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Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 185, No. 2, January 30, pp. 504-510, 1978
Equilibrium and Kinetic Measurements of Carbon Monoxide Binding to Hemoglobin Kansas in the Presence of lnositol Hexaphosphate’ C. L. CASTILLO,*
S. OGAWA,*
* Bell Laboratories, Inc., Murray Hill, New Biomedicinal Chemistry and the Cardiovascular Veterans Administration Received
August
AND
J. M. SALHANYt
Jersey 07974, and i Departments of Biochemistry and Center, University of Nebraska Medical Center and Hospital, Omaha, Nebraska 68105
4, 1977; revised
October
13, 1977
Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun. 49, 1485-1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity. as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO (P,,, = 1.2 mm Hg and K,, = 5.4 x lo” M-l) and almost no cooperativity (n = 1.1) at pH 7, 25°C. The CO “on” and “off’ kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K,, = 5.2 x 10s Mm1 with k, = 1.03 x lo5 Mml.K1 and k,,, = 0.198 s-i) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.
Hemoglobins identified as being in the oxy quaternary structure (R state) by crystallography and/or by nmr2 and CD spectroscopic measurements have been shown to have little or no cooperativity in ligand binding by both equilibrium and kinetic techniques (l-9). The complete ligand binding process to the deoxy quaternary structure (T state) of adult hemoglobin is more difficult to obtain due to the highly cooperative nature of this hemoglobin. Recently, attention has turned to two interesting mutant hemoglobins where structural studies have shown that a fully
liganded deoxy-like quaternary structure can be formed. These two hemoglobins are Hb M Iwate (LY*87 His + Tyr &) and Hb Kansas (cr.& 102 Asn + Thr). There was some disagreement about the presence or absence of cooperative CO binding to the two active sites of Hb M Iwate (10-13). Salhany et al. (14) have recently studied this problem and were able to show that the tetramer binds CO noncooperatively. Similar detailed measurements have not been reported for Hb Kansas. This would seem to be important information to have, since this mutant has four active ligand binding sites and the CO form of the tetramer can be maintained in the deoxy quaternary structure by the addition of IHP (15). Although several reports exist on the CO binding kinetics to this mutant (16-l@, CO binding at equilibrium as well as CO “off’ and “on” kinetics under sufficiently comparable conditions would seem to be required before we could unambiguously conclude that CO binding is or is
1 The portion of this work performed at the University of Nebraska Medical Center was supported by funds from the Cardiovascular Center and the Medical Research Service of the Veterans Administration. 2 Abbreviations used: nmr, nuclear magnetic resonance; CD, circular dichroism; IHP, inositol hexaphosphate; Bistris, bis(2-hydroxyethyl)iminotris(hydroxymethyllmethane; Hb, hemoglobin; Mb, myoglobin. 504 0003~9861/78/1852-0504$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
CO
BINDING
TO HEMOGLOBIN
not cooperative with IHP present. The purpose of this study was to obtain kinetic constants for CO binding and release and to measure CO binding at equilibrium for Hb Kansas + IHP. MATERIALS
AND
METHODS
Hemoglobin Kansas was prepared in the carbon monoxide form, as described previously (15, 191, from freshly drawn blood donated by Mr. David Boyce. Horse myoglobin (Mb) was prepared by chromatography on CM-Sephadex columns after reducing Mb purchased from Sigma (St. Louis, Missouri) in the presence of CO. Gases (CO and NO) were from Matheson (East Rutherford, New Jersey, and Chicago, Illinois). IHP was from Sigma (Type V, sodium salt). The equilibrium CO binding curves were measured by optical absorption spectroscopy after adding known quantities of CO gas successively into a loo-ml tonometer equipped with a 2-mm optical cell. The hemoglobin solution in the tonometer was equilibrated for 30 min in a water bath whose temperature was regulated at 25 + 0.4”C. The sample holder in a Cary 14 spectrophotometer was also maintained at 25°C. For highly concentrated hemoglobin solutions (8-10 mM in hemel, gas mixtures of CO and N, at known ratios were allowed to flow through the tonometer until the gas phase and solution reached equilibrium, all in the same water bath. Deviation of the various spectra from the isosbestics was less than 0.5% of the optical density at that wavelength. Stopped-flow kinetic experiments were performed using a Durrum-Gibson stopped-flow apparatus, interfaced to an On-Line-Instruments-Systems (OLIS, Athens, Georgia) data acquisition system. The system is equipped with a Data General Nova 2/10 computer with 24K of core memory. Typical experiments involved sampling 1000 data points per reaction and accumulating and averaging at least three reactions per experimental condition. The data were analyzed, plotted, and/or read out numerically, all under computer control.
KANSAS
505
deoxy hemes of the (Y and p chains in the tetramer has a maximum, while at 535 nm the spectral difference has an opposite sign (1, 20). Without IHP, the P,,, value in 0.2 M Bistris buffer, pH 7, was 0.14 mm Hg and there was substantial cooperativity (Hill’s constant, n = 2.4). In the presence of 0.4 mM IHP the CO affinity was lowered significantly (P1,2 = 1.2 mm Hg) and the n value dropped to 1.1. Hill’s constant was reproducibly slightly larger than unity under the experimental conditions. The same value of n. was also observed at an 8 to 10 mrvr total heme concentration. Figure 2 shows CO binding kinetics to Hb Kansas + IHP as a function of wavelength. An excellent isosbestic was observed at 424.6 nm (Fig. 2A). When the data at 432 and 417 nm are plotted semilogarithmically (Fig. 2B), homogeneous, wavelength-independent, pseudo-first-order kinetics are observed with a rate constant of 1.03 x lo5 M-“5-I. Gibson et al. (16) have studied the same reaction and obtained a value for this rate constant of 0.9 X lo5 M-’ .s-l at 20°C in 0.05 M Bistris, pH 7, at 7 PM heme. We do not believe that these numbers are significantly different. The slight difference seen may well be due to the difference in temperature between the two experiments. The fact that we were unable to detect any signifi-
RESULTS
Hill plots for CO binding to Hb Kansas at pH 7, and to myoglobin for comparison, are shown in Fig. 1. There was very little wavelength dependence in the value of Y (fraction of CO saturation) for these experiments with Hb Kansas. The value of Y at 590 and 535 nm differed by less than 0.01. It has been previously shown that 590 nm is one of the wavelengths where the optical heterogenerity between the
FIG. 1. Hill plots of CO binding to Mb (A) and to Hb Kansas without (B) and with (C) IHP at pH 7, 25”C, in 0.2 M Bistris buffer. The concentration of Hb Kansas was 0.5 mM in heme, and the IHP concentration was 0.4 mM. The broken line near curve C is a calculated curve for the case where the fourth CO binding is stronger by a factor of 2 than the other three CO binding constants, K, = Vz K,, K, = K2 = K:,.
