- Email: [email protected]
[11] Use of gel electrofocusing in the analysis of hybrid hemoglobins
126
HEMOGLOBIN
CHAINS
AND
HYBRID
[11]
MOLECULES
[1 1] U s e o f G e l E l e c t r o f o c u s i n g i n t h e A n a l y s i s o f H y b r i d Hemoglobins By H. FRANKLIN BUNN A considerable proportion of the vast amount of research on hemoglobin has exploited the fact that it is a multisubunit protein. Structural analysis has shown that each subunit of the a ~ 2 tetramer interacts with the other three subunits at well defined sites. Liganded hemoglobin readily dissociates into a aft dimers at only one of the two possible cleavage planes, the so-called a ~ 2 interface. The bonding energy of intramolecular contacts at the a ~ 2 interface is considerably less than at the ad31 interface (see Turner et al., this volume [37]). The a/3 dimer dissociates into monomers only under more drastic solvent conditions, such as extremes of pH. A considerable amount of information about hemoglobin can be obtained by studying the properties of hybrid tetramers in which subunits from parent hemoglobins are reassembled to form new tetramers. Those that are formed from unlike a/3 dimers are called asymmetrical hybrid tetramers (see Perrella and Rossi-Bernardi, this volume [12]). Those that are formed by dissociation at the aJ3~ interface with subsequent reassembly can be designated symmetrical hybrid tetramers. Finally hybrid hemoglobins can consist of tetramers having heroes of differing oxidation states, so-called valency hybrids (see Cassoly, this volume [10]). The use of gelelectrofocusing in the detection and analysis of these different kinds of hybrid hemoglobins is considered in this chapter.
Symmetrical Hemoglobin Hybrids
(o/2X~2 y, o~2Y~2 x)
The formation of hybrid hemoglobins of the type o~2X~2Yo£2Y~2X from parent hemoglobins o~2x~2x a n d 012x~2y requires mixing the parent hemoglobins under conditions that permit dissociation at the o~81 interface. Upon subsequent reassembly, symmetrical hybrid hemoglobins are formed. 1 a2x~h x + a,y~2 y
H÷ , 2 a x + 2 ~ x + 2 a y + 2 ~
~
a x~y + .~¢x
+ a2x/3sx +
a~
Equivalent amounts of the parent hemoglobins (5-50 mg/ml) are mixed, gassed with carbon monoxide, and incubated in 0.1 M acetate buffer, pH 4.5, at 0° for 18 hr. The hemoglobin mixture is then dialyzed against 0.1 M phosphate buffer pH 7.0 and analyzed by zone electro1 E. R. H u e h n s , E. M. S h o o t e r , and G. H. B e a v e n , Biochem. J. 9 1 , 3 3 1 (1964).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
[11]
ANALYSIS OF HYBRID HEMOGLOBINS
127
A Dean
-azP
-o: 2X
HbX-Hb A--
2
an/ a Can X -a2 ~2 _ ocer~ A 2 2
AX
AA
A
X
A
X
+
+
+
+
FIG. 1. Identification of abnormal subunit in a hemoglobin variant by means of h u m a n canine symmetrical hybrids. Lanes 1 and 2 show abnormal (AX) and normal (AA) hemolysates. The variant (Hb X) comprised about 50% of the total hemoglobin. Lanes 3 and 4 show mixtures of purified Hb A and Hb X with canine hemoglobins, maintained at neutral pH. In lanes 5 and 6 the same mixtures were exposed to low pH (4.5), allowing hybrid hemoglobins to form.
