Proc. Nati. Acad. Sci. USA Vol. 76, No. 10, pp. 4936-4940, October 1979
Biochemistry
13C NMR quantitation of polymer in deoxyhemoglobin S gels (sickle cell anemia/sickle hemoglobin)
CONSTANCE TOM NOGUCHI*, DENNIS A. TORCHIAt, AND ALAN N. SCHECHTER* *Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases; and tLaboratory of Biochemistry, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205
Communicated by C. B. Anfinsen, July 5, 1979
ABSTRACT 13C/'H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 300C, for samples whose hemoglobin concentration ranged from 21 to 32 g/dl. Scalarand dipolar-decoupled spectra and a 13C proton-enhanced dipolar~ecoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of firee molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30'C of 17.3 ± 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes. The mutation of sickle cell hemoglobin (hemoglobin S), g6GlIu-Val, causes concentrated solutions of hemoglobin S to aggregate or gel upon deoxygenation (for review, see ref. 1).
NMR (scalar- and dipolar-decoupled) and proton-enhanced magnetic resonance are useful for observing both free and aggregated or polymerized proteins (8). We have recently reported that these methods can be used to detect both free and polymerized hemoglobin molecules in deoxyhemoglobin S gels and in cells (9). The advantage of such NMR studies over ul-
tracentrifugation sedimentation for determining the fraction of polymer formed is that the sample is not physically altered (or separated into a sol and pellet phase as in ultracentrifugation) and intact erythrocytes as well as hemoglobin S solutions can be studied. We now report the use of '3C/'H magnetic double-resonance spectroscopy to quantitate the fraction of aggregated or polymerized material in deoxyhemoglobin S solutions of varying concentrations. To verify the validity of the NMR spectroscopy, deoxyhemoglobin S solubility data were obtained by using ultracentrifugation sedimentation on the same samples and the data from the two methods were compared within the context of the two-phase model. The correlation of the data from the two methods establishes the usefulness of 13C/IH magnetic double-resonance techniques for characterizing the deoxyhemoglobin S gel and suggests a new method for studying the intracellular gelation of deoxyhemoglobin S. MATERIALS AND METHODS Sample Preparation. Hemoglobin S was prepared from blood samples from an individual homozygous in hemoglobin S and from an individual heterozygous in hemoglobin S by ion exchange chromatography on DEAE-Sephadex A-50 (10). The purified hemoglobin S was concentrated by ultrafiltration and vacuum dialysis and dialyzed into 0.15 M potassium phosphate buffer at pH 7.4. Predetermined amounts of concentrated hemoglobin S solution and buffer were loaded into 8-mm NMR tubes (Wilmad-513A-PP) under nitrogen. For final deoxygenation, freshly made sodium dithionite stock solution was added to give a final dithionite concentration of 0.05-0.08 M. Buffer was added in place of the dithionite solution for the matching control sample. The total sample volume was 0.65 ml. The tubes were sealed with a 5-mm tube cap (Wilmad-504-PP) via a ground glass joint. After the NMR spectra were accumulated, the sample tubes were inverted and immersed in an ice bath, and the solution was allowed to flow into the 5-mm tube cap. By using absorption measurements in the near-infrared (Cary model 17), complete sample deoxygenation and total deoxyhemoglobin S concentration were checked [deoxyhemoglobin S concentration = (A at 910 nm - A at 1090 nm) X 24.56 g/dl
The sickling of erythrocytes characteristic of sickle cell anemia is thought to result from intracellular gelation of deoxyhemoglobin S. Recent biophysical studies of deoxyhemoglobin S solutions have resulted in an analysis of the thermodynamics of gelation and a description of the kinetics of this process in terms of polymer condensation theory. Understanding of the mechanism of intracellular gelation is much less advanced because of lack of methods for studying the process in intact cells. The major method for studying the thermodynamics of deoxyhemoglobin S gels has been ultracentrifugation sedimentation in which the gel is physically separated into a sol phase consisting of free hemoglobin molecules and a pellet phase which contains noncovalently polymerized hemoglobin molecules, in addition to free hemoglobin (2-6). The concentration of the sol phase, which depends on temperature but not on initial deoxyhemoglobin S concentration, has been used as a measure of deoxyhemoglobin S solubility. These results indicate that the polymerization of deoxyhemoglobin S can be described by a two-phase model (7) consisting of deoxyhemoglobin S molecules free in solution in equilibrium with a condensed phase. An alternative method for characterizing deoxyhemoglobin S gels is the use of NMR spectroscopy, especially 13C/IH magnetic double-resonance techniques. Natural abundance 13C
(11)].
l3C NMR. Natural abundance 13C spectra without nuclear Overhauser enhancements were taken of the hemoglobin samples in 8-mm tubes by using a Nicolet TT-14 spectrometer modified for high-power '3C/IH magnetic double-resonance
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Abbreviation: hemoglobin S, sickle cell hemoglobin (36Glu-Val).