506
CASTILLO.
WILlISECONDS
OGAWA.
NILLISROWOS
2. Carbon monoxide binding to hemoglobin Kansas plus IHP. Conditions were 2X, 0.2 M Bistris, pH 7.0; [CO] = 1 mM, [heme] = 45.1 PM, [IHPI = 1 mM, before the mix. The slit width was 0.15 mm and the path length was 2 mm. (A) Time courses of the absorbance changes at 432, 417, and 424.6 nm. The facts that the curves at 432 and 417 nm have the same shape and that there was an excellent isosbestic point at 424.6 nm very strongly suggest that under these conditions no spectral or kinetic heterogeneity in CO binding is present. (B) Normalized time courses of the reactions in A showing that the time courses at these two wavelengths of opposite sign present good pseudo-first-order kinetic behavior with the same second-order rate constant (1.03 x lo5 M-‘.s-‘). FIG.
cant wavelength dependence for Hb Kansas + IHP under our conditions agrees with the equilibrium results discussed above, where no significant difference in the value of Y was noted as a function of wavelength. However, Gibson et aE. (16) did note that CO binding to extremely dilute (3 PM) solutions of Hb Kansas was wavelength dependent in the absence of IHP. As shown previously (18, 211, the CO replacement reaction by NO can be complicated by changes in quaternary structure and attendent nonlinear spectral changes in the NO hemes of the low-affinity state. Phosphate-free adult hemoglobin did not show spectral-kinetic heterogeneity since it did not change quaternary structure according to the nmr and CD spectroscopic measurements (1821). However, both relatively concentrated Hb Kansas (212 PM, before the mix) stripped of phosphate and Hb A + IHP did show spectral-kinetic heterogeneity, and the structural indicators showed that the T state does form with NO as the ligand. At
AND
SALHANY
wavelengths isosbestic for this extraspectral change in the NO hemes, we saw apparently autocatalytic CO release kinetics. With this in mind, we might expect very dilute solutions of stripped Hb Kansas (i.e., the dimer) to show no acceleration and little or no wavelength dependence. The results in Fig. 3 (A and B) seem to confirm these expectations. Figure 3A shows the time course of the spectral change when CO is replaced by NO at 4.1 PM heme (after the mix). We see that homogeneous kinetics occur with only a very slight deviation from isosbesty at 412.8 nm. A semilog plot of the data at 415 and 405.7 nm shows only about a 20% difference in rate. Neither reaction presents any significant deviation from linearity. When the data at 405.7 nm (an isosbestic for the NO color change) are compared with phosphate-free Hb A at the same wavelength and temperature, we see no significant differences. These
;?r;;“m,jo ,bQ zoQ ic yj SECQHOS
0
y;; 40 80 120 IO 200 240 StCQNDS
FIG. 3. The HbCO + HbNO replacement reaction for very dilute, phosphate-free Hb Kansas at several wavelengths. (A) Time course of the absorbance change at 415,412.8, and 405.7 nm. The concentration of heme after the mix was 4.1 pM. The protein was in 0.2 M Bistris, pH 7.0, + 0.1 M NaCl. Temperature = 25 2 1°C. (B) Normalized time course of the reactions at 415 and 405.7 nm in A and of adult Hb under the same solution conditions at 405.7 nm. The reactions for dilute Hb Kansas and Hb A (30 pM after the mix) are the same at 405.7 nm and 25 -t 1°C (0.012 s’). The reaction for Hb Kansas at 415 nm is about 20% faster. There is also a large spectral change in the NO hemes at this wavelength when the tetramer forms. Edelstein’s (22) recent ultracentrifugation measurements have shown that fully saturated Hb NO Kansas will be more associated than Hb CO Kansas under otherwise similar conditions. Thus, we might expect some small fraction of tetramers to form late in this reaction, causing problems in correctly determining the baseline for the reaction at 415 nm.
CO
BINDING
TO
HEMOGLOBIN
507
KANSAS
results suggest that there is little significant difference in the CO off rate between the cx and /3 chains in the dimer of Hb Kansas. At higher protein concentration, the spectral-kinetic heterogeneity observed (18, 21) would seem to be a contribution of tetrameric Hb Kansas and not necessarily chain inequivalence, unless the latter is protein concentration dependent.
At nmr concentrations of heme, Qgawa et al. (15) showed that the addition of IHP caused fully CO-saturated Hb Kansas to switch to the deoxy quaternary structure as judged by changes in several nmr lines. Salhany et al. (18, 21) measured the CO off rate from Hb Kansas + IHP at a lower protein concentration and did observe a considerably faster (factor of -10) CO off rate as compared with the stripped dimer of Hb Kansas or phosphate-free adult Hb. However, the reaction did show some kinetic heterogenity. Owing to this and to the fact that Cl- can critically affect the value of K4,2 for Hb Kansas, even with IHP present (22, 231, it seemed possible that the experiments reported by Salhany et al. (18, 21) for Hb Kansas + IHP could have some dimer contributions. In order to investigate this, and to obtain the value of the CO off rate for the tetramer of Hb Kansas + IHP, we investigated the protein and IHP concentration dependence of the CO + NO replacement reaction for Hb Kansas in 0.2 M Bistris (-0.05 M Cl-) at pH 7 and 25°C. The results in Fig. 4 (A and B) show the CO -+ NO reaction for Hb Kansas at roughly constant initial protein concentration (0.66 to 0.79 mM heme) and variable IHP concentration (0.2 to 15 mM). The reaction does show heterogeneous kinetics when the IHP concentration is about stoichiometric with tetramer (i.e., assuming all of the hemes were in the tetrameric form). Increasing the IHP concentration
above this level causes the reaction to become linear (Fig. 4B, curve D). At 15 mM IHP, the reaction begins to show an initial acceleration. These results have at least two possible interpretations. First, if the protein were all tetrameric and no dimer were present or generated by the
I_-,-~ m::
___-_ !I :I
5’
LT~_
1
;c il r’:lw
__
4.
