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The effect of specific antibody on the oxygen equilibrium of human hemoglobin
BIOCHIMICAET BIOPHYSICAACTA
49
BBA 35317 T H E E F F E C T OF SPECIFIC A N T I B O D Y ON T H E O X Y G E N E Q U I L I B R I U M OF HUMAN H E M O G L O B I N
M O R R I S R E [ C H L I N , L I N D A U D E M AND H E L E N M. R A N N E Y
Department of Medicine, State University of New York at Buffalo School of Medicine, Buffalo, N.Y. ~42o3, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, N . Y . zo46r (U.S.A.) (Received A u g u s t 7th, 1908 )
SUMMARY
Specifically purified anti A 1 hemoglobin antibody has a marked effect on the 0 2 equilibrium of human A1 hemoglobin. The effects noted were an increased 0 2 affinity, decreased value of n, and marked a s y m m e t r y of the 0 2 equilibrium curve. There was substantial preservation of the Bohr effect at all antibody to hemoglobin ratios tested.
INTRODUCTION
The immunochemical study of hemoglobin has been approached as a model system in which the effect of reversible conformational changes on antigenic specificity could be assessed. Such studies initially included the comparison of apohemoglobin and hemoglobin, apomyglobin and myoglobin 1 and more recently a study of the difference between oxy- and deoxyhemoglobin 2 4. These studies have shown t h a t human oxyhemoglobin and deoxyhemoglobin are easily distinguished by their reactivity with specific rabbit antibody to human hemoglobin. These differences were measured by studying the reactions of the two forms of hemoglobin with specific antiserum in quantitative complement fixation experiments. Such differences in the imnmnological behavior of the two forms of hemoglobin were interpreted in terms of the conformational changes known to accompany reactions with 0 2. Thus, since oxy- and deoxyhemoglobin differ immunologically one might expect that specific antibody would affect the 0 2 equilibrium. This report includes our initial studies of the effect of the purified rabbit specific antibody on the 0 2 equilibrium of human hemoglobin. MATERIALS AND METHODS
Antisera to A 1 hemoglobin were obtained from rabbits immunized by a schedule of injections previously described 5. Specific antihemoglobin antibody was purified in the following way. A specific Biochim. Biophys. Acta, 175 (1969) 49 54
5°
M. R E 1 C H I . I N , L. UI)EXI, H. M. I~:\NNEY
precipitate %rmed at equivalence with oxyheinoglobin (as judged by quantitative precipitin data for each serum) was washed 3 :~ with cold saline, and dissolved in o.I M acetic acid, and applied to a Sephadex G-Ioo colunm, 2.5 cm ~. 40.o cm, equilibrated with o.I M acetic acid. Substantial resolution of antibody and antigen was achieved although some heine color was found in the antibody peak. When the material in the excluded volume was neutralized by dialysis against o.i M phosphate buffer (pH 7.o), a small colored precipitate formed. The supernate was clear, colorless, and contained a yield of 7o% of the specific antibody in the original immune precipitate. This soluble material, a 7-S antibody which was active in precipitation and complement fixation reactions, was digested with i°~£, by weight of pepsin in o.oi M cysteine at p H 4.o as described by NISONOFF et al. s. Following digestion for 24 h and alkylation with a 3 × molar excess of iodoacetamide, the pepsin was inactivated by dialysis against pH 8.o borate buffer for 24.o h. Ultracentrifugation revealed a single 3.5-S peak, typical of rabbit Fab fragments. Approx. 95<}.i) of these antihemoglobin Fab fragments were capable of binding hemoglobin as judged by experiments on Sephadex G-Ioo. Concentration of the alkylated Fab fragments was estimated by using an absorbance value of I5.O for a I°,o solution at 28o m# in pH 7.o, o.I M phosphate buffers. The O 2 equilibria were determined by a spectrophotometric method which is a modification 7 of that described by ALLEN, WYMAN AND GUTHEs. The equilibria were all performed in o.I M phosphate buffers at IO°. Only 5 to 6 points were determined for each study in order to minimize formation of methemoglobin; unfortunately sufficient purified antibody was not available for the multiple tonometers necessary for more detailed studies of the saturation curve. RESULTS
Effect of antibody on the oxygen equilibrium Non-immune y-globulin in a mole ratio of 5.7 moles Fab/mole Hb had no effect on the 02 equilibrium (Fig. i). The calculated values for n* and Pl/2* were not different in the presence and absence of non-immune globulin. A titration of hemoglobin with Fab fragments was performed to determine the effect of binding increasing quantities of Fab fragments to the hemoglobin. The complex results of the experiments are illustrated in Fig. 2. At all ratios of Fab to Hb tested there was an effect on both the Pl/z and the value of n and in all cases the equilibrium curve was asymmetrical and the Hill plot was apparently a biphasic curve. The portion of the equilibrium at lower saturation we have designated as Phase I and that at higher saturation, Phase 2. Even at the lowest mole ratio of Fab fragment to hemoglobin (2.2 : I) there was a substantial effect on the value of n and a small increase in O~ affinity. This represented the effect of a single Fab fragment per ~/5 unit. As the quantity of antibody was increased the proportion of the hemoglobin exhibiting higher 02 affinity increased. In addition, as the quantity of antibody was increased the fraction of the equilibrium in the first phase increased and it was the 02 affinity of the first phase that was greatly affected by antibody. In the presence of 2.2, 4.4, and 6.15 moles of Fab per mole Hb the fractional saturation (y) * n is t h e s l o p e o f t h e H I L L p l o t o r l o g Y/i-y/log pO 2 a n d Pl/2 is t h e p O 2 a t w h i c h t h e h e m o g l o b i n is h a l f s a t u r a t e d .