4936
Biochemistry: Noguchi et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
4937 -
experiments in solids. The probe contained an external 2H20 lock, and the sample was maintained at 300C by using,.fiow of temperature-regulated nitrogen gas through the probe in a Dewar flask. The temperature gradient throughout the sample was less than 20C. The probe was tuned to 50 ohms for each sample. A 50-W '3C transmitter (15.09 MHz) gave a 90° rotation of the 13C magnetization in about 5 ,usec. A proton resonant field of 0.7 gauss (^y2H2/2r = 3 kHz) was applied for scalar decoupling and of 13 gauss ('y2H2/27r = 55 kHz) for dipolar decoupling. For the proton-enhanced spectra, the HartmanHahn condition ('yHl = 72H2) was determined as described (9). The sign of the proton spin temperature was alternated in the proton-enhanced experiments to minimize artifacts. Relative signal intensities were determined by direct integration and from difference spectra. In the latter case, one spectrum was multiplied by a scale factor (determined by nulling appropriate pairs of spectra) and then subtracted from a second spectrum. Solubility. Deoxyhemoglobin S solubility was determined by ultracentrifugation as described by Hofrichter et al. (11). Samples were transferred from the NMR tube into 5-mm quartz electron paramagnetic resonance sample tubes (Wilmad PQ-701) under nitrogen; the tubes were sealed with pressure caps (Wilmad-521-PC) and the caps were painted (Glyptol, General Electric). The near-infrared spectra (1300-700 nm) of the sample were identical before and after transfer, indicating that the sample remained fully deoxygenated. The samples were gelled at 30'C and spun at 100,000 X g at 30'C. The solubility or supernatant concentration was determined from absorbance measurements at 910 and 1090 nm as described above.
RESULTS Natural abundance 13C NMR was used to determine the fraction of polymerized deoxyhemoglobin S at 30°C (Fig. 1). For each sample of deoxyhemoglobin S, a 1H scalar-decoupled spectrum, a 'H dipolar-decoupled spectrum, and a protonenhanced spectrum were recorded. A 'H scalar-decoupled spectrum of the matching oxygenated control (made up identically but with buffer in place of sodium dithionite solution) was also recorded (Fig. 1, curve a). We assign resonances in the spectra as follows: carbonyl carbons at 15-25 ppm; aromatic carbons at 60-75 ppm; aliphatic carbons (primarily backbone a-carbons) at 120-150 ppm; and side-chain aliphatic carbons at 150-200 ppm. For the oxygenated control, the intensity of the scalar-decoupled spectrum, which monitors only rotationally free material (identified with free hemoglobin in solution), was directly proportional to the total hemoglobin S (Fig. 2) because no gel was present. The scalar-decoupled spectrum (Fig. 1, curve b) of deoxyhemoglobin S at the same concentration was diminished due to the immobile material in the gel (identified with polymerized molecules). The dipolar-decoupled spectrum (Fig. 1, curve c) which is sensitive to both mobile and immobile hemoglobin had more intensity in the aliphatic region. However, the intensity still was less than the oxygenated control: only 75-80% of the polymer carbon signal was observed in the dipolar-decoupled spectrum because of incomplete spin-lattice relaxation and because of electron-nucleus dipolar broadening (9). The difference between the dipolar decoupled spectrum and the scalar-decoupled spectrum of deoxyhemoglobin S (Fig. 1, curve d) represents the spectrum of polymerized molecules. The signal intensity of the proton-enhanced spectrum (Fig. 1, curve e) also was proportional to the amount of polymerized deoxyhemoglobin S (Fig. 3) but was 2.5 + 0.5 times greater than the signal intensity of the difference spectrum (Fig. 1, curve
A
"'I aa
b
c
d
el
4
II
0
100
200
ppm
FIG. 1. '3C/PH double magnetic resonance spectra for hemoglobin S (28 g/dl) at 30°C. Curve a is the 1H scalar-decoupled spectrum for the oxygenated control sample. The 1H scalar-decoupled spectrum monitors only free or unaggregated material. The 1H dipolar-decoupled spectrum for this sample was similar because there was no polymerized hemoglobin present. (The 1H dipolar-decoupled spectrum is proportional to the amount of total hemoglobin present, free and aggregated). Curves b through e are spectra of the deoxygenated sample. Curve b is the 1H scalar-decoupled spectrum for the deoxyhemoglobin S sample. The intensity is diminished compared to the oxygenated sample spectrum (curve a) due to the aggregation or polymerization of some of the hemoglobin S in the gel state. Curve c is the 1H dipolar-decoupled spectrum of the deoxyhemoglobin S sample. Curve d is the difference spectrum between the 1H dipolardecoupled spectrum and the 1H scalar-decoupled spectrum, which is a measure of the amount of aggregated or polymerized deoxyhemoglobin S. Curve e is the proton-enhanced spectrum which is also proportional to the amount of polymerized or aggregated hemoglobin. However, this spectrum is about 2.5 times greater in intensity than curve d. Spectroscopic parameters are the same as given in ref. 9; chemical shift is based on CS2.
