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A calorimetric study of the recombination of the isolated α and β chains of human hemoglobin
64
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35600 A CALORIMETRIC STUDY OF THE RECOMBINATION
OF
T H E I S O L A T E D a A N D fl C H A I N S O F H U M A N H E M O G L O B I N
\V. J. E V A N S ' , L. F O R L A N I , M. B R U N O R I , J. W Y M A N AND E. A N T O N I N I
Institute of Biochemistry and Center for Molecular Biology of the C.N.R., University of Rome, and Regina Elena Institute for Cancer Research, Rome (Italy) (Received J a n u a r y i3th, 197 o)
SUMMARY
The e n t h a l p y change a c c o m p a n y i n g r e c o m b i n a t i o n of a S H o 2 and flSHo2 chains of h u m a n hemoglobin has been m e a s u r e d calorimetrically. The overall process is e x o t h e r m a l , with a m i n i m a l ~]H in the n e i g h b o r h o o d of --7000 cal/mole (of dimer). This value a p p e a r s to be s o m e w h a t d e p e n d e n t on the m o l a r r a t i o of aSHo 2 and flSUo 2 chains.
INTRODUCTION
Since the d e v e l o p m e n t of a procedure for isolating the a and t3 chains of h u m a n hemoglobin in their n a t i v e s t a t e 1, studies on their functional a n d s t r u c t u r a l p r o p e r t i e s h a v e been v e r y n u m e r o u s (see ref. 2 for a review). This p a p e r is concerned with direct calorimetric m e a s u r e m e n t s of the h e a t of r e c o m b i n a t i o n of the liganded chains to form t e t r a m e r i c hemoglobin according to the overall reaction scheme: 4a +/3 4 +2
(a 2fl2)
(1)
The subject acquires special interest in view of evidence which indicates t h a t the heat of dissociation of the t e t r a m e r i c molecule into its dimers is either zero or v e r y small s . MATERIALS AND METHODS
H u m a n hemoglobin was p r e p a r e d according to the toluene procedure 4 or the (NH4)2SO 4 procedure 5. The a a n d /5 chains were p r e p a r e d using the p - h y d r o x y m e r c u r i b e n z o a t e (PHMB) p r o c e d u r e according to B u c c I AND FRONTICELLI 1. The isolated chains were c o n c e n t r a t e d (up to i - l O .3 M in heme) either b y p r e c i p i t a t i o n with (NH4)2SO 4 at 9o% s a t u r a t i o n or b y r a p i d dialysis u n d e r v a c u u m . There was Abbreviation: P H M B , p - h y d r o x y m e r c u r i b e n z o a t e . " Present address: U.S. D e p a r t m e n t of Agriculture, Southern Utilization Research and D c v e l o p m e n t Division of New Orleans, U.S.A.
Biochim. Biophys. Acta, 214 (197o) 64 68
RECOMBINATION OF (l AND ~ CHAINS OF HEMOGLOBIN
()5
no spectroscopic difference between the chains as obtained by the two methods of concentration, and they behaved alike in the calorimetric experiments described below. Bound hydroxymercuribenzoate (PHMB) was generally removed by treatment with I-dodecanethiol 6, and in a few experiments by sodium thioglycollate. After regeneration the purity and the extent of recombination of the SH chains was examined by starch gel electrophoresis ~. The two chains were always dialyzed for one day against the appropriate solvent, usually o.I M potassium phosphate + o.I M NaC1 at pH 6.8. Most of the experiments were performed with oxygenated chains, although two of them were carried out with the carbon monoxide derivative. Measurements were made at 25 °. The time necessary to reach initial temperature equilibrium was between 9° and 12o rain. During this period the isolated chains sometimes developed a precipitate and gave spectroscopic evidence of the formation of hemichromogen. In this event the evolution of heat, after the reactants were mixed, continued for a very long time, and all such experiments were discarded. More stable material was obtained by heating the regenerated and concentrated solutions at 35-4 o° for about I rain and then centrifuging off the precipitate. This procedure was always used except for a few controls. It finds its justification in a number of control experiments which indicate that the functional and structural properties of the reassembled hemoglobin obtained from chains treated in this way are identical with those of the native molecule (unpublished experiments from this laboratory). Two microcatorimeters of the heat conduction type were used. As described elsewhere 8,9 they lend themselves to automatic integration of