phoresis. Because of its high resolution and reproducibility, gel electrofocusing is an ideal analytical system for detecting the hybrid hemoglobins. The preparation of symmetrical hemoglobin hybrids has been useful in the analysis of subunit composition. For example, this approach will determine whether the abnormality in an unidentified hemoglobin variant lies in the a chain or/3 chain. The analysis of human variants is usually done by forming hybrids with canine hemoglobin. Canine hemoglobin is prepared from a hemolysate of dog blood. Since no minor hemoglobin components are present, no further purification is necessary. Canine hemoglobin tends to precipitate during storage. As discussed by Bucci (this volume [7]), the a and/3 chains of human hemoglobin differ widely in charge with isoelectric points of approximately 8 and 6, respectively. In contrast, the subunits of canine hemoglobin differ only slightly in charge. Therefore, the identity of human-canine hybrid hemoglobin can be readily established by a comparison with the electrophoretic properties of the
128
HEMOGLOBINCHAINS AND HYBRID MOLECULES
[11]
parent hemoglobins. Figure 1 shows an example in which the abnormality of an unidentified hemoglobin variant was shown to reside in the/3 chain. As shown in lanes 3 and 4, human Hb A had an isoelectric point very close to that of canine hemoglobin. In contrast, the variant hemoglobin had an isoelectric point about 0.1 pH unit higher than human Hb A and canine hemoglobin. The pattern obtained after hybrids were formed is shown in lanes 5 and 6. The composition of the hybrid hemoglobin bands can be inferred from the relative charges of the human and canine subunits. Thus, the hybrid band with the high isoelectric point must be o/2A/32Can and that with the low isoelectric point is otsCan/32A. The human hemoglobin variant shown in Fig. 1 yields two hybrid bands, one with an isoelectric point identical to that of the hybrid otsA/32 Can and the other having an isoelectric point about 0.1 pH unit higher than a2can/3s g. Therefore, the relative positive charge of this variant can be attributed entirely to the fl chain. Preparative quantities of symmetrical hybrid hemoglobins can be isolated by column chromatography. One of the most interesting experimental applications is in the study of the sickling phenomenon. Benesch et al. 2 have prepared hybrids of the form ax/3 s from mixtures of various human a chain variants with Hb S (otd326 Val). Measurements of the solubility and gelation of these hybrid hemoglobins in comparison to that of native Hb S has provided valuable new information on the a chain contact sites that are involved in the polymerization of Hb S. Thus far there has been excellent agreement between the assignments derived from these experiments and the contact sites determined by X-ray crystallography.
Asymmetrical Hemoglobin Hybrids (o~Xo/y~x/3y) W h e n two unlike hemoglobins of differing charge (asx/3sx and asY/32Y), are mixed and separated by conventional analytic techniques such as zone electrophoresis or ion exchange chromatography, only two components are detected. However, failure to detect the asymmetric hybrid tetramer (axay/3x/3y) cannot be considered as evidence against its existence. During the separation procedure, the hybrid hemoglobin dissociates at the a ~ 2 interface and forms dimmers of unlike charge
During the separation, these dimers sort with like dimers. The asymmetri2R. E. Benesch, S. Jung, R. Benesch,J. Mack, and R. G. Schneider, Nature (London) 260, 219 (1976).
[11]
129
ANALYSIS OF HYBRID HEMOGLOBINS
--A 2 S
A
~! i~i!4 ~!! ~ ¸
/
20'
40'
60'
FIG. 2. Gel electrofocusing patterns of a mixture of equivalent amount of carboxyhemoglobins S and A. Photographs of the gels were obtained 20, 40, and 60 min after application of the sample. During this time the asymmetrical hybrid (C~2flA/3s) gradually disappeared.
cal hybrid dissipates, and only the two parent hemoglobins can be detected. As shown in Fig. 2, if the analytic procedure is both fast and of sufficiently high resolution, the asymmetric hybrid may be visualized for a limited period of time. Here equivalent amounts of carboxyhemoglobins A (o£2A~gA) and S (a~A/gzs) were mixed and analyzed by gel electrofocusing. Twenty minutes after application, a prominent middle band, the asymmetric hybrid ad3Afl s, could be seen. By 40 min, the hybrid band had become much fainter, and by 60 rain it could no longer be seen. In order to demonstrate stable asymmetric hybrids, it is necessary to inhibit the rate of dissociation of the hybrid tetramer at the a~fl2 interface. This can be done by employing low temperature a (see also Perrella and Rossi-Bernardi, this volume [12]), by crosslinking reagents, 4 or by taking 3 M. Perella, M. Samaja, and L. Rosi-Bernardi, J. Biol. Chem. 254, 8748 (1979). 4 R. W. Macleod and R. J. Hill, J. Biol. Chem. 248, 100 (1973).