d) because of the intensity enhancement provided by crosspolarization. This enhancement factor was measured by nulling the proton-enhanced spectrum with the difference spectrum. The data in Fig. 3 can be extrapolated to zero polymer to obtain an estimate of deoxyhemoglobin S solubility. A linear regression analysis of the data gives zero polymer at a hemoglobin concentration of 17.3 b 2 g/dl. The fraction of free deoxyhemoglobin S is taken to be the ratio of the integrated intensity of the aliphatic region of the deoxyhemoglobin S scalar-decoupled spectrum to the integrated intensity of the oxyhemoglobin S scalar-decoupled
4938
Proc. Natl. Acad. Sci. USA, 76 (1979)
Biochemistry: Noguchi et al.
C
CD
0.8
26 Hemoglobin, g/dl
22
30
FIG. 2. Integrated intensity of the1H scalar-decoupled spectrum for the oxygenated control as a function of hemoglobin S concentration. The integrated intensity was obtained by numerical integration of the aliphatic region with the Nicolet software. The results are normalized to the maximum obtained (31.2 g/dl, oxygenated sample). Scaling each spectrum to this maximum until a null difference spectrum is obtained gives similar results. The straight line represents a linear regression analysis of the data (relative intensity = 0.081 + 0.030[HbS]; r2 = 0.949).
spectrum.t The polymer fraction is 1 minus the free fraction (Fig. 4). The polymer fraction increased with increasing deoxyhemoglobin S concentration, from 0.22 at 20.7 g/dl to 0.52 at 31.7 g/dl. The value of the fraction of free deoxyhemoglobin S was also calculated from the two scalar-decoupled spectra by using the Nicolet software for directly obtaining null difference spectra. The results of these methods fall within 10% of each other. Lastly, the fraction of polymerized deoxyhemoglobin S was obtained by using ultracentrifugation sedimentation which separates the gel into a sol phase and a pellet (3). The pellet is assumed to be composed of polymerized hemoglobin as well as free hemoglobin equal in concentration to the sol phase. By using a polymer concentration of 70 g/dl [obtained from studies of deoxyhemoglobin A and S mixtures (12)] and the sol phase concentration, the fraction of polymerized deoxyhemoglobin S was determined by assuming a two-phase model. When the NMR spectral experiments were completed, the sol phase concentration was determined after ultracentrifugation sedimentation, and the fraction of polymerized deoxyhemoglobin S was calculated. The calculated fraction of polymerized hemoglobin varied from 0.31 at 20.7 g/dl to 0.68 at 31.7 g/dl by this method. These uncorrected values have an absolute average 0.15 greater than the values obtained from the NMR spectroscopy, well beyond the uncertainties of the measurements. The apparent discrepancy between the two methods can be accounted for, in part, by considering the concentration gradient introduced during ultracentrifugation sedimentation of concentrated hemoglobin solutions. For example, for the sedimentation tubes with a sample volume of 350 Al and an oxyhemoglobin S solution of 12 g/dl, spinning under conditions t These methods are equivalent to method A in ref. 9. As was indicated in that paper,
we no
longer
use
method B [ratio of
integrated aliphatic
intensity in the difference spectrum (Fig. 1, curve d) to that in the oxygenated control (Fig. 1, curve a)l because incomplete spin-lattice relaxation and paramagnetic broadening cause underestimation of
polymer in that method.