Biochim. Bioph3,s. Acla, i 7 5 (1969) 4 9 54
EFFECT OF ANTIBODY ON HEMOGLOBIN
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Fig. I. 0 - - 0 , H b A w i t h y-globulin, p H 6.99, moles F a b / m o l e s H b = 5 . 7 : I . P112 = 4.13, n = 2.47. M e t h e m o g l o b i n : initial, o % , final 13%. 0 - - 0 , H b A control, p H 6.97. P x / 2 = 4.13, n = 2.47. M e t h e m o g l o b i n : initial, o % ; final, 8 % . Fig. 2. Curv e i, H b A w i t h a n t i b o d y 87B 7, p H 6.98, moles F a b / m o l e s H b -- 2.2:1. F i r s t p h a s e of c u r v e : P l / 2 - - 3-4 °, n 0.83. Second p h a s e : P l / ~ - - 2.47, n -- 1.52. M e t h e m o g l o b i n : i n i t i a l , 0 % ; final, i i ~ o . Curve 2, H b A w i t h a n t i b o d y 87B 7, p H 6.98, moles F a b / m o l e s H b -- 4.4:1. F i r s t p h a s e of c u r v e : P l / ~ - - 2.45, n = 0.62. Second p h a s e : P l / ~ - - 2.22, n -- 1.43. M e t h e m o g l o b i n : initial, o% ; final, 11%. Curve 3, H b A w i t h a n t i b o d y 87B7, p H 6.99, moles F a b / m o l e s H b -6.15:1. F i r s t p h a s e of c u r v e : Pl/.2 - - 1-64, n -- 0.67. Second p h a s e : P l / 2 = 2.11, n = 1.4o. Methem o g l o b i n : initial, 1 . 8 % ; final, 2 4 % .
observed in the first phase was 33, 47, and 6o%, respectively. The values for P112 were 3.4 o, 2.45, and 1.64 in the first phase, but differed little inter se in the second phase. Indeed it appeared that once some critical saturation characteristic of each ratio of antibody to hemoglobin was reached, the remainder of the saturation curve was not altered significantly by different amounts of antibody. Another feature of these experiments was that the slope of the first phase of the Hill plot was always less than I and the slope of the second phase was greater than i. The proportion of methemoglobin (in samples of pH below 7.o) was estimated from the data of BENESCH, MACDUFF AND BENESCH17: in most of the runs, little or no methemoglobin was present at the beginning of the experiments. At the conclusion of the 02 equilibria studies, II-24~o (av. 15%) methemoglobin was found in the experiments in which specific antisera were added, and 13% in the control experiment in which ;~-globulin was used. While these methemoglobin values were high, they did not appear sufficient to account for the decreased values of n. Lastly we prepared Fab fragments from another rabbit antiserum to human hemoglobin (82B7) and tested the effect of a single Fab to Hb mole ratio of 3-4. Although there were quantitative differences in the effect of this preparation, the features noted before were also present here: asymmetry of the Oz equilibrium curve with resolution into two phases in the Hill plot, increased O~ affinity and a substantial decrease in the value of n.
Effect of antibody on the Bohr effect Control experiments with non-immune 7-globulin demonstrated the absence of Biochim.
Biophys.