130
H E M O G L O B I N C H A I N S A N D HYBRID M O L E C U L E S
[11]
advantage of the fact that deoxyhemoglobin dissociates into aft dimers far less readily than liganded hemoglobin) ,6 Thus deoxygenation of a mixture of oxyhemoglobins "freezes" the hybrid tetramer and allows it to be separated from the parent hemoglobins. This experimental approach depends upon maintaining hemoglobins in the fully deoxygenated state. Isoelectric focusing in cylindrical gels is an ideal method because the apparatus lends itself to working under strict anaerobic conditions. Cylinders (0.3 x 10 cm) containing 4% polymerized acrylamide gel and 2% Ampholine (pH 6-8) are prepared as previously described 6 and placed in the apparatus that cools them at 4 °. The anolyte (0.02 M H3PO4) and the catholyte (0.01 M NaOH) are gassed with nitrogen to remove dissolved oxygen. After these solutions are transferred into the apparatus, a slow stream of nitrogen is continuously passed into the catholyte, on top. A current of 1 mA per gel is applied; after a 20-min period of prefocusing, 10 ml of 0.1% sodium dithionite, prepared anaerobically, is added to the catholyte. The negatively charged dithionite anions pass through the gels, purging them of traces of dissolved oxygen. A small glass vial, containing an equimolar mixture of the parent oxyhemoglobins (10 mg/ml) and 5% Ampholine in 0.05 M phosphate buffer pH 7.0, is sealed with a rubber septum and deoxygenated by gassing with hydrated nitrogen. A 0.2 equivalent amount of sodium dithionite is added to the hemoglobin solution. By means of an airtight microsyringe, 0.010 ml of the deoxygenated hemoglobin solution is passed through a hole in the top lid of the apparatus and applied to the cylindrical gel. About 90 min after reapplication of the current, the hemoglobin bands have focused and can be removed for photography, spectral analysis, staining, etc. Figure 3 shows the analysis of a mixture containing equivalent amounts of hemoglobins S and A. When the hemoglobin mixture was in the oxy form (upper panel), no hybrid species could be detected once the hemoglobins had begun to focus at their isoelectric points, for reasons discussed above. In contrast, if the same mixture was deoxygenated and applied to anaerobic gels, a prominent and stable middle band appeared, which comprised up to 50% of the total and on direct anal~,ses was shown to have the composition OtgflAfls. AS expected, when Hb A and Hb S were deoxygenated prior to mixing, no hybrid was detected. This experimental approach has been useful in studying interactions between hemoglobin subunits. It has been applied to the measurement of the rate of dissociation of deoxyhemoglobin into aft dimers, to the study of hemoglobin variants that have structural abnormalities at the a ~ 2 in5 C. M. Park, Ann. N. Y. Acad. Sci. 209, 237 (1973). 6 H. F. Bunn and M. McDonough, Biochemistry 13, 988 (1974).
[11]
ANALYSIS OF HYBRID HEMOGLOBINS
13 1
FIG. 3. Gel electrofocusing patterns of a mixture of equivalent amounts of Hb S and Hb A after equilibrium had been attained. No hybrid species could be seen when oxyhemoglobins were analyzed under anaerobic conditions. When this mixture was deoxygenated and analyzed anaerobically, a stable hybrid hemoglobin (ctd3Afls) was readily demonstrated. If Hb A and Hb S were each deoxygenated prior to mixing (lower right), no hybrid was seen.