the electronmotive force-time curves; moreover they have the advantage of securing complete mixing of equal volumes of reagents and of requiring only small amounts of material. Calibration of the two instruments was performed as reported before s& The volume of material normally used was 1.5 ml for the larger calorimeter and o.5 ml for the smaller one. Spectrophotometric measurements were carried out with a Cary model 14 recording spectrophotometer. The concentrations, given in heine equivalents, were obtained using published values for the extinction coefficients~°. Starch gel electrophoresis was performed with the discontinuous buffer system according to POULIK~. RESULTS
Since the heat evolved on mixing the regenerated ~ and fl chains appears to be dependent upon the presence of an excess of/3 chains over ¢ chains, we have divided the experiments into two groups: (i) experiments performed with the ratio [fl~/Ea] = I ± 0.2; and (ii) experiments performed with the ratio [fl]/Ea] >~ 2. Table I summarizes the results obtained when [/~]/Ea~ --- I. The overall enthalpy change, reported in Column 6 of the table, was obtained from the experimental heat by taking account of the volume and the concentration of the chain that was present in smaller amount. These results give a value of the overall enthalpy change, obtained upon mixing nearly equimolar amounts of the ~ and /~ chains, / ] H --7IOO ~ 2580 cal/mole (of dimer) at pH 6.8 and 25 °. This value is the average of Biochim. Biophys. Acta, 214 (i97o) 64-68
w . J . EVANS et al.
66 TABLE
I
V A L U E S OF /] H FOR T H E R E C O M B I N A T I O N OF a AND /~ C H A I N S
Conditions: 25% o , I M potassium phosphate buffer + o . I M N a C 1 ( p H 6.8). R a t i o [fl]/[a] = i / = o . 2 . (a) = results obtained with the small calorimeter; (b) = results obtained with the large calorimeter. I
Prep. No.
2 [a] >( z o 4
3 [fl] × lO 4
4
5
Ratio
Experimental heat
[fl7l[ct]
(l~cal) 4 5
6 8 9 IO
II 14
15
4.35 4.35 IO.OO
5.51 5.51 11.88
IO.OO
II.98
IO.OO IO.OO 1.1o 1I.I 4 5 "I7 5.17 1.45 1.45 9.43 9.43 3 .62 3 .62 8.47 6.30 3.51 3.26 6.73 3.48
9.42 9.52 0.87 10.14 5'77 5.77 1.81 1.8I 9.30 9.3 o 3-59 3-59 8.15 6.06 3.63 3.04 7.24 3.5 °
1.27 1-27 1.19 1.19 o.94 o.94 0.79 O.91 t. I2 I.I2 1.25 1.25 0.99 o.99 0.99 0.99 0.96 I.IO 1.o4 0.93 1.o8 i.oo
i 4 2 8 2 7 8 I 2 3 IO 1 3 12 II 4 2 9 3
427 257 824 133 339 916 514 899 824 427 763 687 315 165 332 958 931 ooo 945 75 ° 7 lo 281
(a) (b) (a) (b) (a) (a) (t1) (b) (a) (b) (a) (b) (a) (b) (a) (b) (b) (b) (b) (b) (b) (b)
6 -- A H (cal/mole of dimer) 6 6 5 5 4 5 3 5 3 3 IO 12 7 7 7 7 IO II 9 6 9 6
5oo 52o 65 ° 420 96o
6oo 94 ° 850 19o 13o 53 ° 353 128 287 423 53 ° 577 640 200 030 618 286
16
22 experiments obtained with IO different preparations. The results reported in Table I clearly indicate that the two calorimeters gave the same answer within rather small errors; moreover the different experiments performed with the same preparation gave fairly consistent results. Therefore it would seem that the large scatter in the values of A H must result primarily from unknown differences between the various chain preparations used. It is significant that a control experiment performed with a chain preparation, which, as judged by kinetic and electrophoretie analyses, failed to recombine, gave zero heat. The experiments performed at two other pH's (6 and 7.5), as well as those performed with the carbon monoxide derivative and the ratio [/3]/[al =~ I, gave the same results. Table II reports similar results obtained under conditions in which the ratio [/3]/[a] >~ 2. Here the overall enthalpy change, obtained from II experiments performed with 7 different chain preparations, is, as shown by statistical analysis, significantly larger, namely A H = --1276o ~ 35oo cal/mole (of dimer). Owing to the rather large errors it was not possible to analyze in detail the dependence of A H on tile ratio [/5]/[a]. Only a few experiments were carried out with an excess of a chains over /5 chains; the enthalpy change was then, within errors, similar to the average value obtained when [/3]/[a] --- i. B i o c h i m . B i o p h y s . Acta, 2 1 4 (197 o) 6 4 - 6 8
67
R E C O M B I N A T I O N OF a AND fl CHAINS OF HEMOGLOBIN TABLE
I1
VALUES OF zL] H FOR THE RECOMBINATION OF C~ AND [J CHAINS C o n d i t i o n s as in T a b l e I, e x c e p t t h a t r a t i o [fl]/[a~ >~ 2.