2.
FIG. 4. CO + NO replacement reaction for Hb Kansas + IHP as a function of IHP concentration. The initial protein concentrations were kept nearly constant (0.66 to 0.79 mM) and IHP concentration was varied: (A) 0.2 mrq (B) 0.95 mrq (C) 2 mM; (D) 6 mM; and (El 15 mM. The reaction was studied at 589 nm in 0.2 M Bistris, pH 7, at 25 2 1°C. The concentrations are before the mix values.
mix, it could be suggested that the chains in the tetramer become inequivalent upon the addition of IHP at “stoichiometric” levels. Further addition of IHP could lead to binding to a hypothetical second site which predominately would affect CO release from the slow chain. The net result would be apparent kinetic equivalence between the chains at high IHP. Unfortunately, studying this reaction as a function of wavelength cannot be used to test this rather complex possibility due to the nonlinear spectral change which occurs in the NO heme, even in nitrosyl Hb Kansas (18, 21). A second and perhaps more plausible interpretation would be that 0.2 mM IHP simply does not provide enough binding energy to sufficiently increase the tetramer population of the CO form of this hemoglobin under our specific solution conditions (0.2 M Bistris., pH 7? -0.05 M Cl-). If dimers are contributing to the heterogeneity seen in Fig. 4, we might expect the slow phase of the reaction to have a rate comparable to that of the dimer and, more important, we should see the fraction of slow phase decrease as the concentration of IHP increases. This effect is seen most clearly in the results shown in Fig. 5, where the initial protein concentration was about 0.14 to 0.2 mM, with IHP first at 0.05 and then at 1 mM. Curve A shows
508
CASTILLO,
OGAWA,
a marked slow phase with a rate constant (0.029 s-l) similar to that of the dimer (Fig. 3). The slow phase accounts for about 38% of the reaction under these particular conditions. It should also be noted that there is a marked lag period in curve A, The presence of this lag period and its length were protein concentration dependent at constant IHP (results not shown). Raising the IHP concentration by a factor of 20 under these conditions caused a marked reduction in the fraction of slow phase (-13% slow phase) and an apparent reduction in the length of the lag period. Although we have not attempted to quantitatively interpret these rather complex reactions, the fact that the lag period and fraction of slow phase are both protein and IHP concentration dependent seems to qualitatively suggest that they are due to the presence of dimers. The similarity of the rate constant for the slow phase to that for the dimer would also be consistent with this interpretation. Unfortunately, the best experimental test of this proposal is not technically possible to perform. The test would be to increase the heme concentration beyond the 1 mM level after the mix at stoichiometric IHP concentrations. The difficulty is partly one of the high absorptions which these samples would have, as well as the limitation in NO concentration (1 mM). Based on the present interpretation, we would suggest that
FIG. 5. The CO + NO replacement reaction for Hb Kansas + IHP at about the same protein concentration (0.198 + 0.139 rnM in heme) and at 0.05 mM (A) and 1 mM (B) IHP, before the mix. The reactions (A and B) were carried out at 589 nm in a 2-cm cell at 25 f 1°C in 0.2 M Bistris, pH 7.
AND
SALHANY
the most valid measurement of the CO off rate from the tetrumer of Hb Kansas + IHP would be given by the results in Fig. 4, curve C or D (2-6 mM IHP, 0.795 mM heme, before the mix), which show largely homogeneous kinetics and a rate constant of 0.198 s-l at 25°C. This value is somewhat larger than that reported previously (18, 21). We do not know the exact reason for this difference. It is possible that the chloride concentration was higher than that estimated in the previous experiments. This would tend to increase K4,2 and thereby decrease the initial concentration 01’ tetramers, even with IHP present (22, 23). DISCUSSION
Previous nmr studies on Hb Kansas showed that the addition of IHP could produce a switch in quaternary structure at pH 7, even with four CO molecules bound (15, 24). Additional structural evidence that Hb Kansas can be maintained in the deoxy quaternary structure after binding CO came from the recent X-ray crystallographic study by Anderson (25). He showed that binding CO to crystals of deoxy Hb Kansas did not cause the crystals to crack, quite unlike the classical crystal cracking experiments of Haurowitz (26). Anderson also noted that the degree of tertiary disturbance with ligation of the T state was related more to the ligation of the particular subunit and appeared not to affect the structure of its neighbors. He pointed out that this would be in keeping with an allosteric interpretation of ligand binding to hemoglobin. Our results suggest that within the tetramer of Hb Kansas + IHP, CO binds to all subunits with a very low affinity, with little or no cooperativity, and with no evidence for significant chain inequivalence. When the CO “on” rate constant and the CO “off’ rate constant for the tetramer + IHP are used to calculate the equilibrium constant, we obtain a value which is in excellent agreement with the direct CO binding equilibrium measurements at high protein concentration (Table I). It should be noted that this agreement occurs despite the fact that the CO
CO
BINDING
TO
HEMOGLOBIN TABLE
EQUILIBRIUM
AND
KINETIC
CONSTANTS
Hb Kansas (dimer) Hb A stripped Hb A stripped (first step) Hb Kansas + IHP Hb M Iwate
+ IHP
I
FOR VARIOUS
R T
9.3 x 10”” 11 x 10”” 2 x 10””
0.012b 0.0120 0.09’
TJ
1.03 x 105b
0.198b
T”
1.9 x loj’
a J. M. Salhany, unpublished flash photolysis NaCl, pH 7, 25 ? 1°C. * From this study. ’ Calculated from the kinetic measurements. d Reference 28. p Reference 27. f Reference 15. Y Measured equilibrium constant. * Reference 29. I Reference 14.