132
HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[11]
A2 (*,,
FIG. 4. Separation of partially oxidized (a+/32 and ot2B~) and fully oxidized (Ot2/32 + + ) hemoglobins from oxyhemoglobin (t~d32).In the middle panel, the hemoglobin was treated with 0.5 equivalent of K3Fe(CNh per heme; in the right-hand panel, the hemoglobin was treated with 1.0 equivalents of K3Fe(CN)6 per berne. terface, and to ascertain w h e t h e r two hemoglobins coexist within the s a m e red cell. V a l e n c y H y b r i d s (a+fl2 a n d ~t2fl~) The study o f valency hybrids, particularly those that contain heine in the c y a n m e t form have p r o v e d to be v e r y useful as models for intermediate states o f ligation. I f the heine is oxidized but not bound to an anionic ligand such as cyanide, it will be m o r e positively charged than unoxidized heme. Accordingly, fully and partially oxidized hemoglobin can be separated from unoxidized hemoglobin by ion-exchange c h r o m a t o g r a p h y / r T. H. J. Huisman, Arch. Biochem. Biophys. 113, 427 (1966).
[12]
HEMOGLOBIN HYBRID FORMATION
133
However, much higher resolution can be achieved by gel electrofocusing. 8 Figure 4 shows an analysis of human Hb A that had been partially oxidized by treatment with 0.5 equivalent of K3Fe(CN)e per heme equivalent (middle panel) or fully oxidized by treatment with 1.0 equivalent of K3Fe(CN)6 (right-hand panel). Methemoglobin ~a+°+~2~,~has j an isoelectric point of 7.20, 0.25 unit higher than that of oxyhemoglobin ( a ~ ) . In addition, two clearly separated bands are apparent midway between oxyhemoglobin and methemoglobin. Spectral analysis showed that these two bands were each half oxidized. Subsequent analyses have shown that the upper band is ot~fl2 and the lower is a 2/32. + This finding is a graphic demonstration of the well established differences between the heme environments of the a chains and/3 chains. 8 H. F. Bunn and J. W. Drysdale, Biochim. Biophys. Acta 229, 51 (1971).
[12] D e t e c t i o n
of Hemoglobin Hybrid Subzero Temperature
B y M I C H E L E PERRELLA a n d
Formation
at
L U I G I ROSSI-BERNARDI
The study of hybrid species (asymmetric hybrids) in mixtures of unlike tetrameric hemoglobins can yield significant information on the structure -function relationship of hemoglobin. Studies of asymmetric hybrid formation have been carded out, for instance, to determine the plane of cleavage of the hemoglobin tetramer 1 and the rate of the hemoglobin tetramer dissociationY 'a The formation of hybrid molecules between Hb S and minor hemoglobin components present in the red cells of sickle cell blood, such as Hb A, Hb A2, and Hb F, has been recognized as an important factor in the inhibition of Hb S gelling promoted by these minor components.4,5 Intermediates in the reactions of hemoglobin with ligands or with oxidants can also be considered hybrid molecules. In this case the dimers forming the tetrameric hybrid belong to the same hemoglobin species but exist in different liganded or valency states (ligand and valency hybrids). Hybridization reactions that occur via dimerization of tetramers and subsequent reassociation of dimers (symmetrical hydrids; this volume C. M. Park, J. Biol. Chem. 245, 5390 (1970). 2 H. F. Bunn and M. McDonough, Biochemistry 13, 988 (1974). 3 M. Perrella, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 254, 8748 (1979). 4 R. M. Bookchin, R. L. Nagel, and T. Balazs, Nature (London) 256, 667 (1975). 5 M. A. Goldberg, M. A. Husson, and H. F. Bunn, J. Biol. Chem. 252, 3414 (1977).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