Acta,
175 (1969) 49-54
M. R E I ( H I A N ,
52
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any non-specific effect of ().5 moles of Fab fragmentsper m{)le ()f hem<,globin. With 6. 5 moles of Fab there was a 3-fold increase in 02 affinity at pH 7.82 colnpared tl) pH 6.84. This compares to a 3-fold increase fl)r hemoglobin in phosphate lmffer ahme. We then measured the effect of pH on the O,, equilibrium of the helnoglobin-antibody complex. In Fig. 3 is illustrated the effect of 6.5 moles of Fab per mole Hb prepared from serum 89Bo on the O 2 equilibrium of hemoglobin at pH 7.00 and 7.94. The effects noted before were all present at both pH values and because of the heterogeneity of the plots it is difficult to make quantitative comparison between the Bohr effect of hemoglobin-antibody complex and hemoglobin alone. A substantial Bohr effect remained at both antibody to hemoglobin ratios tested. The overall O2 affinity was 2.6 times as great at pH 7.94 as at pH 7.o. This compared to a 3-fold increase in the control for a slightly larger change in pH from 7.82 to 6.84 . 10.0 2 8.0 6.0
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F i g . 3. O - - O , H b A w i t h a n t i b o d y 8 9 B o , p H 7.00, m o l e s F a b / m o l e s H b 6. 5 : i. F i r s t p h a s c o f c u r v e : Pl/2 1.68, n 0 . 8 3 . S e c o n d p h a s e : t)1/2 -- 2.14, n - - 1.52. @ - - , ~ ) , H b A w i t h a n t i b o d y 8 9 B O , p H 7-94, m o l e s F a b / m o l e s H b = 6. 5 : i. F i r s t p h a s e o f c u r v e : Pt/2 o.66, n - - o . 8 7 . S e c o n d p h a s e : Pl/2 o . 8 t , n , 1.14 . F i g . 4. curve: A B 87, phase:
Q - - - O , H b A w i t h a n t i b o d y A B 87, p H 6 . 8 8 , m o l e s F a b / m o l e s H b 4 . o : [. F i r s t p h a s e o f fit~2 = 3 . 6 ° , n o . 6 1 . S e c o n d p h a s e : Pl/2 - 1-84, n - - 1.29. @ @, H I ) A w i t h a n t i b o d y p H 7.83, m o l e s F a b / m o l e s H b -- 4.o : 1. F i r s t p h a s e o f c u r v e : Pl/~ o . 7 3 , n - - o. 72. S e c o n d Pt/2 o . 7 3 , ~z . . 1.36.
Tile effect of Fab fragments prepared from serum 87B 7 were similar to those from serum 89Bo. In this experiment (see Fig. 4) a marked sensitivity of the 02 equilibrium to pH was noted in the presence of 4.o moles of Fab per mole hemoglobin. This is so even though the affinity at both pH values was substantially increased and the marked a s y m m e t r y as well as a decreased value of n were present. Again comparing the P~/2 at the t w o pH values the overall affinity was 2.4 times as high at pH 7.83 as at pH 6.88. Here again in the presence of 4.o moles Fab per mole hemoglobin the Bohr effect was largely preserved in the face of substantial increase in 02 affinity, decreased values of n and biphasic equilibrium curves.
Biochim. Biophys. Acla, 175 ( I 9 6 9 ) 49 54
EFFECT OF ANTIBODY ON HEMOGLOBIN
~3
DISCUSSION
Specific antibody to hemoglobin has a profound effect on the affinity of hemoglobin for 02 and markedly affects the shape of the O2 equilibrium curve. Interestingly, there is a substantial preservation of the Bohr effect even at the highest mole ratios of antibody employed. If one thinks of specific antibody as a ligand in the same way as 02, protons, bromthymol blue, etc. the effect of antibody on the 02 affinity is predictable on the basis of the reciprocal effects known to affect such ligand-hemoglobin systems. For example, as oxyhemoglobin has less affinity for protons than does deoxyhemoglobin, at acid pH hemoglobin has less affinity for 02 than at a more alkaline pH. Sinfilarly, deoxyhemoglobin binds bromthymol blue more firnfly than oxyhemoglobin and as expected hemoglobin has a lower affinity for 02 in the presence of bromthymol blue than in its absence 9. Thus, since oxyhemoglobin reacts better with specific antibody than deoxyhemoglobin, it is not surprising that specific antibody increases the oxygen affinity of human hemoglobin. This might be another reflection of the generalization that any ligand which reacts better with oxyhemoglobin than deoxyhemoglobin increases oxygen affinity (haptoglobin 1°, specific antibody, p-hydroxymercuribenzoaten, 12) while those that react better with deoxyhemoglobin lower the 02 affinity (Frotons, b r c m t h y m o l blue 9, 2,3-diphosphoglycerate13). M()re interesting perhaps is the marked effect of specific antibody on the shape of the equilibrium curve. The asymmetric curves can be resolved into at least 2 comFonents in the Hill plot. Moreover, the first phase is characterized by a value of ~z less than I and the second by a value of n greater than I. Values of n less than I are seen where there is functional heterogeneity of the sites which is not normally present. In an analogous situation the oxidation-reduction equilibrium of hemoglobin bound to haptoglobin is less than I when roughly half of hemoglobin is ferri and the other half is ferro. This heterogeneity is likely due to the a and fl chains behaving as independent redox systems when