results;
off rate was measured by replacement with NO. The results presented in Table I also illustrate the correlation between the CO binding properties and the structural measurements for the selected hemoglobins shown to be in either the R or T quaternary structures. Hb M Iwate and Hb Kansas + IHP, both of which are in T even when fully liganded (Table I), show quite similar CO binding properties under reasonably comparable conditions. All of the kinetic constants are at least an order of magnitude or more different from the same kinetic constants for the noncooperative dimer of Hb Kansas or stripped adult HbCO in the R state. Furthermore, the affinities are about two orders of magnitude different. Direct comparison with the deoxy quaternary structure of stripped Hb A over its entire ligation manifold is not possible, but clearly the predominant affinity difference for CO binding calculated from the available kinetic data does appear to hold. One problem is in the determination of 1 for the T state of Hb A in the absence of phosphate. Sharma et al. (27) have recently measured I,. Their value does appear to be somewhat lower (factor of -2) than that for Hb Kansas + IHP or Hb M Iwate (Table I). It is clear from the results in Table I that those
4
WM
AT 25 ? 2°C
1 (S ‘)
S ‘)
(M-’
ligation
HEMOGLOBINS
I’
Quaternary structure
509
KANSAS
L (M-‘1 7.8 x 108’ 9.2 x IOR’ 22.2 x lo”’ 5.2 5.4 8.3 9.5
0.23’ heme,
1 mM CO in 0.2
M
Bistris
x x x x
105’ lO”,J 105’ lo”V + 0.1 M
hemoglobins identified as having the deoxy quaternary structure even when fully liganded show a very low CO affinity (-two orders of magnitude) and no evidence for significant cooperativity in CO binding. ACKNOWLEDGMENTS We thank Ms. Becky Jacobs for her aid in the preparation of this manuscript and Mr. John Friel for his skillful preparation of the illustrations presented in this report. REFERENCES 1. ANTONINI, E., AND BRUNORI, M. (1971) Hemoglobin and Myoglobin in their Reaction with Ligands, p. 296, North-Holland, Amsterdam. 2. KILMARTIN, J. V., HEWITT, J. A., AND WOOTON, J. F. (1975) J. Mol. Biol. 93, 2033218. 3. KILMARTIN, J. V., IMAI, K., AND JONES, R. T. (1975) in Erythrocyte Structure and Function (Brewer, G. J., ed.), pp. 21-35, Alan R. Liss, New York. 4. BUNN, H. F., WOHL, R. C., BRADLEY, T. B., COOLEY, M., AND GIBSON, Q. H. (1974) J. Biol. Chem. 249, 7402-7409. 5. HEWITT, J. A., AND GIBSON, Q. H. (19731 J. Mol. Biol. 74, 489-498. 6. GIBSON, Q. H., AND NAGEL, R. L. (1974) J. Biol. Chem. 249, 7255-7259. 7. MOFFAT, K., OLSON, J. S., GIBSON, Q. H., AND KILMARTIN, J. V. (1973) J. Biol. Chem. 248, 6387-6393.
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CASTILLO,
OGAWA,
8. SHULMAN, R. G., OGAWA, S., AND HOPFIELD, J. J. (1972) Arch. Biochem. Biophys. 151, 68-74. 9. IMAI, K. (1973) Biochemistry 12, 798-808. 10. GERSONDE, K., OVERKAMP, M., SICK, H., TRITTELVITZ, E., &ND JUNGE, W. (1973) Eur. J. Biochem. 39, 403-412. 11. GIBSON, Q. H., HELLER, P., AND YAKULIS, V. (1966) J. Biol. Chem. 241, 1650-1651. 12. HAYASHI, N., MOTOKAWA, Y., AND KIKUCHI, G. (1966) J. Bid. Chem. 241, 79-84. 13. NISHIKURA, K., SUGITA, Y., NAGAI, M., AND YONEYAMA, Y. (1975) J. Biol. Chem. 250, 6679-6685. 14. SALHANY, J. M., CASTILLO, C. L., AND OGAWA, S. (1976) Biochemistry 15, 5344-5349. 15. OGAWA, S., MAYER, A., AND SHULMAN, R. G. (1972) Biochem. Biophys. Res. Commun. 49, 1485-1491. 16. GIBSON, Q. H., RIGGS, A., AND IMAMURA, T. (1973) J. Biol. Chem. 248, 5976-5986. 17. HOPFIELD, J. J., OGAWA, S., AND SHULMAN, R. G. (1972) Biochem. Biophys. Res. Commun. 49, 1480-1484. 18. SALHANY, J. M., OGAWA, S., AND SHULMAN, R.
AND
SALHANY
G. (1975) Biochemistry 14, 2180-2190. 19. BONAVENTURA, J., AND RIGGS, A. (1968) J. Biol. Chem. 243, 980-991. 20. SUGITA, Y. (1975) J. Biol. Chem. 250, 1251-1256. 21. SALHANY, J. M., OGAWA, S., AND SHULMAN, R. G. (1974)Proc. Nut. Acad. Sci. USA 71, 33593362. 22. EDELSTEIN, S. J. (1975) Annu. Rev. Biochem. 44, 209-232. 23. ATHA, D. H., AND RIGGS, A. (1976) J. Biol. Chem. 251, 5537-5543. 24. SHULMAN, R. G., OGAWA, S., MAYER, A., AND CASTILLO, C. L. (1973) Ann. N.Y. Acad. Sci. 222, S-20. 25. ANDERSON, L. (1975) J. Mol. Biol. 94, 33-49. 26. HAUROWITZ, F. (1938) Z. Physiol. Chem. 254, 266. 27. SHARMA, V. S., SCHMIDT, M. R., AND RANNEY, H. M. (1976) J. Biol. Chem. 251,4267-4272. 28. SAWICKI, C. A., AND GIBSON, Q. H. (1976) J. Biol. Chem. 251, 1533-1542. 29. MAYER, A., OGAWA, S., SHULMAN, R. G., AND GERSONDE, K. (1973) J. Mol. Biol. 81, 187197.