I
2
3
Prep. ~\rO.
:'c L .... X I01
,"fi]
4"49 3.87 3.7 ° 1.96 I"70 1.7 ° 3.18 3.18 3.78 2-54 1.41
11"45 7.83 8.25 9-56 10"43 10.43 10.37 IO.37 9.42 8.72 9.3 °
I 2 3 7
II 15 IO
X
I0 4
4
5
6
Ratio ,fi]l[cz/
Experimental heat (peal)
A~H (cal/mole q/dimer)
2.54
I995 2949 2993 255 ° 1345 2878 I533 4582 4776 4o63 133o
8 9o0 15 280 i 6 18o 13 o o o 15 829 I I 284 9 640 9 6o0 ,S 423 13184
2.02 3.23 4.88 6.13 6.13 3.26 3.26 2.49 3.43 6.59
(a) (a) (a) (a) (a) (b) (a) (b) (b) (b) (a)
19ooo
CONCLUSION
As regards the differences of zl H between the various preparations, we have no explanation to offer; nor is there any clear explanation for the effect of an excess of/3 chains in raising the value of A H, though it is tempting to associate these effects with the fact that the /3 chains are present as tetramers, while the a chains are essentially monomers. Electrophoretic analysis was used to test the extent of recombination of the chains and was always performed on an aliquot of the same material used in the calorimetric measurements. In a few experiments, in which the same control was performed directly on the material recovered from the calorimeter cell at the end of the measurement, no modification of the electrophoretic pattern was observed. However, although the electrophoretic experiments reveal the extent of recombination of the chains, t h e y unfortunately give no information concerning the speed of the process or the presence of other associated processes which might contribute significantly to the measured heat. In spite of various perplexities and uncertainties, and notwithstanding the large scatter of the observations, there seems to be no doubt that the overall recombination of the isolated a and/3 chains is an exothermal process, with a minimal value of AH in the neighbourhood of - - 7 o o o cal/mole of dimer. REFERENCES I l'~. BUCCI AND C. FRONTICELLI, J. Biol. Chem., 240 (I965) P C 5 5 I . 2 t';. ANTONINI ANn M. BRUNORI, A n n . Rev. Bioehem., 39 (197 o) in t h e press. 3 g . CHIANCONE, L. M. GILBERT, G. A. GILBERT AND G. L. KELLETT, .J. Biol. Chem., 243 (1968) 1212. 4 K. g . DRABKIN, J. Biol. Chem., 164 (1946) 7o3 . 5 A. R o s s I FANELLI, E. ANTONINI AND A. CAPUTO, J. Biol. Chem., 236 (1961) 391. 6 E. C. DE RENZO, C. IOPPOLO, G. AMICONI, E. ANTONINI AND J. WYMAN, .J. Biol. Chem., 242 (1967) 485 ° . 7 H. D. POULIK, Nature, 18o (1957) 1477.
Biochim. Biophys. Acta, z~ 4 (I97 o) 04 68
68
w.J.
EVANS et al.
8 W. J. EVANS, E. J. McCOURTENEY AND W. B. CARNEY,Anal. Chem., 4 ° (I968) 262. 9 W. J. EVANS, in H. D. BROWN, Biochemical Calorimetry, A c a d e m i c Press, New York, 1969 p. "a57. io E. ANTONINI, Physiol. Rev., 45 (1965) 123.