V
30
26 22 Hemoglobin, g/dl
18
FIG. 3. Integrated intensity at 30'C of the "3C proton-enhanced spectrum as a function of deoxyhemoglobin S concentration. The total integrated intensity of the 13C proton-enhanced spectrum including the carbonyl carbons was obtained by using the Nicolet software. The results are normalized to the maximal value obtained (31.2 g/dl deoxygenated sample). The relative intensity is proportional to the amount of aggregated or polymerized material present. The straight line represents a linear regression analysis of the data (relative intensity = -1.16 + 0.067 X hemoglobin concentration (g/dl); r2 = 0.945).
used for solubility studies results in a noticeable concentration gradient such that the mean (+SEM) hemoglobin concentration is 8.9 i 0.29 g/dl for the top lOO1,l and 10.0 i 0.1 g/dl for the top 250 ,l, as determined by the cyanmethemoglobin method at 540 nm (13).
0.60
(U
E 0o4k
0.2
2 22
26 Hemoglobin, g/dl
30
FIG. 4. Fraction of polymerized deoxyhemoglobin S as a function of deoxyhemoglobin S concentration at 300C. 0, Results obtained from 13C NMR by determining first the fraction of free hemoglobin from comparison between the 'H scalar-decoupled spectra of the deoxy-sample and the matching oxygenated control; O, results obtained from ultracentrifugation sedimentation on the same samples after the NMR data were collected, assuming a polymer concentration of 70 g/dl. The solid line represents the predicted fraction of polymer, assuming this polymer concentration and a solubility of 16.4 g/dl. The dashed line, which best fits the NMR data, extrapolates to a solubility of 18.4 g/dl.
Biochemistry: Noguchi et al. DISCUSSION NMR spectroscopy has been used to study sickle hemoglobin and to follow the gelation process. 1H NMR has been used to demonstrate several surface histidine residues which differ between oxy- and deoxyhemoglobin S (14, 15). Water 1H NMR relaxation methods have been used to follow the process of polymerization, in hemoglobin solutions and in cells (16-19). In contrast to these earlier studies, the 13C/IH magnetic double-resonance spectroscopic methods discussed here can be used to quantitate directly the fraction of polymerized hemoglobin molecules and thus can give useful direct information about the thermodynamic and kinetic properties of the polymerization process. In order to show the usefulness of the NMR spectroscopy for such quantitation, the NMR data were compared to data from sedimentation within the framework of a two-phase model. The two-phase model has been used extensively in the analysis of the thermodynamics of gelation (3-6). Our own measurements of 13C NMR linewidths of deoxyhemoglobin S gels, indicating the coexistance of an isotropically mobile phase and a polymerized or crystalline-like phase, are also consistent with a two-phase model (9). The two-phase model assumes that the deoxyhemoglobin S gel is composed of free hemoglobin of concentration C., and of polymer of concentration Cp. The total concentration of deoxyhemoglobin S, CT, can then be separated into the condensed phase and the free phase (CT = CP VP + C. V, in which vP and v, are the volume fractions of polymerized and free deoxyhemoglobin S). The fraction of polymerized deoxyhemoglobin S, FP = CP VP/CT, and the fraction of free deoxyhemoglobin S, F, = C, V,/CT, can also be completely expressed in terms of CT, CP, and C, to yield FP = Cp (CT - C,)/[CT(Cp - C,,)] and F, = C, (Cp - CT)/[CT(CP - C,.)]. The concentration of deoxyhemoglobin S in the polymer state, C., has been determined to be 70 g/dl (12), which is close to the value of 69.6 g/dl, the hemoglobin concentration of deoxyhemoglobin S crystals (20). In ultracentrifugation sedimentation studies the supernatant concentration is assumed to be the deoxyhemoglobin S solubility, C., or free homoglobin concentration (3, 4). By using that value for C,,, the total deoxyhemoglobin S concentration, CT, which is measured directly, and the above value for CP, the fraction of polymerized and free hemoglobin (FP and F,J, respectively) can be determined. In NMR studies, the fraction of polymerized and free hemoglobin are measured and are used to determine C,. If the two-phase model is valid, the NMR and ultracentrifuge sedimentation experiments should give similar values for the amount of polymerized and free material for any deoxyhemoglobin S sample. We find that, in the concentration range 24-32 g/dl, the polymer fraction obtained by using uncorrected ultracentrifugation sedimentation data (Fig. 4) is on the average 0.15 higher than that obtained by using 13C NMR data. For example, at a total deoxyhemoglobin S