Equilibrium and Kinetic Measurements of Carbon Monoxide Binding to Hemoglobin Kansas in the Presence of lnositol Hexaphosphate’ C. L. CASTILLO,*
S. OGAWA,*
* Bell Laboratories, Inc., Murray Hill, New Biomedicinal Chemistry and the Cardiovascular Veterans Administration Received
August
AND
J. M. SALHANYt
Jersey 07974, and i Departments of Biochemistry and Center, University of Nebraska Medical Center and Hospital, Omaha, Nebraska 68105
4, 1977; revised
October
13, 1977
Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun. 49, 1485-1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity. as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO (P,,, = 1.2 mm Hg and K,, = 5.4 x lo” M-l) and almost no cooperativity (n = 1.1) at pH 7, 25°C. The CO “on” and “off’ kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K,, = 5.2 x 10s Mm1 with k, = 1.03 x lo5 Mml.K1 and k,,, = 0.198 s-i) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.
Hemoglobins identified as being in the oxy quaternary structure (R state) by crystallography and/or by nmr2 and CD spectroscopic measurements have been shown to have little or no cooperativity in ligand binding by both equilibrium and kinetic techniques (l-9). The complete ligand binding process to the deoxy quaternary structure (T state) of adult hemoglobin is more difficult to obtain due to the highly cooperative nature of this hemoglobin. Recently, attention has turned to two interesting mutant hemoglobins where structural studies have shown that a fully
liganded deoxy-like quaternary structure can be formed. These two hemoglobins are Hb M Iwate (LY*87 His + Tyr &) and Hb Kansas (cr.& 102 Asn + Thr). There was some disagreement about the presence or absence of cooperative CO binding to the two active sites of Hb M Iwate (10-13). Salhany et al. (14) have recently studied this problem and were able to show that the tetramer binds CO noncooperatively. Similar detailed measurements have not been reported for Hb Kansas. This would seem to be important information to have, since this mutant has four active ligand binding sites and the CO form of the tetramer can be maintained in the deoxy quaternary structure by the addition of IHP (15). Although several reports exist on the CO binding kinetics to this mutant (16-l@, CO binding at equilibrium as well as CO “off’ and “on” kinetics under sufficiently comparable conditions would seem to be required before we could unambiguously conclude that CO binding is or is
1 The portion of this work performed at the University of Nebraska Medical Center was supported by funds from the Cardiovascular Center and the Medical Research Service of the Veterans Administration. 2 Abbreviations used: nmr, nuclear magnetic resonance; CD, circular dichroism; IHP, inositol hexaphosphate; Bistris, bis(2-hydroxyethyl)iminotris(hydroxymethyllmethane; Hb, hemoglobin; Mb, myoglobin. 504 0003~9861/78/1852-0504$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
CO
BINDING
TO HEMOGLOBIN
not cooperative with IHP present. The purpose of this study was to obtain kinetic constants for CO binding and release and to measure CO binding at equilibrium for Hb Kansas + IHP. MATERIALS
AND
METHODS
Hemoglobin Kansas was prepared in the carbon monoxide form, as described previously (15, 191, from freshly drawn blood donated by Mr. David Boyce. Horse myoglobin (Mb) was prepared by chromatography on CM-Sephadex columns after reducing Mb purchased from Sigma (St. Louis, Missouri) in the presence of CO. Gases (CO and NO) were from Matheson (East Rutherford, New Jersey, and Chicago, Illinois). IHP was from Sigma (Type V, sodium salt). The equilibrium CO binding curves were measured by optical absorption spectroscopy after adding known quantities of CO gas successively into a loo-ml tonometer equipped with a 2-mm optical cell. The hemoglobin solution in the tonometer was equilibrated for 30 min in a water bath whose temperature was regulated at 25 + 0.4”C. The sample holder in a Cary 14 spectrophotometer was also maintained at 25°C. For highly concentrated hemoglobin solutions (8-10 mM in hemel, gas mixtures of CO and N, at known ratios were allowed to flow through the tonometer until the gas phase and solution reached equilibrium, all in the same water bath. Deviation of the various spectra from the isosbestics was less than 0.5% of the optical density at that wavelength. Stopped-flow kinetic experiments were performed using a Durrum-Gibson stopped-flow apparatus, interfaced to an On-Line-Instruments-Systems (OLIS, Athens, Georgia) data acquisition system. The system is equipped with a Data General Nova 2/10 computer with 24K of core memory. Typical experiments involved sampling 1000 data points per reaction and accumulating and averaging at least three reactions per experimental condition. The data were analyzed, plotted, and/or read out numerically, all under computer control.
KANSAS
505
deoxy hemes of the (Y and p chains in the tetramer has a maximum, while at 535 nm the spectral difference has an opposite sign (1, 20). Without IHP, the P,,, value in 0.2 M Bistris buffer, pH 7, was 0.14 mm Hg and there was substantial cooperativity (Hill’s constant, n = 2.4). In the presence of 0.4 mM IHP the CO affinity was lowered significantly (P1,2 = 1.2 mm Hg) and the n value dropped to 1.1. Hill’s constant was reproducibly slightly larger than unity under the experimental conditions. The same value of n. was also observed at an 8 to 10 mrvr total heme concentration. Figure 2 shows CO binding kinetics to Hb Kansas + IHP as a function of wavelength. An excellent isosbestic was observed at 424.6 nm (Fig. 2A). When the data at 432 and 417 nm are plotted semilogarithmically (Fig. 2B), homogeneous, wavelength-independent, pseudo-first-order kinetics are observed with a rate constant of 1.03 x lo5 M-“5-I. Gibson et al. (16) have studied the same reaction and obtained a value for this rate constant of 0.9 X lo5 M-’ .s-l at 20°C in 0.05 M Bistris, pH 7, at 7 PM heme. We do not believe that these numbers are significantly different. The slight difference seen may well be due to the difference in temperature between the two experiments. The fact that we were unable to detect any signifi-
RESULTS
Hill plots for CO binding to Hb Kansas at pH 7, and to myoglobin for comparison, are shown in Fig. 1. There was very little wavelength dependence in the value of Y (fraction of CO saturation) for these experiments with Hb Kansas. The value of Y at 590 and 535 nm differed by less than 0.01. It has been previously shown that 590 nm is one of the wavelengths where the optical heterogenerity between the
FIG. 1. Hill plots of CO binding to Mb (A) and to Hb Kansas without (B) and with (C) IHP at pH 7, 25”C, in 0.2 M Bistris buffer. The concentration of Hb Kansas was 0.5 mM in heme, and the IHP concentration was 0.4 mM. The broken line near curve C is a calculated curve for the case where the fourth CO binding is stronger by a factor of 2 than the other three CO binding constants, K, = Vz K,, K, = K2 = K:,.
