Biophys. Acta 490, 164 (1977). 2~ M. R. Waterman and T. Yonetani, J. Biol. Chem. 245, 5847 (1970). s6 M. R. Waterman, R. Gondko, and T. Yonetani, Arch. Biochem. Biophys. 145, 448 (1971). 27 K. H. Winterhaltcr and D. A. Deranlcau, Biochemistry 6, 3136 (1967). 2s j. j. Leonard, T. Yonctani, and J. B. Callis, Biochemistry 13, 1460 (1974).
[10]
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121
a(Zn)~8(Fe)z and a(Fe)~(Zn)z, and porphyrin-zinc hybrid hemoglobins, a(des-Fe)d3(Zn)2 and a(Zn)di(des-Fe)2, were prepared and their fluorescent properties were studied. Acknowledgments The authors thank to Drs. K. Imai and H. Yamamotofor suggestionsand discussions. This workhas been supportedby ResearchGrant HL-14508fromthe NationalHeart, Lung, and BloodInstituteand ResearchGrants PCM79-22841 and OIP 75-10059fromthe National Science Foundation.
[10] P r e p a r a t i o n o f G l o b i n - H e m o g l o b i n Hybrids: Artificially Prepared and Naturally Occurring Semihemoglobins B y R O B E R T CASSOLY
Semihemoglobins are derivatives of human hemoglobin (Hb) that carry half the normal number of hemes, i.e., heme on only one kind of chain, the complementary subunit being heme free. These compounds are called semihemoglobins a, 1-4/3, 5 or y,6 according to the nature of the heme-carrying chain. They can be considered as symmetrical globin-Hb hybrids and represent a good model for the study of the properties of individual chains within a tetramer with limited interference from the other type of subunit. Semihemoglobins are also used to prepare Hb hybrids carrying different prosthetic groups in a straightforward way. The interest in these compounds increased after the discovery of a naturally occurring semihemoglobin a (Hb Gun Hill) whose 13 chain is unable to bind heme owing to deletion of five amino acids. 7 Several pathological semihemoglobins have since been characterized. In this chapter, a general method for preparing semihemoglobins is described. The implications of these hybrids in the study of Hb properties
1 R. Banerjee and R. Cassoly, Biochim. Biophys. Acta 133, 545 (1967). 2 R. Cassoly, E. Bucci, M. Iwatsubo, and R. Banerjee, Biochim. Biophys. Acta 133, 557 (1967). a K. H. Winterhaiter and D. A. Deranleau, Biochemistry, 6, 3136-3143 (1967). 4 K. H. Winterhalter, C. Ioppolo, and E. Antonini, Biochemistry 10, 3790 (1971). R. Cassoly, Biochim. Biophys. Acta 168, 370 (1968). 6 R. Gondko, M. J. Obreska, and M. R. Waterman, Biochem. Biophys. Res. Commun. 56, 444 (1974). 7 T. B. Bradley, Jr., R. C. Wohl, and R. F. Rieder, Science 157, 1581 (1967).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181976-0