Biochim. Biophys. Acta, 214 (I97 o) 64-68
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35600 A CALORIMETRIC STUDY OF THE RECOMBINATION
OF
T H E I S O L A T E D a A N D fl C H A I N S O F H U M A N H E M O G L O B I N
\V. J. E V A N S ' , L. F O R L A N I , M. B R U N O R I , J. W Y M A N AND E. A N T O N I N I
Institute of Biochemistry and Center for Molecular Biology of the C.N.R., University of Rome, and Regina Elena Institute for Cancer Research, Rome (Italy) (Received J a n u a r y i3th, 197 o)
SUMMARY
The e n t h a l p y change a c c o m p a n y i n g r e c o m b i n a t i o n of a S H o 2 and flSHo2 chains of h u m a n hemoglobin has been m e a s u r e d calorimetrically. The overall process is e x o t h e r m a l , with a m i n i m a l ~]H in the n e i g h b o r h o o d of --7000 cal/mole (of dimer). This value a p p e a r s to be s o m e w h a t d e p e n d e n t on the m o l a r r a t i o of aSHo 2 and flSUo 2 chains.
INTRODUCTION
Since the d e v e l o p m e n t of a procedure for isolating the a and t3 chains of h u m a n hemoglobin in their n a t i v e s t a t e 1, studies on their functional a n d s t r u c t u r a l p r o p e r t i e s h a v e been v e r y n u m e r o u s (see ref. 2 for a review). This p a p e r is concerned with direct calorimetric m e a s u r e m e n t s of the h e a t of r e c o m b i n a t i o n of the liganded chains to form t e t r a m e r i c hemoglobin according to the overall reaction scheme: 4a +/3 4 +2
(a 2fl2)
(1)
The subject acquires special interest in view of evidence which indicates t h a t the heat of dissociation of the t e t r a m e r i c molecule into its dimers is either zero or v e r y small s . MATERIALS AND METHODS
H u m a n hemoglobin was p r e p a r e d according to the toluene procedure 4 or the (NH4)2SO 4 procedure 5. The a a n d /5 chains were p r e p a r e d using the p - h y d r o x y m e r c u r i b e n z o a t e (PHMB) p r o c e d u r e according to B u c c I AND FRONTICELLI 1. The isolated chains were c o n c e n t r a t e d (up to i - l O .3 M in heme) either b y p r e c i p i t a t i o n with (NH4)2SO 4 at 9o% s a t u r a t i o n or b y r a p i d dialysis u n d e r v a c u u m . There was Abbreviation: P H M B , p - h y d r o x y m e r c u r i b e n z o a t e . " Present address: U.S. D e p a r t m e n t of Agriculture, Southern Utilization Research and D c v e l o p m e n t Division of New Orleans, U.S.A.
Biochim. Biophys. Acta, 214 (197o) 64 68
RECOMBINATION OF (l AND ~ CHAINS OF HEMOGLOBIN
()5
no spectroscopic difference between the chains as obtained by the two methods of concentration, and they behaved alike in the calorimetric experiments described below. Bound hydroxymercuribenzoate (PHMB) was generally removed by treatment with I-dodecanethiol 6, and in a few experiments by sodium thioglycollate. After regeneration the purity and the extent of recombination of the SH chains was examined by starch gel electrophoresis ~. The two chains were always dialyzed for one day against the appropriate solvent, usually o.I M potassium phosphate + o.I M NaC1 at pH 6.8. Most of the experiments were performed with oxygenated chains, although two of them were carried out with the carbon monoxide derivative. Measurements were made at 25 °. The time necessary to reach initial temperature equilibrium was between 9° and 12o rain. During this period the isolated chains sometimes developed a precipitate and gave spectroscopic evidence of the formation of hemichromogen. In this event the evolution of heat, after the reactants were mixed, continued for a very long time, and all such experiments were discarded. More stable material was obtained by heating the regenerated and concentrated solutions at 35-4 o° for about I rain and then centrifuging off the precipitate. This procedure was always used except for a few controls. It finds its justification in a number of control experiments which indicate that the functional and structural properties of the reassembled hemoglobin obtained from chains treated in this way are identical with those of the native molecule (unpublished experiments from this laboratory). Two microcatorimeters of the heat conduction type were used. As described elsewhere 8,9 they lend themselves to automatic integration of the electronmotive force-time curves; moreover they have the advantage of securing complete mixing of equal volumes of reagents and of requiring only small amounts of material. Calibration of the two instruments was performed as reported before s& The volume of material normally used was 1.5 ml for the larger calorimeter and o.5 ml for the smaller one. Spectrophotometric measurements were carried out with a Cary model 14 recording spectrophotometer. The concentrations, given in heine equivalents, were obtained using published values for the extinction coefficients~°. Starch gel electrophoresis was performed with the discontinuous buffer system according to POULIK~. RESULTS