506
CASTILLO.
WILlISECONDS
OGAWA.
NILLISROWOS
2. Carbon monoxide binding to hemoglobin Kansas plus IHP. Conditions were 2X, 0.2 M Bistris, pH 7.0; [CO] = 1 mM, [heme] = 45.1 PM, [IHPI = 1 mM, before the mix. The slit width was 0.15 mm and the path length was 2 mm. (A) Time courses of the absorbance changes at 432, 417, and 424.6 nm. The facts that the curves at 432 and 417 nm have the same shape and that there was an excellent isosbestic point at 424.6 nm very strongly suggest that under these conditions no spectral or kinetic heterogeneity in CO binding is present. (B) Normalized time courses of the reactions in A showing that the time courses at these two wavelengths of opposite sign present good pseudo-first-order kinetic behavior with the same second-order rate constant (1.03 x lo5 M-‘.s-‘). FIG.
cant wavelength dependence for Hb Kansas + IHP under our conditions agrees with the equilibrium results discussed above, where no significant difference in the value of Y was noted as a function of wavelength. However, Gibson et aE. (16) did note that CO binding to extremely dilute (3 PM) solutions of Hb Kansas was wavelength dependent in the absence of IHP. As shown previously (18, 211, the CO replacement reaction by NO can be complicated by changes in quaternary structure and attendent nonlinear spectral changes in the NO hemes of the low-affinity state. Phosphate-free adult hemoglobin did not show spectral-kinetic heterogeneity since it did not change quaternary structure according to the nmr and CD spectroscopic measurements (1821). However, both relatively concentrated Hb Kansas (212 PM, before the mix) stripped of phosphate and Hb A + IHP did show spectral-kinetic heterogeneity, and the structural indicators showed that the T state does form with NO as the ligand. At
AND
SALHANY
wavelengths isosbestic for this extraspectral change in the NO hemes, we saw apparently autocatalytic CO release kinetics. With this in mind, we might expect very dilute solutions of stripped Hb Kansas (i.e., the dimer) to show no acceleration and little or no wavelength dependence. The results in Fig. 3 (A and B) seem to confirm these expectations. Figure 3A shows the time course of the spectral change when CO is replaced by NO at 4.1 PM heme (after the mix). We see that homogeneous kinetics occur with only a very slight deviation from isosbesty at 412.8 nm. A semilog plot of the data at 415 and 405.7 nm shows only about a 20% difference in rate. Neither reaction presents any significant deviation from linearity. When the data at 405.7 nm (an isosbestic for the NO color change) are compared with phosphate-free Hb A at the same wavelength and temperature, we see no significant differences. These
;?r;;“m,jo ,bQ zoQ ic yj SECQHOS
0
y;; 40 80 120 IO 200 240 StCQNDS
FIG. 3. The HbCO + HbNO replacement reaction for very dilute, phosphate-free Hb Kansas at several wavelengths. (A) Time course of the absorbance change at 415,412.8, and 405.7 nm. The concentration of heme after the mix was 4.1 pM. The protein was in 0.2 M Bistris, pH 7.0, + 0.1 M NaCl. Temperature = 25 2 1°C. (B) Normalized time course of the reactions at 415 and 405.7 nm in A and of adult Hb under the same solution conditions at 405.7 nm. The reactions for dilute Hb Kansas and Hb A (30 pM after the mix) are the same at 405.7 nm and 25 -t 1°C (0.012 s’). The reaction for Hb Kansas at 415 nm is about 20% faster. There is also a large spectral change in the NO hemes at this wavelength when the tetramer forms. Edelstein’s (22) recent ultracentrifugation measurements have shown that fully saturated Hb NO Kansas will be more associated than Hb CO Kansas under otherwise similar conditions. Thus, we might expect some small fraction of tetramers to form late in this reaction, causing problems in correctly determining the baseline for the reaction at 415 nm.
CO
BINDING
TO
HEMOGLOBIN
507
KANSAS
results suggest that there is little significant difference in the CO off rate between the cx and /3 chains in the dimer of Hb Kansas. At higher protein concentration, the spectral-kinetic heterogeneity observed (18, 21) would seem to be a contribution of tetrameric Hb Kansas and not necessarily chain inequivalence, unless the latter is protein concentration dependent.
At nmr concentrations of heme, Qgawa et al. (15) showed that the addition of IHP caused fully CO-saturated Hb Kansas to switch to the deoxy quaternary structure as judged by changes in several nmr lines. Salhany et al. (18, 21) measured the CO off rate from Hb Kansas + IHP at a lower protein concentration and did observe a considerably faster (factor of -10) CO off rate as compared with the stripped dimer of Hb Kansas or phosphate-free adult Hb. However, the reaction did show some kinetic heterogenity. Owing to this and to the fact that Cl- can critically affect the value of K4,2 for Hb Kansas, even with IHP present (22, 231, it seemed possible that the experiments reported by Salhany et al. (18, 21) for Hb Kansas + IHP could have some dimer contributions. In order to investigate this, and to obtain the value of the CO off rate for the tetramer of Hb Kansas + IHP, we investigated the protein and IHP concentration dependence of the CO + NO replacement reaction for Hb Kansas in 0.2 M Bistris (-0.05 M Cl-) at pH 7 and 25°C. The results in Fig. 4 (A and B) show the CO -+ NO reaction for Hb Kansas at roughly constant initial protein concentration (0.66 to 0.79 mM heme) and variable IHP concentration (0.2 to 15 mM). The reaction does show heterogeneous kinetics when the IHP concentration is about stoichiometric with tetramer (i.e., assuming all of the hemes were in the tetrameric form). Increasing the IHP concentration
above this level causes the reaction to become linear (Fig. 4B, curve D). At 15 mM IHP, the reaction begins to show an initial acceleration. These results have at least two possible interpretations. First, if the protein were all tetrameric and no dimer were present or generated by the
I_-,-~ m::
___-_ !I :I
5’
LT~_
1
;c il r’:lw
__
4.