concentration of 28 g/dl, 13C NMR yields a polymer fraction of 0.44 whereas ultracentrifugation sedimentation yields a polymer fraction of 0.56. The differences between sedimentation and NMR measurements can be examined in terms of the differences in the free hemoglobin concentration C,. C, is taken to be the supernatant concentration in sedimentation and was found to be 16.4 g/dl at 30°C under these conditions (21, 22). From Fig. 3, we estimate a solubility (no polymer present) of 17.3 g/dl. Our own experiments confirm the observations by others that the sedimenation process underestimates solubility and overestimates polymer, either by packing free hemoglobin molecules into the pellet or by forming new polymer at the top of the pellet (23, 24). Behe and Englander (23) estimated this ef-
Proc. Natl. Acad. Sci. USA 76(1979)
4939
feet at 5% [as did B. Magdoff-Fairchild (personal communication)]. Although our measurements suggest an effect several times higher, nonideality (25) would decrease this effect at the higher concentrations in which the NMR experiments were done. Thus, the value of 17.3 g/dl would fit both our data from proton-enhanced spectra and corrected ultracentrifugation runs. The solid line in Fig. 4, representing the polymer fraction, was calculated by using the 16.4 g/dl value and a polymer concentration of 70 g/dl; the dashed line, which better fits the NMR data, requires a solubility of 18.4 g/dl. Use of the 17.3 g/dl solubility value would lower the ultracentrifugation data almost to within the error limits of the NMR data. Several errors could arise in the process of collecting the 13C NMR data (9). Paramagnetism of deoxyhemoglobin S or paramagnetism due to the presence of sodium dithionite could decrease the signal intensity via dipolar broadening. Another source of error would exist if the exchange rate between free and bound (polymerized) deoxyhemoglobin S molecules were sufficiently slow as to broaden the signal peaks. However, these sources of error would result in loss of intensity for the free hemoglobin fraction, resulting in an overestimate of the polymer fraction. If the polymer contributed to the scalar-decoupled spectrum, this would result in an underestimate of the polymer fraction. We believe this is unlikely, however, because one would then expect a greater intensity contribution from the mobile side-chain carbon atoms than from the backbone carbon atoms and we find no difference in their relative intensities in the oxy- and deoxyhemoglobin spectra (Fig. 1, curves a and
b). §
Another possible source of discrepancy between the two methods relates to the assumed value of the polymer concentration. However, it would take significantly higher values, to reconcile the two sets of data, and the value we have used (12) is close to the maximum, which seems reasonable (27). A failure of the approximations of the two-phase model (7) could also be envisioned to account for our results. In any case, the residual divergence of data of the two methods is small enough to justify further equilibrium and kinetic studies of sickle hemoglobin polymerization. The major significance of the use of '3C/'H magnetic double-resonance spectroscopy is likely to be in the study of the mechanism of cell sickling. Methods of studying intracellular gelation have relied upon techniques such as light and electron microscopy, birefringence, and filterability (1), as well as water proton relaxation methods (16-19). None of these methods" gives a simple, linear measurement of the polymerization process and thus have been of limited utility in studying the mechanism of sickling or the effects of sickling inhibitors (1). We have shown that these new 13C NMR methods can be used to study intracellular sickle hemoglobin polymerization (9), and measurements of the extent of polymerization as a function of PO2, pH, temperature, and concentration of various covalent and noncovalent inhibitors are now possible. § The intensity of the scalar-decoupled spectrum of deoxyhemoglobin S was independent of Y2H2 in the range 1-3 kHz, indicating that intensity from anisotropically mobile groups in the polymer was not visualized in the scalar-decoupled spectrum. The absence of scalardecoupled spectral intensity in collagen provides further evidence that such groups are not detected in scalar-decoupled spectra (26). Smaller values of polymer concentration used previously (3, 4) were based on the assumption that the pellet contained no free monomer. Only now are reliable methods for measuring the kinetics of polymerization of sickle hemoglobin in single cells being developed
(28).
4940
Biochemistry: Noguchi et al.