bound to haptoglobin reflecting the intrinsic difference in the redox properties of the isolated chains 14. The probable heterogeneity in our case could result from a number of factors. Possible causes might be that more antibody might be directed against one chain and then when one type of chain has antibody bound and the other does not, they have markedly different 02 affinities. In another vein there is evidence from the immunological study of m u t a n t hemoglobins that specific antibody reacts with m a n y widely separated sites on the surface of both a and fl polypeptide chains. Each of these might have a somewhat different effect on the 02 affinity and the summation of such effects would be the measured equilibrium curve. The induction of methemoglobin in the course of the experiment could also lower the value of n below i but this is not adequate to explain our data. The behavior of the curves at higher saturation in the presence of antibody is quite different than at lower saturations and does exhibit some interaction. Interestingly, the behavior at high saturations, while different from normal hemoglobin, seems almost independent of the quantity of antibody bound. It seems possible that specific antibody has a marked effect on the binding of the first two O 2 molecules but a much smaller effect on the binding of the last two. Many of these effects are capable of further analysis at the immunological level and such studies are in progress. For example, we now know that a mole of tetrameric Biochim. Biophys. ,4cta, 175 (1969) 49 54
54
M. REICHLIN, L. UI)EM, H. M. RANNEY
hemoglobin can bind many more than 6 moles of Fab. Serum 87t37 has tile potential of binding at least 12 moles of Fab fragments per mole of tetrameric hemoglobin. Certainly, the effect of covering all the antigenic sites might reveal some property of the equilibrium which will enlarge our understanding of the present data. Similarly it is possible to prepare pure anti a and pure anti fi antibodies and the relative effects of binding on one or the other of the chains will tell us if either chain has a predominant role in mediating the properties we have described. And lastly, it is possible but not yet practical that antibodies can be prepared against a single antigenic determinant in a known location of the hemoglobin molecule. Such experiments could tell us something of the fine structural basis of the effects described. Studies in other immunological systems have demonstrated the feasibility of the latter approach as. Lastly, it is interesting that while there are profound effects on the equilibrium curve of hemoglobin effected by binding specific antibody there was substantial preservation of the Bohr effect. The dissociation of the effect on Oz affinity and the value of n from the Bohr effect are reminiscent of the O 2 equilibrium of hemoglobin Chesapeake 16. By further study and analysis of this system we would hope to further evaluate both the effects of conformation changes on immunological reactivity and hopefully localize sites of such structure changes by appropriately purified antibodies. ACKNOWLEDGEMENTS
This investigation was supported by Grants AM lO428 and AM o45o2 from the U.S. Public Health Service and by a grant from the Life Insurance Medical Research Fund. M.R. is the recipient of a Career Development Award (5K3-AM-2o-729) from the National Institutes of Health. H.M.R. is a Career Scientist (Contract 1-392) of the Health Research Council of the City of New York. REFERENCES i M. REICHLIN, M. HAY AND L. LEVINE, Biochemistry, 2 (1963) 971. 2 M. REICHLIN, E. B u c c I , E. ANTONINI, J. WYMAN AND A. ROSsI-FANELLI, J. Mol. Biol., 9 (1964) 785 . 3 M. REICHLIN, E. B u c c I , J. \VYMAN, E. ANTONINI AND A. ROSsI-FANELLI, dr . Mol. Biol., II (1965) 775. 4 M. REICHLIN, l~. BOCCI, C. FRONTICELLI, J. WYMAN, E. ANTONINI, C. IOPPOLO AND a . ROSSIFANELLI, J. Ntol. Biol., 17 (1966) 18. 5 M. REICHLIN, M. HAY AND L. LEVINE, lmmunochemist(~,, i (1964) 21. 6 A. NISONOFF, F. C. WtSSLER, L. N. LIPMAN AND D. L. WOERNLEY, Arch. Biochem. Biophys., 89 (I96O) 230. 7 H. M. RANNEY, R. \V. BRIEHL AND A. S. JACOBS, J. Biol., Chem., 240 (1965) 2442. 8 D. W. ALLEN, K. V. GUTHE AND J. WYMAN, J. Biol. Chem., 187 (195 o) 393. 9 E. ANTONINI, J. WYMAN, R. MORETTI AND A. ROSSI-FANELLI, Biochim. Biophys. Aeta, 71 (1963) 124 • i o R. L. NAGEL, J. B. WITTENBERG AND H. M. RANNEY, Biochim. Biophys. Acta, IOO (1965) 280. I I S. A. MORELL, V. E. AYERS, P. HOFFMAN AND F. TAKETA, Proc. Natl. Acad. Sci. U.S., 48 (1962) lO57. 12 t . E. BENESCH AND R. BENESCH, Biochemistry, i (1962) 735. 13 R. BENESCH AND R. E. BENESCH, Federation Proc., 27 (1968) 339. 14 M. BRUNORI, A. ALFSEN, U. SAGGESE, E. ANTONINI AND J. WYMAN, J. Biol. Chem., 243 (1968) 2950. 15 A. NISONOFF, E. MARGOLIASH AND M. REICHLIN, in preparation. 16 ]R. L. NAGEL, Q. H. GIBSON AND S. CHARACHE, Biochemistry, 6 (1967) 2395. 17 R. BENESCH, G. MAcDuFF AND R. E. BENESCH, Anal. Biochem., i i (1965) 81.