2.
FIG. 4. CO + NO replacement reaction for Hb Kansas + IHP as a function of IHP concentration. The initial protein concentrations were kept nearly constant (0.66 to 0.79 mM) and IHP concentration was varied: (A) 0.2 mrq (B) 0.95 mrq (C) 2 mM; (D) 6 mM; and (El 15 mM. The reaction was studied at 589 nm in 0.2 M Bistris, pH 7, at 25 2 1°C. The concentrations are before the mix values.
mix, it could be suggested that the chains in the tetramer become inequivalent upon the addition of IHP at “stoichiometric” levels. Further addition of IHP could lead to binding to a hypothetical second site which predominately would affect CO release from the slow chain. The net result would be apparent kinetic equivalence between the chains at high IHP. Unfortunately, studying this reaction as a function of wavelength cannot be used to test this rather complex possibility due to the nonlinear spectral change which occurs in the NO heme, even in nitrosyl Hb Kansas (18, 21). A second and perhaps more plausible interpretation would be that 0.2 mM IHP simply does not provide enough binding energy to sufficiently increase the tetramer population of the CO form of this hemoglobin under our specific solution conditions (0.2 M Bistris., pH 7? -0.05 M Cl-). If dimers are contributing to the heterogeneity seen in Fig. 4, we might expect the slow phase of the reaction to have a rate comparable to that of the dimer and, more important, we should see the fraction of slow phase decrease as the concentration of IHP increases. This effect is seen most clearly in the results shown in Fig. 5, where the initial protein concentration was about 0.14 to 0.2 mM, with IHP first at 0.05 and then at 1 mM. Curve A shows
508
CASTILLO,
OGAWA,
a marked slow phase with a rate constant (0.029 s-l) similar to that of the dimer (Fig. 3). The slow phase accounts for about 38% of the reaction under these particular conditions. It should also be noted that there is a marked lag period in curve A, The presence of this lag period and its length were protein concentration dependent at constant IHP (results not shown). Raising the IHP concentration by a factor of 20 under these conditions caused a marked reduction in the fraction of slow phase (-13% slow phase) and an apparent reduction in the length of the lag period. Although we have not attempted to quantitatively interpret these rather complex reactions, the fact that the lag period and fraction of slow phase are both protein and IHP concentration dependent seems to qualitatively suggest that they are due to the presence of dimers. The similarity of the rate constant for the slow phase to that for the dimer would also be consistent with this interpretation. Unfortunately, the best experimental test of this proposal is not technically possible to perform. The test would be to increase the heme concentration beyond the 1 mM level after the mix at stoichiometric IHP concentrations. The difficulty is partly one of the high absorptions which these samples would have, as well as the limitation in NO concentration (1 mM). Based on the present interpretation, we would suggest that
FIG. 5. The CO + NO replacement reaction for Hb Kansas + IHP at about the same protein concentration (0.198 + 0.139 rnM in heme) and at 0.05 mM (A) and 1 mM (B) IHP, before the mix. The reactions (A and B) were carried out at 589 nm in a 2-cm cell at 25 f 1°C in 0.2 M Bistris, pH 7.
AND
SALHANY
the most valid measurement of the CO off rate from the tetrumer of Hb Kansas + IHP would be given by the results in Fig. 4, curve C or D (2-6 mM IHP, 0.795 mM heme, before the mix), which show largely homogeneous kinetics and a rate constant of 0.198 s-l at 25°C. This value is somewhat larger than that reported previously (18, 21). We do not know the exact reason for this difference. It is possible that the chloride concentration was higher than that estimated in the previous experiments. This would tend to increase K4,2 and thereby decrease the initial concentration 01’ tetramers, even with IHP present (22, 23). DISCUSSION
Previous nmr studies on Hb Kansas showed that the addition of IHP could produce a switch in quaternary structure at pH 7, even with four CO molecules bound (15, 24). Additional structural evidence that Hb Kansas can be maintained in the deoxy quaternary structure after binding CO came from the recent X-ray crystallographic study by Anderson (25). He showed that binding CO to crystals of deoxy Hb Kansas did not cause the crystals to crack, quite unlike the classical crystal cracking experiments of Haurowitz (26). Anderson also noted that the degree of tertiary disturbance with ligation of the T state was related more to the ligation of the particular subunit and appeared not to affect the structure of its neighbors. He pointed out that this would be in keeping with an allosteric interpretation of ligand binding to hemoglobin. Our results suggest that within the tetramer of Hb Kansas + IHP, CO binds to all subunits with a very low affinity, with little or no cooperativity, and with no evidence for significant chain inequivalence. When the CO “on” rate constant and the CO “off’ rate constant for the tetramer + IHP are used to calculate the equilibrium constant, we obtain a value which is in excellent agreement with the direct CO binding equilibrium measurements at high protein concentration (Table I). It should be noted that this agreement occurs despite the fact that the CO
CO
BINDING
TO
HEMOGLOBIN TABLE
EQUILIBRIUM
AND
KINETIC
CONSTANTS
Hb Kansas (dimer) Hb A stripped Hb A stripped (first step) Hb Kansas + IHP Hb M Iwate
+ IHP
I
FOR VARIOUS
R T
9.3 x 10”” 11 x 10”” 2 x 10””
0.012b 0.0120 0.09’
TJ
1.03 x 105b
0.198b
T”
1.9 x loj’
a J. M. Salhany, unpublished flash photolysis NaCl, pH 7, 25 ? 1°C. * From this study. ’ Calculated from the kinetic measurements. d Reference 28. p Reference 27. f Reference 15. Y Measured equilibrium constant. * Reference 29. I Reference 14.