1. Dean, J. & Schechter, A. N. (1978) N. Engl. J. Med. 299, 752-763; 804-811; 863-870. 2. Bertles, J. F., Rabinowitz, R. & D6bler, J. (1970) Science 169, 375-377. 3. Hofrichter, J., Ross, P. D., Eaton, W. A. (1976) in Proceedings of the Symposium on Molecular and Cellular Aspects of Sickle Cell Disease, DHEW Publication No. (NIH)76-1007, ed. Hercules, J. I., Cottam, G. L., Waterman, M. R. & Schechter, A. N. (GPO, Washington, DC), pp. 185-223. 4. Ross, P. D., Hofrichter, J. & Eaton, W. A. (1977) J. Mol. Biol. 115, 111-134. 5. Magdoff-Fairchild, B., Poillon, W. N., Li, T.-I & Bertles, J. F. (1976) Proc. Natl. Acad. Sci. USA 73,990-994. 6. Pumphrey, J. G. & Steinhardt, J. (1976) J. Mol. Biol. 112, 359-375. 7. Minton, A. P. (1974) J. Mol. Biol. 82,483-498. 8. Torchia, D. A. & VanderHart, D. C. (1979) in Topics in Carbon-13 NMR Spectroscopy, ed. Levy, G. C. (Wiley, New York), Vol. 3, pp. 325-360. 9. Sutherland, J. We H., Egan, W., Schechter, A. N. & Torchia, D. A. (1979) Biochemistry 18, 1797-1803. 10. Huisman, T. H. J. & Dozy, A. M. (1965) J. Chromatogr. 19, 160-169. 11. Hofrichter, J., Ross, P. D. & Eaton, W. A. (1976) Proc. Natl. Acad. Sci. USA 73,3035-3039. 12. Sunshine, H. R., Hofrichter, J. & Eaton, W. R. (1979) J. Mol. Biol., in press. 13. Van Assendelft, 0. W. (1970) Spectrophotometry of Haemoglobin Derivatives (Royal Vanogram, Assen, Netherlands), pp. 110-112.
Proc. Natl. Acad. Sci. USA 76 (1979) 14. Ho, C., & Russu, L. M. (1978) in Biochemical and Clinical Aspects of Hemoglobin Abnormalities, ed. Caughey, W. S. (Academic, New York), pp. 179-194. 15. Russu, I. M. & Ho, C. (1979) Blophys. J. 25, 127a. 16. Zipp, A., James, T. L., Kuntz, I. D. (1976) Biochim. Blophys. Acta 428,291-303. 17. Lindstrom, T. R., Koenig, S. H., Boussios, T. & Bertles, J. F. (1976) Biophys. J. 16,679-689. 18. Eaton, W. A., Hofrichter, J., Ross, P. D., Tschuden, R. G. & Becker, E. D. (1976) Biochem. Biophys. Res. Commun. 69, 538-547. 19. Cottam, G. L., Shibata, K. & Waterman, M. R. (1978) in Biochemical and Clinical Aspects of Hemoglobin Abnormalities, ed. Caughey, W. S. (Academic, New York), pp. 695-715. 20. Wishner, B. C., Ward, K. B., Lattman, E. E. & Love, W. E. (1975) J. Mol. Biol. 98, 161-178. 21. Noguchi, C. T. & Schechter, A. N. (1977) Biochem. Biophys. Res. Commun. 74, 637-642. 22. Noguchi, C. T. & Schechter, A. N. (1978) Biochemistry 17, 5455-5459. 23. Behe, M. J. & Englander, S. W. (1978) Biophys. J. 23,129-145. 24. Chung, L. L. & Magdoff-Fairchild, B. (1978) Arch. Biochem. Biophys. 189,535-539. 25. Ross, P. D., Briehl, R. W. & Minton, A. P. (1978) Biopolymers 17, 2285-2288. 26. Jelinski, L. W. & Torchia, D. A. (1979) J. Mol. Biol., in press. 27. Hofrichter, J. (1979) J. Mol. Biol. 128,335-370. 28. Sunshine, H. R., Ferrone, F. A., Hofrichter, J. H. & Eaton, W. A. (1979) in Development of Therapeutics Agents for Sickle Cell Disease, eds. Rosa, J., Beuzard, Y. & Hercules, J. (Elsevier/ North-Holland, Amsterdam), pp. 131-146.
Biochemistry
13C NMR quantitation of polymer in deoxyhemoglobin S gels (sickle cell anemia/sickle hemoglobin)
CONSTANCE TOM NOGUCHI*, DENNIS A. TORCHIAt, AND ALAN N. SCHECHTER* *Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases; and tLaboratory of Biochemistry, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205
Communicated by C. B. Anfinsen, July 5, 1979
ABSTRACT 13C/'H magnetic double-resonance spectroscopy has been used to quantitate the amount of polymerized hemoglobin S in deoxygenated gels at 300C, for samples whose hemoglobin concentration ranged from 21 to 32 g/dl. Scalarand dipolar-decoupled spectra and a 13C proton-enhanced dipolar~ecoupled spectrum were recorded for each sample as was a scalar-decoupled spectrum for a matching oxyhemoglobin S control. The difference between the oxyhemoglobin S and deoxyhemoglobin S scalar-decoupled spectra was used to determine the polymer fraction, and this value was compared with the polymer fraction determined by using ultracentrifugation sedimentation on the same sample (assuming a two-phase model). The polymer fraction value determined by uncorrected sedimentation averaged 0.15 more than the value obtained from NMR. The discrepancy between the two techniques was largely removed when the analysis of the sedimentation data included a correction for depletion of hemoglobin in the supernatant or sol phase due to sedimentation of firee molecules. The best fit to both the sedimentation and NMR data was obtained by using a solubility of deoxyhemoglobin S at 30'C of 17.3 ± 1 g/dl. These results indicate that the NMR techniques, which do not require separation of the sample into a sol phase and a pellet phase, provide quantitative information about the deoxyhemoglobin S polymer and will be useful for studies of sickle erythrocytes. The mutation of sickle cell hemoglobin (hemoglobin S), g6GlIu-Val, causes concentrated solutions of hemoglobin S to aggregate or gel upon deoxygenation (for review, see ref. 1).