Since the heat evolved on mixing the regenerated ~ and fl chains appears to be dependent upon the presence of an excess of/3 chains over ¢ chains, we have divided the experiments into two groups: (i) experiments performed with the ratio [fl~/Ea] = I ± 0.2; and (ii) experiments performed with the ratio [fl]/Ea] >~ 2. Table I summarizes the results obtained when [/~]/Ea~ --- I. The overall enthalpy change, reported in Column 6 of the table, was obtained from the experimental heat by taking account of the volume and the concentration of the chain that was present in smaller amount. These results give a value of the overall enthalpy change, obtained upon mixing nearly equimolar amounts of the ~ and /~ chains, / ] H --7IOO ~ 2580 cal/mole (of dimer) at pH 6.8 and 25 °. This value is the average of Biochim. Biophys. Acta, 214 (i97o) 64-68
w . J . EVANS et al.
66 TABLE
I
V A L U E S OF /] H FOR T H E R E C O M B I N A T I O N OF a AND /~ C H A I N S
Conditions: 25% o , I M potassium phosphate buffer + o . I M N a C 1 ( p H 6.8). R a t i o [fl]/[a] = i / = o . 2 . (a) = results obtained with the small calorimeter; (b) = results obtained with the large calorimeter. I
Prep. No.
2 [a] >( z o 4
3 [fl] × lO 4
4
5
Ratio
Experimental heat
[fl7l[ct]
(l~cal) 4 5
6 8 9 IO
II 14
15
4.35 4.35 IO.OO
5.51 5.51 11.88
IO.OO
II.98
IO.OO IO.OO 1.1o 1I.I 4 5 "I7 5.17 1.45 1.45 9.43 9.43 3 .62 3 .62 8.47 6.30 3.51 3.26 6.73 3.48
9.42 9.52 0.87 10.14 5'77 5.77 1.81 1.8I 9.30 9.3 o 3-59 3-59 8.15 6.06 3.63 3.04 7.24 3.5 °
1.27 1-27 1.19 1.19 o.94 o.94 0.79 O.91 t. I2 I.I2 1.25 1.25 0.99 o.99 0.99 0.99 0.96 I.IO 1.o4 0.93 1.o8 i.oo
i 4 2 8 2 7 8 I 2 3 IO 1 3 12 II 4 2 9 3
427 257 824 133 339 916 514 899 824 427 763 687 315 165 332 958 931 ooo 945 75 ° 7 lo 281
(a) (b) (a) (b) (a) (a) (t1) (b) (a) (b) (a) (b) (a) (b) (a) (b) (b) (b) (b) (b) (b) (b)
6 -- A H (cal/mole of dimer) 6 6 5 5 4 5 3 5 3 3 IO 12 7 7 7 7 IO II 9 6 9 6
5oo 52o 65 ° 420 96o
6oo 94 ° 850 19o 13o 53 ° 353 128 287 423 53 ° 577 640 200 030 618 286
16
22 experiments obtained with IO different preparations. The results reported in Table I clearly indicate that the two calorimeters gave the same answer within rather small errors; moreover the different experiments performed with the same preparation gave fairly consistent results. Therefore it would seem that the large scatter in the values of A H must result primarily from unknown differences between the various chain preparations used. It is significant that a control experiment performed with a chain preparation, which, as judged by kinetic and electrophoretie analyses, failed to recombine, gave zero heat. The experiments performed at two other pH's (6 and 7.5), as well as those performed with the carbon monoxide derivative and the ratio [/3]/[al =~ I, gave the same results. Table II reports similar results obtained under conditions in which the ratio [/3]/[a] >~ 2. Here the overall enthalpy change, obtained from II experiments performed with 7 different chain preparations, is, as shown by statistical analysis, significantly larger, namely A H = --1276o ~ 35oo cal/mole (of dimer). Owing to the rather large errors it was not possible to analyze in detail the dependence of A H on tile ratio [/5]/[a]. Only a few experiments were carried out with an excess of a chains over /5 chains; the enthalpy change was then, within errors, similar to the average value obtained when [/3]/[a] --- i. B i o c h i m . B i o p h y s . Acta, 2 1 4 (197 o) 6 4 - 6 8
67
R E C O M B I N A T I O N OF a AND fl CHAINS OF HEMOGLOBIN TABLE
I1
VALUES OF zL] H FOR THE RECOMBINATION OF C~ AND [J CHAINS C o n d i t i o n s as in T a b l e I, e x c e p t t h a t r a t i o [fl]/[a~ >~ 2.