HEMOGLOBIN
CHAINS
AND
HYBRID
[11]
MOLECULES
[1 1] U s e o f G e l E l e c t r o f o c u s i n g i n t h e A n a l y s i s o f H y b r i d Hemoglobins By H. FRANKLIN BUNN A considerable proportion of the vast amount of research on hemoglobin has exploited the fact that it is a multisubunit protein. Structural analysis has shown that each subunit of the a ~ 2 tetramer interacts with the other three subunits at well defined sites. Liganded hemoglobin readily dissociates into a aft dimers at only one of the two possible cleavage planes, the so-called a ~ 2 interface. The bonding energy of intramolecular contacts at the a ~ 2 interface is considerably less than at the ad31 interface (see Turner et al., this volume [37]). The a/3 dimer dissociates into monomers only under more drastic solvent conditions, such as extremes of pH. A considerable amount of information about hemoglobin can be obtained by studying the properties of hybrid tetramers in which subunits from parent hemoglobins are reassembled to form new tetramers. Those that are formed from unlike a/3 dimers are called asymmetrical hybrid tetramers (see Perrella and Rossi-Bernardi, this volume [12]). Those that are formed by dissociation at the aJ3~ interface with subsequent reassembly can be designated symmetrical hybrid tetramers. Finally hybrid hemoglobins can consist of tetramers having heroes of differing oxidation states, so-called valency hybrids (see Cassoly, this volume [10]). The use of gelelectrofocusing in the detection and analysis of these different kinds of hybrid hemoglobins is considered in this chapter.
Symmetrical Hemoglobin Hybrids
(o/2X~2 y, o~2Y~2 x)
The formation of hybrid hemoglobins of the type o~2X~2Yo£2Y~2X from parent hemoglobins o~2x~2x a n d 012x~2y requires mixing the parent hemoglobins under conditions that permit dissociation at the o~81 interface. Upon subsequent reassembly, symmetrical hybrid hemoglobins are formed. 1 a2x~h x + a,y~2 y
H÷ , 2 a x + 2 ~ x + 2 a y + 2 ~
~
a x~y + .~¢x
+ a2x/3sx +
a~
Equivalent amounts of the parent hemoglobins (5-50 mg/ml) are mixed, gassed with carbon monoxide, and incubated in 0.1 M acetate buffer, pH 4.5, at 0° for 18 hr. The hemoglobin mixture is then dialyzed against 0.1 M phosphate buffer pH 7.0 and analyzed by zone electro1 E. R. H u e h n s , E. M. S h o o t e r , and G. H. B e a v e n , Biochem. J. 9 1 , 3 3 1 (1964).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0
[11]
ANALYSIS OF HYBRID HEMOGLOBINS
127
A Dean
-azP
-o: 2X
HbX-Hb A--
2
an/ a Can X -a2 ~2 _ ocer~ A 2 2
AX
AA
A
X
A
X
+
+
+
+
FIG. 1. Identification of abnormal subunit in a hemoglobin variant by means of h u m a n canine symmetrical hybrids. Lanes 1 and 2 show abnormal (AX) and normal (AA) hemolysates. The variant (Hb X) comprised about 50% of the total hemoglobin. Lanes 3 and 4 show mixtures of purified Hb A and Hb X with canine hemoglobins, maintained at neutral pH. In lanes 5 and 6 the same mixtures were exposed to low pH (4.5), allowing hybrid hemoglobins to form.