NMR (scalar- and dipolar-decoupled) and proton-enhanced magnetic resonance are useful for observing both free and aggregated or polymerized proteins (8). We have recently reported that these methods can be used to detect both free and polymerized hemoglobin molecules in deoxyhemoglobin S gels and in cells (9). The advantage of such NMR studies over ul-
tracentrifugation sedimentation for determining the fraction of polymer formed is that the sample is not physically altered (or separated into a sol and pellet phase as in ultracentrifugation) and intact erythrocytes as well as hemoglobin S solutions can be studied. We now report the use of '3C/'H magnetic double-resonance spectroscopy to quantitate the fraction of aggregated or polymerized material in deoxyhemoglobin S solutions of varying concentrations. To verify the validity of the NMR spectroscopy, deoxyhemoglobin S solubility data were obtained by using ultracentrifugation sedimentation on the same samples and the data from the two methods were compared within the context of the two-phase model. The correlation of the data from the two methods establishes the usefulness of 13C/IH magnetic double-resonance techniques for characterizing the deoxyhemoglobin S gel and suggests a new method for studying the intracellular gelation of deoxyhemoglobin S. MATERIALS AND METHODS Sample Preparation. Hemoglobin S was prepared from blood samples from an individual homozygous in hemoglobin S and from an individual heterozygous in hemoglobin S by ion exchange chromatography on DEAE-Sephadex A-50 (10). The purified hemoglobin S was concentrated by ultrafiltration and vacuum dialysis and dialyzed into 0.15 M potassium phosphate buffer at pH 7.4. Predetermined amounts of concentrated hemoglobin S solution and buffer were loaded into 8-mm NMR tubes (Wilmad-513A-PP) under nitrogen. For final deoxygenation, freshly made sodium dithionite stock solution was added to give a final dithionite concentration of 0.05-0.08 M. Buffer was added in place of the dithionite solution for the matching control sample. The total sample volume was 0.65 ml. The tubes were sealed with a 5-mm tube cap (Wilmad-504-PP) via a ground glass joint. After the NMR spectra were accumulated, the sample tubes were inverted and immersed in an ice bath, and the solution was allowed to flow into the 5-mm tube cap. By using absorption measurements in the near-infrared (Cary model 17), complete sample deoxygenation and total deoxyhemoglobin S concentration were checked [deoxyhemoglobin S concentration = (A at 910 nm - A at 1090 nm) X 24.56 g/dl
The sickling of erythrocytes characteristic of sickle cell anemia is thought to result from intracellular gelation of deoxyhemoglobin S. Recent biophysical studies of deoxyhemoglobin S solutions have resulted in an analysis of the thermodynamics of gelation and a description of the kinetics of this process in terms of polymer condensation theory. Understanding of the mechanism of intracellular gelation is much less advanced because of lack of methods for studying the process in intact cells. The major method for studying the thermodynamics of deoxyhemoglobin S gels has been ultracentrifugation sedimentation in which the gel is physically separated into a sol phase consisting of free hemoglobin molecules and a pellet phase which contains noncovalently polymerized hemoglobin molecules, in addition to free hemoglobin (2-6). The concentration of the sol phase, which depends on temperature but not on initial deoxyhemoglobin S concentration, has been used as a measure of deoxyhemoglobin S solubility. These results indicate that the polymerization of deoxyhemoglobin S can be described by a two-phase model (7) consisting of deoxyhemoglobin S molecules free in solution in equilibrium with a condensed phase. An alternative method for characterizing deoxyhemoglobin S gels is the use of NMR spectroscopy, especially 13C/IH magnetic double-resonance techniques. Natural abundance 13C
(11)].
l3C NMR. Natural abundance 13C spectra without nuclear Overhauser enhancements were taken of the hemoglobin samples in 8-mm tubes by using a Nicolet TT-14 spectrometer modified for high-power '3C/IH magnetic double-resonance
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Abbreviation: hemoglobin S, sickle cell hemoglobin (36Glu-Val).