Bioehim. Biophys. Aeta, 175 (1969) 49-54
49
BBA 35317 T H E E F F E C T OF SPECIFIC A N T I B O D Y ON T H E O X Y G E N E Q U I L I B R I U M OF HUMAN H E M O G L O B I N
M O R R I S R E [ C H L I N , L I N D A U D E M AND H E L E N M. R A N N E Y
Department of Medicine, State University of New York at Buffalo School of Medicine, Buffalo, N.Y. ~42o3, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, N . Y . zo46r (U.S.A.) (Received A u g u s t 7th, 1908 )
SUMMARY
Specifically purified anti A 1 hemoglobin antibody has a marked effect on the 0 2 equilibrium of human A1 hemoglobin. The effects noted were an increased 0 2 affinity, decreased value of n, and marked a s y m m e t r y of the 0 2 equilibrium curve. There was substantial preservation of the Bohr effect at all antibody to hemoglobin ratios tested.
INTRODUCTION
The immunochemical study of hemoglobin has been approached as a model system in which the effect of reversible conformational changes on antigenic specificity could be assessed. Such studies initially included the comparison of apohemoglobin and hemoglobin, apomyglobin and myoglobin 1 and more recently a study of the difference between oxy- and deoxyhemoglobin 2 4. These studies have shown t h a t human oxyhemoglobin and deoxyhemoglobin are easily distinguished by their reactivity with specific rabbit antibody to human hemoglobin. These differences were measured by studying the reactions of the two forms of hemoglobin with specific antiserum in quantitative complement fixation experiments. Such differences in the imnmnological behavior of the two forms of hemoglobin were interpreted in terms of the conformational changes known to accompany reactions with 0 2. Thus, since oxy- and deoxyhemoglobin differ immunologically one might expect that specific antibody would affect the 0 2 equilibrium. This report includes our initial studies of the effect of the purified rabbit specific antibody on the 0 2 equilibrium of human hemoglobin. MATERIALS AND METHODS
Antisera to A 1 hemoglobin were obtained from rabbits immunized by a schedule of injections previously described 5. Specific antihemoglobin antibody was purified in the following way. A specific Biochim. Biophys. Acta, 175 (1969) 49 54
5°
M. R E 1 C H I . I N , L. UI)EXI, H. M. I~:\NNEY
precipitate %rmed at equivalence with oxyheinoglobin (as judged by quantitative precipitin data for each serum) was washed 3 :~ with cold saline, and dissolved in o.I M acetic acid, and applied to a Sephadex G-Ioo colunm, 2.5 cm ~. 40.o cm, equilibrated with o.I M acetic acid. Substantial resolution of antibody and antigen was achieved although some heine color was found in the antibody peak. When the material in the excluded volume was neutralized by dialysis against o.i M phosphate buffer (pH 7.o), a small colored precipitate formed. The supernate was clear, colorless, and contained a yield of 7o% of the specific antibody in the original immune precipitate. This soluble material, a 7-S antibody which was active in precipitation and complement fixation reactions, was digested with i°~£, by weight of pepsin in o.oi M cysteine at p H 4.o as described by NISONOFF et al. s. Following digestion for 24 h and alkylation with a 3 × molar excess of iodoacetamide, the pepsin was inactivated by dialysis against pH 8.o borate buffer for 24.o h. Ultracentrifugation revealed a single 3.5-S peak, typical of rabbit Fab fragments. Approx. 95<}.i) of these antihemoglobin Fab fragments were capable of binding hemoglobin as judged by experiments on Sephadex G-Ioo. Concentration of the alkylated Fab fragments was estimated by using an absorbance value of I5.O for a I°,o solution at 28o m# in pH 7.o, o.I M phosphate buffers. The O 2 equilibria were determined by a spectrophotometric method which is a modification 7 of that described by ALLEN, WYMAN AND GUTHEs. The equilibria were all performed in o.I M phosphate buffers at IO°. Only 5 to 6 points were determined for each study in order to minimize formation of methemoglobin; unfortunately sufficient purified antibody was not available for the multiple tonometers necessary for more detailed studies of the saturation curve. RESULTS
Effect of antibody on the oxygen equilibrium Non-immune y-globulin in a mole ratio of 5.7 moles Fab/mole Hb had no effect on the 02 equilibrium (Fig. i). The calculated values for n* and Pl/2* were not different in the presence and absence of non-immune globulin. A titration of hemoglobin with Fab fragments was performed to determine the effect of binding increasing quantities of Fab fragments to the hemoglobin. The complex results of the experiments are illustrated in Fig. 2. At all ratios of Fab to Hb tested there was an effect on both the Pl/z and the value of n and in all cases the equilibrium curve was asymmetrical and the Hill plot was apparently a biphasic curve. The portion of the equilibrium at lower saturation we have designated as Phase I and that at higher saturation, Phase 2. Even at the lowest mole ratio of Fab fragment to hemoglobin (2.2 : I) there was a substantial effect on the value of n and a small increase in O~ affinity. This represented the effect of a single Fab fragment per ~/5 unit. As the quantity of antibody was increased the proportion of the hemoglobin exhibiting higher 02 affinity increased. In addition, as the quantity of antibody was increased the fraction of the equilibrium in the first phase increased and it was the 02 affinity of the first phase that was greatly affected by antibody. In the presence of 2.2, 4.4, and 6.15 moles of Fab per mole Hb the fractional saturation (y) * n is t h e s l o p e o f t h e H I L L p l o t o r l o g Y/i-y/log pO 2 a n d Pl/2 is t h e p O 2 a t w h i c h t h e h e m o g l o b i n is h a l f s a t u r a t e d .