I
2
3
Prep. ~\rO.
:'c L .... X I01
,"fi]
4"49 3.87 3.7 ° 1.96 I"70 1.7 ° 3.18 3.18 3.78 2-54 1.41
11"45 7.83 8.25 9-56 10"43 10.43 10.37 IO.37 9.42 8.72 9.3 °
I 2 3 7
II 15 IO
X
I0 4
4
5
6
Ratio ,fi]l[cz/
Experimental heat (peal)
A~H (cal/mole q/dimer)
2.54
I995 2949 2993 255 ° 1345 2878 I533 4582 4776 4o63 133o
8 9o0 15 280 i 6 18o 13 o o o 15 829 I I 284 9 640 9 6o0 ,S 423 13184
2.02 3.23 4.88 6.13 6.13 3.26 3.26 2.49 3.43 6.59
(a) (a) (a) (a) (a) (b) (a) (b) (b) (b) (a)
19ooo
CONCLUSION
As regards the differences of zl H between the various preparations, we have no explanation to offer; nor is there any clear explanation for the effect of an excess of/3 chains in raising the value of A H, though it is tempting to associate these effects with the fact that the /3 chains are present as tetramers, while the a chains are essentially monomers. Electrophoretic analysis was used to test the extent of recombination of the chains and was always performed on an aliquot of the same material used in the calorimetric measurements. In a few experiments, in which the same control was performed directly on the material recovered from the calorimeter cell at the end of the measurement, no modification of the electrophoretic pattern was observed. However, although the electrophoretic experiments reveal the extent of recombination of the chains, t h e y unfortunately give no information concerning the speed of the process or the presence of other associated processes which might contribute significantly to the measured heat. In spite of various perplexities and uncertainties, and notwithstanding the large scatter of the observations, there seems to be no doubt that the overall recombination of the isolated a and/3 chains is an exothermal process, with a minimal value of AH in the neighbourhood of - - 7 o o o cal/mole of dimer. REFERENCES I l'~. BUCCI AND C. FRONTICELLI, J. Biol. Chem., 240 (I965) P C 5 5 I . 2 t';. ANTONINI ANn M. BRUNORI, A n n . Rev. Bioehem., 39 (197 o) in t h e press. 3 g . CHIANCONE, L. M. GILBERT, G. A. GILBERT AND G. L. KELLETT, .J. Biol. Chem., 243 (1968) 1212. 4 K. g . DRABKIN, J. Biol. Chem., 164 (1946) 7o3 . 5 A. R o s s I FANELLI, E. ANTONINI AND A. CAPUTO, J. Biol. Chem., 236 (1961) 391. 6 E. C. DE RENZO, C. IOPPOLO, G. AMICONI, E. ANTONINI AND J. WYMAN, .J. Biol. Chem., 242 (1967) 485 ° . 7 H. D. POULIK, Nature, 18o (1957) 1477.
Biochim. Biophys. Acta, z~ 4 (I97 o) 04 68
68
w.J.
EVANS et al.
8 W. J. EVANS, E. J. McCOURTENEY AND W. B. CARNEY,Anal. Chem., 4 ° (I968) 262. 9 W. J. EVANS, in H. D. BROWN, Biochemical Calorimetry, A c a d e m i c Press, New York, 1969 p. "a57. io E. ANTONINI, Physiol. Rev., 45 (1965) 123.
Biochim. Biophys. Acta, 214 (I97 o) 64-68