phoresis. Because of its high resolution and reproducibility, gel electrofocusing is an ideal analytical system for detecting the hybrid hemoglobins. The preparation of symmetrical hemoglobin hybrids has been useful in the analysis of subunit composition. For example, this approach will determine whether the abnormality in an unidentified hemoglobin variant lies in the a chain or/3 chain. The analysis of human variants is usually done by forming hybrids with canine hemoglobin. Canine hemoglobin is prepared from a hemolysate of dog blood. Since no minor hemoglobin components are present, no further purification is necessary. Canine hemoglobin tends to precipitate during storage. As discussed by Bucci (this volume [7]), the a and/3 chains of human hemoglobin differ widely in charge with isoelectric points of approximately 8 and 6, respectively. In contrast, the subunits of canine hemoglobin differ only slightly in charge. Therefore, the identity of human-canine hybrid hemoglobin can be readily established by a comparison with the electrophoretic properties of the
128
HEMOGLOBINCHAINS AND HYBRID MOLECULES
[11]
parent hemoglobins. Figure 1 shows an example in which the abnormality of an unidentified hemoglobin variant was shown to reside in the/3 chain. As shown in lanes 3 and 4, human Hb A had an isoelectric point very close to that of canine hemoglobin. In contrast, the variant hemoglobin had an isoelectric point about 0.1 pH unit higher than human Hb A and canine hemoglobin. The pattern obtained after hybrids were formed is shown in lanes 5 and 6. The composition of the hybrid hemoglobin bands can be inferred from the relative charges of the human and canine subunits. Thus, the hybrid band with the high isoelectric point must be o/2A/32Can and that with the low isoelectric point is otsCan/32A. The human hemoglobin variant shown in Fig. 1 yields two hybrid bands, one with an isoelectric point identical to that of the hybrid otsA/32 Can and the other having an isoelectric point about 0.1 pH unit higher than a2can/3s g. Therefore, the relative positive charge of this variant can be attributed entirely to the fl chain. Preparative quantities of symmetrical hybrid hemoglobins can be isolated by column chromatography. One of the most interesting experimental applications is in the study of the sickling phenomenon. Benesch et al. 2 have prepared hybrids of the form ax/3 s from mixtures of various human a chain variants with Hb S (otd326 Val). Measurements of the solubility and gelation of these hybrid hemoglobins in comparison to that of native Hb S has provided valuable new information on the a chain contact sites that are involved in the polymerization of Hb S. Thus far there has been excellent agreement between the assignments derived from these experiments and the contact sites determined by X-ray crystallography.
Asymmetrical Hemoglobin Hybrids (o~Xo/y~x/3y) W h e n two unlike hemoglobins of differing charge (asx/3sx and asY/32Y), are mixed and separated by conventional analytic techniques such as zone electrophoresis or ion exchange chromatography, only two components are detected. However, failure to detect the asymmetric hybrid tetramer (axay/3x/3y) cannot be considered as evidence against its existence. During the separation procedure, the hybrid hemoglobin dissociates at the a ~ 2 interface and forms dimmers of unlike charge
During the separation, these dimers sort with like dimers. The asymmetri2R. E. Benesch, S. Jung, R. Benesch,J. Mack, and R. G. Schneider, Nature (London) 260, 219 (1976).
[11]
129
ANALYSIS OF HYBRID HEMOGLOBINS
--A 2 S
A
~! i~i!4 ~!! ~ ¸
/
20'
40'
60'
FIG. 2. Gel electrofocusing patterns of a mixture of equivalent amount of carboxyhemoglobins S and A. Photographs of the gels were obtained 20, 40, and 60 min after application of the sample. During this time the asymmetrical hybrid (C~2flA/3s) gradually disappeared.
cal hybrid dissipates, and only the two parent hemoglobins can be detected. As shown in Fig. 2, if the analytic procedure is both fast and of sufficiently high resolution, the asymmetric hybrid may be visualized for a limited period of time. Here equivalent amounts of carboxyhemoglobins A (o£2A~gA) and S (a~A/gzs) were mixed and analyzed by gel electrofocusing. Twenty minutes after application, a prominent middle band, the asymmetric hybrid ad3Afl s, could be seen. By 40 min, the hybrid band had become much fainter, and by 60 rain it could no longer be seen. In order to demonstrate stable asymmetric hybrids, it is necessary to inhibit the rate of dissociation of the hybrid tetramer at the a~fl2 interface. This can be done by employing low temperature a (see also Perrella and Rossi-Bernardi, this volume [12]), by crosslinking reagents, 4 or by taking 3 M. Perella, M. Samaja, and L. Rosi-Bernardi, J. Biol. Chem. 254, 8748 (1979). 4 R. W. Macleod and R. J. Hill, J. Biol. Chem. 248, 100 (1973).