Biochim. Bioph3,s. Acla, i 7 5 (1969) 4 9 54
EFFECT OF ANTIBODY ON HEMOGLOBIN
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Fig. I. 0 - - 0 , H b A w i t h y-globulin, p H 6.99, moles F a b / m o l e s H b = 5 . 7 : I . P112 = 4.13, n = 2.47. M e t h e m o g l o b i n : initial, o % , final 13%. 0 - - 0 , H b A control, p H 6.97. P x / 2 = 4.13, n = 2.47. M e t h e m o g l o b i n : initial, o % ; final, 8 % . Fig. 2. Curv e i, H b A w i t h a n t i b o d y 87B 7, p H 6.98, moles F a b / m o l e s H b -- 2.2:1. F i r s t p h a s e of c u r v e : P l / 2 - - 3-4 °, n 0.83. Second p h a s e : P l / ~ - - 2.47, n -- 1.52. M e t h e m o g l o b i n : i n i t i a l , 0 % ; final, i i ~ o . Curve 2, H b A w i t h a n t i b o d y 87B 7, p H 6.98, moles F a b / m o l e s H b -- 4.4:1. F i r s t p h a s e of c u r v e : P l / ~ - - 2.45, n = 0.62. Second p h a s e : P l / ~ - - 2.22, n -- 1.43. M e t h e m o g l o b i n : initial, o% ; final, 11%. Curve 3, H b A w i t h a n t i b o d y 87B7, p H 6.99, moles F a b / m o l e s H b -6.15:1. F i r s t p h a s e of c u r v e : Pl/.2 - - 1-64, n -- 0.67. Second p h a s e : P l / 2 = 2.11, n = 1.4o. Methem o g l o b i n : initial, 1 . 8 % ; final, 2 4 % .
observed in the first phase was 33, 47, and 6o%, respectively. The values for P112 were 3.4 o, 2.45, and 1.64 in the first phase, but differed little inter se in the second phase. Indeed it appeared that once some critical saturation characteristic of each ratio of antibody to hemoglobin was reached, the remainder of the saturation curve was not altered significantly by different amounts of antibody. Another feature of these experiments was that the slope of the first phase of the Hill plot was always less than I and the slope of the second phase was greater than i. The proportion of methemoglobin (in samples of pH below 7.o) was estimated from the data of BENESCH, MACDUFF AND BENESCH17: in most of the runs, little or no methemoglobin was present at the beginning of the experiments. At the conclusion of the 02 equilibria studies, II-24~o (av. 15%) methemoglobin was found in the experiments in which specific antisera were added, and 13% in the control experiment in which ;~-globulin was used. While these methemoglobin values were high, they did not appear sufficient to account for the decreased values of n. Lastly we prepared Fab fragments from another rabbit antiserum to human hemoglobin (82B7) and tested the effect of a single Fab to Hb mole ratio of 3-4. Although there were quantitative differences in the effect of this preparation, the features noted before were also present here: asymmetry of the Oz equilibrium curve with resolution into two phases in the Hill plot, increased O~ affinity and a substantial decrease in the value of n.
Effect of antibody on the Bohr effect Control experiments with non-immune 7-globulin demonstrated the absence of Biochim.
Biophys.