results;
off rate was measured by replacement with NO. The results presented in Table I also illustrate the correlation between the CO binding properties and the structural measurements for the selected hemoglobins shown to be in either the R or T quaternary structures. Hb M Iwate and Hb Kansas + IHP, both of which are in T even when fully liganded (Table I), show quite similar CO binding properties under reasonably comparable conditions. All of the kinetic constants are at least an order of magnitude or more different from the same kinetic constants for the noncooperative dimer of Hb Kansas or stripped adult HbCO in the R state. Furthermore, the affinities are about two orders of magnitude different. Direct comparison with the deoxy quaternary structure of stripped Hb A over its entire ligation manifold is not possible, but clearly the predominant affinity difference for CO binding calculated from the available kinetic data does appear to hold. One problem is in the determination of 1 for the T state of Hb A in the absence of phosphate. Sharma et al. (27) have recently measured I,. Their value does appear to be somewhat lower (factor of -2) than that for Hb Kansas + IHP or Hb M Iwate (Table I). It is clear from the results in Table I that those
4
WM
AT 25 ? 2°C
1 (S ‘)
S ‘)
(M-’
ligation
HEMOGLOBINS
I’
Quaternary structure
509
KANSAS
L (M-‘1 7.8 x 108’ 9.2 x IOR’ 22.2 x lo”’ 5.2 5.4 8.3 9.5
0.23’ heme,
1 mM CO in 0.2
M
Bistris
x x x x
105’ lO”,J 105’ lo”V + 0.1 M
hemoglobins identified as having the deoxy quaternary structure even when fully liganded show a very low CO affinity (-two orders of magnitude) and no evidence for significant cooperativity in CO binding. ACKNOWLEDGMENTS We thank Ms. Becky Jacobs for her aid in the preparation of this manuscript and Mr. John Friel for his skillful preparation of the illustrations presented in this report. REFERENCES 1. ANTONINI, E., AND BRUNORI, M. (1971) Hemoglobin and Myoglobin in their Reaction with Ligands, p. 296, North-Holland, Amsterdam. 2. KILMARTIN, J. V., HEWITT, J. A., AND WOOTON, J. F. (1975) J. Mol. Biol. 93, 2033218. 3. KILMARTIN, J. V., IMAI, K., AND JONES, R. T. (1975) in Erythrocyte Structure and Function (Brewer, G. J., ed.), pp. 21-35, Alan R. Liss, New York. 4. BUNN, H. F., WOHL, R. C., BRADLEY, T. B., COOLEY, M., AND GIBSON, Q. H. (1974) J. Biol. Chem. 249, 7402-7409. 5. HEWITT, J. A., AND GIBSON, Q. H. (19731 J. Mol. Biol. 74, 489-498. 6. GIBSON, Q. H., AND NAGEL, R. L. (1974) J. Biol. Chem. 249, 7255-7259. 7. MOFFAT, K., OLSON, J. S., GIBSON, Q. H., AND KILMARTIN, J. V. (1973) J. Biol. Chem. 248, 6387-6393.
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CASTILLO,
OGAWA,
8. SHULMAN, R. G., OGAWA, S., AND HOPFIELD, J. J. (1972) Arch. Biochem. Biophys. 151, 68-74. 9. IMAI, K. (1973) Biochemistry 12, 798-808. 10. GERSONDE, K., OVERKAMP, M., SICK, H., TRITTELVITZ, E., &ND JUNGE, W. (1973) Eur. J. Biochem. 39, 403-412. 11. GIBSON, Q. H., HELLER, P., AND YAKULIS, V. (1966) J. Biol. Chem. 241, 1650-1651. 12. HAYASHI, N., MOTOKAWA, Y., AND KIKUCHI, G. (1966) J. Bid. Chem. 241, 79-84. 13. NISHIKURA, K., SUGITA, Y., NAGAI, M., AND YONEYAMA, Y. (1975) J. Biol. Chem. 250, 6679-6685. 14. SALHANY, J. M., CASTILLO, C. L., AND OGAWA, S. (1976) Biochemistry 15, 5344-5349. 15. OGAWA, S., MAYER, A., AND SHULMAN, R. G. (1972) Biochem. Biophys. Res. Commun. 49, 1485-1491. 16. GIBSON, Q. H., RIGGS, A., AND IMAMURA, T. (1973) J. Biol. Chem. 248, 5976-5986. 17. HOPFIELD, J. J., OGAWA, S., AND SHULMAN, R. G. (1972) Biochem. Biophys. Res. Commun. 49, 1480-1484. 18. SALHANY, J. M., OGAWA, S., AND SHULMAN, R.
AND
SALHANY
G. (1975) Biochemistry 14, 2180-2190. 19. BONAVENTURA, J., AND RIGGS, A. (1968) J. Biol. Chem. 243, 980-991. 20. SUGITA, Y. (1975) J. Biol. Chem. 250, 1251-1256. 21. SALHANY, J. M., OGAWA, S., AND SHULMAN, R. G. (1974)Proc. Nut. Acad. Sci. USA 71, 33593362. 22. EDELSTEIN, S. J. (1975) Annu. Rev. Biochem. 44, 209-232. 23. ATHA, D. H., AND RIGGS, A. (1976) J. Biol. Chem. 251, 5537-5543. 24. SHULMAN, R. G., OGAWA, S., MAYER, A., AND CASTILLO, C. L. (1973) Ann. N.Y. Acad. Sci. 222, S-20. 25. ANDERSON, L. (1975) J. Mol. Biol. 94, 33-49. 26. HAUROWITZ, F. (1938) Z. Physiol. Chem. 254, 266. 27. SHARMA, V. S., SCHMIDT, M. R., AND RANNEY, H. M. (1976) J. Biol. Chem. 251,4267-4272. 28. SAWICKI, C. A., AND GIBSON, Q. H. (1976) J. Biol. Chem. 251, 1533-1542. 29. MAYER, A., OGAWA, S., SHULMAN, R. G., AND GERSONDE, K. (1973) J. Mol. Biol. 81, 187197.