130
H E M O G L O B I N C H A I N S A N D HYBRID M O L E C U L E S
[11]
advantage of the fact that deoxyhemoglobin dissociates into aft dimers far less readily than liganded hemoglobin) ,6 Thus deoxygenation of a mixture of oxyhemoglobins "freezes" the hybrid tetramer and allows it to be separated from the parent hemoglobins. This experimental approach depends upon maintaining hemoglobins in the fully deoxygenated state. Isoelectric focusing in cylindrical gels is an ideal method because the apparatus lends itself to working under strict anaerobic conditions. Cylinders (0.3 x 10 cm) containing 4% polymerized acrylamide gel and 2% Ampholine (pH 6-8) are prepared as previously described 6 and placed in the apparatus that cools them at 4 °. The anolyte (0.02 M H3PO4) and the catholyte (0.01 M NaOH) are gassed with nitrogen to remove dissolved oxygen. After these solutions are transferred into the apparatus, a slow stream of nitrogen is continuously passed into the catholyte, on top. A current of 1 mA per gel is applied; after a 20-min period of prefocusing, 10 ml of 0.1% sodium dithionite, prepared anaerobically, is added to the catholyte. The negatively charged dithionite anions pass through the gels, purging them of traces of dissolved oxygen. A small glass vial, containing an equimolar mixture of the parent oxyhemoglobins (10 mg/ml) and 5% Ampholine in 0.05 M phosphate buffer pH 7.0, is sealed with a rubber septum and deoxygenated by gassing with hydrated nitrogen. A 0.2 equivalent amount of sodium dithionite is added to the hemoglobin solution. By means of an airtight microsyringe, 0.010 ml of the deoxygenated hemoglobin solution is passed through a hole in the top lid of the apparatus and applied to the cylindrical gel. About 90 min after reapplication of the current, the hemoglobin bands have focused and can be removed for photography, spectral analysis, staining, etc. Figure 3 shows the analysis of a mixture containing equivalent amounts of hemoglobins S and A. When the hemoglobin mixture was in the oxy form (upper panel), no hybrid species could be detected once the hemoglobins had begun to focus at their isoelectric points, for reasons discussed above. In contrast, if the same mixture was deoxygenated and applied to anaerobic gels, a prominent and stable middle band appeared, which comprised up to 50% of the total and on direct anal~,ses was shown to have the composition OtgflAfls. AS expected, when Hb A and Hb S were deoxygenated prior to mixing, no hybrid was detected. This experimental approach has been useful in studying interactions between hemoglobin subunits. It has been applied to the measurement of the rate of dissociation of deoxyhemoglobin into aft dimers, to the study of hemoglobin variants that have structural abnormalities at the a ~ 2 in5 C. M. Park, Ann. N. Y. Acad. Sci. 209, 237 (1973). 6 H. F. Bunn and M. McDonough, Biochemistry 13, 988 (1974).
[11]
ANALYSIS OF HYBRID HEMOGLOBINS
13 1
FIG. 3. Gel electrofocusing patterns of a mixture of equivalent amounts of Hb S and Hb A after equilibrium had been attained. No hybrid species could be seen when oxyhemoglobins were analyzed under anaerobic conditions. When this mixture was deoxygenated and analyzed anaerobically, a stable hybrid hemoglobin (ctd3Afls) was readily demonstrated. If Hb A and Hb S were each deoxygenated prior to mixing (lower right), no hybrid was seen.