Acta,
175 (1969) 49-54
M. R E I ( H I A N ,
52
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any non-specific effect of ().5 moles of Fab fragmentsper m{)le ()f hem<,globin. With 6. 5 moles of Fab there was a 3-fold increase in 02 affinity at pH 7.82 colnpared tl) pH 6.84. This compares to a 3-fold increase fl)r hemoglobin in phosphate lmffer ahme. We then measured the effect of pH on the O,, equilibrium of the helnoglobin-antibody complex. In Fig. 3 is illustrated the effect of 6.5 moles of Fab per mole Hb prepared from serum 89Bo on the O 2 equilibrium of hemoglobin at pH 7.00 and 7.94. The effects noted before were all present at both pH values and because of the heterogeneity of the plots it is difficult to make quantitative comparison between the Bohr effect of hemoglobin-antibody complex and hemoglobin alone. A substantial Bohr effect remained at both antibody to hemoglobin ratios tested. The overall O2 affinity was 2.6 times as great at pH 7.94 as at pH 7.o. This compared to a 3-fold increase in the control for a slightly larger change in pH from 7.82 to 6.84 . 10.0 2 8.0 6.0
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F i g . 3. O - - O , H b A w i t h a n t i b o d y 8 9 B o , p H 7.00, m o l e s F a b / m o l e s H b 6. 5 : i. F i r s t p h a s c o f c u r v e : Pl/2 1.68, n 0 . 8 3 . S e c o n d p h a s e : t)1/2 -- 2.14, n - - 1.52. @ - - , ~ ) , H b A w i t h a n t i b o d y 8 9 B O , p H 7-94, m o l e s F a b / m o l e s H b = 6. 5 : i. F i r s t p h a s e o f c u r v e : Pt/2 o.66, n - - o . 8 7 . S e c o n d p h a s e : Pl/2 o . 8 t , n , 1.14 . F i g . 4. curve: A B 87, phase:
Q - - - O , H b A w i t h a n t i b o d y A B 87, p H 6 . 8 8 , m o l e s F a b / m o l e s H b 4 . o : [. F i r s t p h a s e o f fit~2 = 3 . 6 ° , n o . 6 1 . S e c o n d p h a s e : Pl/2 - 1-84, n - - 1.29. @ @, H I ) A w i t h a n t i b o d y p H 7.83, m o l e s F a b / m o l e s H b -- 4.o : 1. F i r s t p h a s e o f c u r v e : Pl/~ o . 7 3 , n - - o. 72. S e c o n d Pt/2 o . 7 3 , ~z . . 1.36.
Tile effect of Fab fragments prepared from serum 87B 7 were similar to those from serum 89Bo. In this experiment (see Fig. 4) a marked sensitivity of the 02 equilibrium to pH was noted in the presence of 4.o moles of Fab per mole hemoglobin. This is so even though the affinity at both pH values was substantially increased and the marked a s y m m e t r y as well as a decreased value of n were present. Again comparing the P~/2 at the t w o pH values the overall affinity was 2.4 times as high at pH 7.83 as at pH 6.88. Here again in the presence of 4.o moles Fab per mole hemoglobin the Bohr effect was largely preserved in the face of substantial increase in 02 affinity, decreased values of n and biphasic equilibrium curves.
Biochim. Biophys. Acla, 175 ( I 9 6 9 ) 49 54
EFFECT OF ANTIBODY ON HEMOGLOBIN
~3
DISCUSSION
Specific antibody to hemoglobin has a profound effect on the affinity of hemoglobin for 02 and markedly affects the shape of the O2 equilibrium curve. Interestingly, there is a substantial preservation of the Bohr effect even at the highest mole ratios of antibody employed. If one thinks of specific antibody as a ligand in the same way as 02, protons, bromthymol blue, etc. the effect of antibody on the 02 affinity is predictable on the basis of the reciprocal effects known to affect such ligand-hemoglobin systems. For example, as oxyhemoglobin has less affinity for protons than does deoxyhemoglobin, at acid pH hemoglobin has less affinity for 02 than at a more alkaline pH. Sinfilarly, deoxyhemoglobin binds bromthymol blue more firnfly than oxyhemoglobin and as expected hemoglobin has a lower affinity for 02 in the presence of bromthymol blue than in its absence 9. Thus, since oxyhemoglobin reacts better with specific antibody than deoxyhemoglobin, it is not surprising that specific antibody increases the oxygen affinity of human hemoglobin. This might be another reflection of the generalization that any ligand which reacts better with oxyhemoglobin than deoxyhemoglobin increases oxygen affinity (haptoglobin 1°, specific antibody, p-hydroxymercuribenzoaten, 12) while those that react better with deoxyhemoglobin lower the 02 affinity (Frotons, b r c m t h y m o l blue 9, 2,3-diphosphoglycerate13). M()re interesting perhaps is the marked effect of specific antibody on the shape of the equilibrium curve. The asymmetric curves can be resolved into at least 2 comFonents in the Hill plot. Moreover, the first phase is characterized by a value of ~z less than I and the second by a value of n greater than I. Values of n less than I are seen where there is functional heterogeneity of the sites which is not normally present. In an analogous situation the oxidation-reduction equilibrium of hemoglobin bound to haptoglobin is less than I when roughly half of hemoglobin is ferri and the other half is ferro. This heterogeneity is likely due to the a and fl chains behaving as independent redox systems when bound to haptoglobin reflecting the intrinsic difference in the redox properties of the isolated chains 14. The probable heterogeneity in our case could result from a number of factors. Possible causes might be that more antibody might be directed against one chain and then when one type of chain has antibody bound and the other does not, they have markedly different 02 affinities. In another vein there is evidence from the immunological study of m u t a n t hemoglobins that specific antibody reacts with m a n y widely separated sites on the surface of both a and fl polypeptide chains. Each of these might have a somewhat different effect on the 02 affinity and the summation of such effects would be the measured equilibrium curve. The induction of methemoglobin in the course of the experiment could also lower the value of n below i but this is not adequate to explain our data. The behavior of the curves at higher saturation in the presence of antibody is quite different than at lower saturations and does exhibit some interaction. Interestingly, the behavior at high saturations, while different from normal hemoglobin, seems almost independent of the quantity of antibody bound. It seems possible that specific antibody has a marked effect on the binding of the first two O 2 molecules but a much smaller effect on the binding of the last two. Many of these effects are capable of further analysis at the immunological level and such studies are in progress. For example, we now know that a mole of tetrameric Biochim. Biophys. ,4cta, 175 (1969) 49 54
54
M. REICHLIN, L. UI)EM, H. M. RANNEY
hemoglobin can bind many more than 6 moles of Fab. Serum 87t37 has tile potential of binding at least 12 moles of Fab fragments per mole of tetrameric hemoglobin. Certainly, the effect of covering all the antigenic sites might reveal some property of the equilibrium which will enlarge our understanding of the present data. Similarly it is possible to prepare pure anti a and pure anti fi antibodies and the relative effects of binding on one or the other of the chains will tell us if either chain has a predominant role in mediating the properties we have described. And lastly, it is possible but not yet practical that antibodies can be prepared against a single antigenic determinant in a known location of the hemoglobin molecule. Such experiments could tell us something of the fine structural basis of the effects described. Studies in other immunological systems have demonstrated the feasibility of the latter approach as. Lastly, it is interesting that while there are profound effects on the equilibrium curve of hemoglobin effected by binding specific antibody there was substantial preservation of the Bohr effect. The dissociation of the effect on Oz affinity and the value of n from the Bohr effect are reminiscent of the O 2 equilibrium of hemoglobin Chesapeake 16. By further study and analysis of this system we would hope to further evaluate both the effects of conformation changes on immunological reactivity and hopefully localize sites of such structure changes by appropriately purified antibodies. ACKNOWLEDGEMENTS
This investigation was supported by Grants AM lO428 and AM o45o2 from the U.S. Public Health Service and by a grant from the Life Insurance Medical Research Fund. M.R. is the recipient of a Career Development Award (5K3-AM-2o-729) from the National Institutes of Health. H.M.R. is a Career Scientist (Contract 1-392) of the Health Research Council of the City of New York. REFERENCES i M. REICHLIN, M. HAY AND L. LEVINE, Biochemistry, 2 (1963) 971. 2 M. REICHLIN, E. B u c c I , E. ANTONINI, J. WYMAN AND A. ROSsI-FANELLI, J. Mol. Biol., 9 (1964) 785 . 3 M. REICHLIN, E. B u c c I , J. \VYMAN, E. ANTONINI AND A. ROSsI-FANELLI, dr . Mol. Biol., II (1965) 775. 4 M. REICHLIN, l~. BOCCI, C. FRONTICELLI, J. WYMAN, E. ANTONINI, C. IOPPOLO AND a . ROSSIFANELLI, J. Ntol. Biol., 17 (1966) 18. 5 M. REICHLIN, M. HAY AND L. LEVINE, lmmunochemist(~,, i (1964) 21. 6 A. NISONOFF, F. C. WtSSLER, L. N. LIPMAN AND D. L. WOERNLEY, Arch. Biochem. Biophys., 89 (I96O) 230. 7 H. M. RANNEY, R. \V. BRIEHL AND A. S. JACOBS, J. Biol., Chem., 240 (1965) 2442. 8 D. W. ALLEN, K. V. GUTHE AND J. WYMAN, J. Biol. Chem., 187 (195 o) 393. 9 E. ANTONINI, J. WYMAN, R. MORETTI AND A. ROSSI-FANELLI, Biochim. Biophys. Aeta, 71 (1963) 124 • i o R. L. NAGEL, J. B. WITTENBERG AND H. M. RANNEY, Biochim. Biophys. Acta, IOO (1965) 280. I I S. A. MORELL, V. E. AYERS, P. HOFFMAN AND F. TAKETA, Proc. Natl. Acad. Sci. U.S., 48 (1962) lO57. 12 t . E. BENESCH AND R. BENESCH, Biochemistry, i (1962) 735. 13 R. BENESCH AND R. E. BENESCH, Federation Proc., 27 (1968) 339. 14 M. BRUNORI, A. ALFSEN, U. SAGGESE, E. ANTONINI AND J. WYMAN, J. Biol. Chem., 243 (1968) 2950. 15 A. NISONOFF, E. MARGOLIASH AND M. REICHLIN, in preparation. 16 ]R. L. NAGEL, Q. H. GIBSON AND S. CHARACHE, Biochemistry, 6 (1967) 2395. 17 R. BENESCH, G. MAcDuFF AND R. E. BENESCH, Anal. Biochem., i i (1965) 81.
Bioehim. Biophys. Aeta, 175 (1969) 49-54