132
HEMOGLOBIN CHAINS AND HYBRID MOLECULES
[11]
A2 (*,,
FIG. 4. Separation of partially oxidized (a+/32 and ot2B~) and fully oxidized (Ot2/32 + + ) hemoglobins from oxyhemoglobin (t~d32).In the middle panel, the hemoglobin was treated with 0.5 equivalent of K3Fe(CNh per heme; in the right-hand panel, the hemoglobin was treated with 1.0 equivalents of K3Fe(CN)6 per berne. terface, and to ascertain w h e t h e r two hemoglobins coexist within the s a m e red cell. V a l e n c y H y b r i d s (a+fl2 a n d ~t2fl~) The study o f valency hybrids, particularly those that contain heine in the c y a n m e t form have p r o v e d to be v e r y useful as models for intermediate states o f ligation. I f the heine is oxidized but not bound to an anionic ligand such as cyanide, it will be m o r e positively charged than unoxidized heme. Accordingly, fully and partially oxidized hemoglobin can be separated from unoxidized hemoglobin by ion-exchange c h r o m a t o g r a p h y / r T. H. J. Huisman, Arch. Biochem. Biophys. 113, 427 (1966).
[12]
HEMOGLOBIN HYBRID FORMATION
133
However, much higher resolution can be achieved by gel electrofocusing. 8 Figure 4 shows an analysis of human Hb A that had been partially oxidized by treatment with 0.5 equivalent of K3Fe(CN)e per heme equivalent (middle panel) or fully oxidized by treatment with 1.0 equivalent of K3Fe(CN)6 (right-hand panel). Methemoglobin ~a+°+~2~,~has j an isoelectric point of 7.20, 0.25 unit higher than that of oxyhemoglobin ( a ~ ) . In addition, two clearly separated bands are apparent midway between oxyhemoglobin and methemoglobin. Spectral analysis showed that these two bands were each half oxidized. Subsequent analyses have shown that the upper band is ot~fl2 and the lower is a 2/32. + This finding is a graphic demonstration of the well established differences between the heme environments of the a chains and/3 chains. 8 H. F. Bunn and J. W. Drysdale, Biochim. Biophys. Acta 229, 51 (1971).
[12] D e t e c t i o n
of Hemoglobin Hybrid Subzero Temperature
B y M I C H E L E PERRELLA a n d
Formation
at
L U I G I ROSSI-BERNARDI
The study of hybrid species (asymmetric hybrids) in mixtures of unlike tetrameric hemoglobins can yield significant information on the structure -function relationship of hemoglobin. Studies of asymmetric hybrid formation have been carded out, for instance, to determine the plane of cleavage of the hemoglobin tetramer 1 and the rate of the hemoglobin tetramer dissociationY 'a The formation of hybrid molecules between Hb S and minor hemoglobin components present in the red cells of sickle cell blood, such as Hb A, Hb A2, and Hb F, has been recognized as an important factor in the inhibition of Hb S gelling promoted by these minor components.4,5 Intermediates in the reactions of hemoglobin with ligands or with oxidants can also be considered hybrid molecules. In this case the dimers forming the tetrameric hybrid belong to the same hemoglobin species but exist in different liganded or valency states (ligand and valency hybrids). Hybridization reactions that occur via dimerization of tetramers and subsequent reassociation of dimers (symmetrical hydrids; this volume C. M. Park, J. Biol. Chem. 245, 5390 (1970). 2 H. F. Bunn and M. McDonough, Biochemistry 13, 988 (1974). 3 M. Perrella, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 254, 8748 (1979). 4 R. M. Bookchin, R. L. Nagel, and T. Balazs, Nature (London) 256, 667 (1975). 5 M. A. Goldberg, M. A. Husson, and H. F. Bunn, J. Biol. Chem. 252, 3414 (1977).
METHODS IN ENZYMOLOGY, VOL. 76
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181976-0