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Proc. Natl. Acad. Sci. USA 76 (1979)
4937 -
experiments in solids. The probe contained an external 2H20 lock, and the sample was maintained at 300C by using,.fiow of temperature-regulated nitrogen gas through the probe in a Dewar flask. The temperature gradient throughout the sample was less than 20C. The probe was tuned to 50 ohms for each sample. A 50-W '3C transmitter (15.09 MHz) gave a 90° rotation of the 13C magnetization in about 5 ,usec. A proton resonant field of 0.7 gauss (^y2H2/2r = 3 kHz) was applied for scalar decoupling and of 13 gauss ('y2H2/27r = 55 kHz) for dipolar decoupling. For the proton-enhanced spectra, the HartmanHahn condition ('yHl = 72H2) was determined as described (9). The sign of the proton spin temperature was alternated in the proton-enhanced experiments to minimize artifacts. Relative signal intensities were determined by direct integration and from difference spectra. In the latter case, one spectrum was multiplied by a scale factor (determined by nulling appropriate pairs of spectra) and then subtracted from a second spectrum. Solubility. Deoxyhemoglobin S solubility was determined by ultracentrifugation as described by Hofrichter et al. (11). Samples were transferred from the NMR tube into 5-mm quartz electron paramagnetic resonance sample tubes (Wilmad PQ-701) under nitrogen; the tubes were sealed with pressure caps (Wilmad-521-PC) and the caps were painted (Glyptol, General Electric). The near-infrared spectra (1300-700 nm) of the sample were identical before and after transfer, indicating that the sample remained fully deoxygenated. The samples were gelled at 30'C and spun at 100,000 X g at 30'C. The solubility or supernatant concentration was determined from absorbance measurements at 910 and 1090 nm as described above.
RESULTS Natural abundance 13C NMR was used to determine the fraction of polymerized deoxyhemoglobin S at 30°C (Fig. 1). For each sample of deoxyhemoglobin S, a 1H scalar-decoupled spectrum, a 'H dipolar-decoupled spectrum, and a protonenhanced spectrum were recorded. A 'H scalar-decoupled spectrum of the matching oxygenated control (made up identically but with buffer in place of sodium dithionite solution) was also recorded (Fig. 1, curve a). We assign resonances in the spectra as follows: carbonyl carbons at 15-25 ppm; aromatic carbons at 60-75 ppm; aliphatic carbons (primarily backbone a-carbons) at 120-150 ppm; and side-chain aliphatic carbons at 150-200 ppm. For the oxygenated control, the intensity of the scalar-decoupled spectrum, which monitors only rotationally free material (identified with free hemoglobin in solution), was directly proportional to the total hemoglobin S (Fig. 2) because no gel was present. The scalar-decoupled spectrum (Fig. 1, curve b) of deoxyhemoglobin S at the same concentration was diminished due to the immobile material in the gel (identified with polymerized molecules). The dipolar-decoupled spectrum (Fig. 1, curve c) which is sensitive to both mobile and immobile hemoglobin had more intensity in the aliphatic region. However, the intensity still was less than the oxygenated control: only 75-80% of the polymer carbon signal was observed in the dipolar-decoupled spectrum because of incomplete spin-lattice relaxation and because of electron-nucleus dipolar broadening (9). The difference between the dipolar decoupled spectrum and the scalar-decoupled spectrum of deoxyhemoglobin S (Fig. 1, curve d) represents the spectrum of polymerized molecules. The signal intensity of the proton-enhanced spectrum (Fig. 1, curve e) also was proportional to the amount of polymerized deoxyhemoglobin S (Fig. 3) but was 2.5 + 0.5 times greater than the signal intensity of the difference spectrum (Fig. 1, curve
A
"'I aa
b
c
d
el
4
II
0
100
200
ppm
FIG. 1. '3C/PH double magnetic resonance spectra for hemoglobin S (28 g/dl) at 30°C. Curve a is the 1H scalar-decoupled spectrum for the oxygenated control sample. The 1H scalar-decoupled spectrum monitors only free or unaggregated material. The 1H dipolar-decoupled spectrum for this sample was similar because there was no polymerized hemoglobin present. (The 1H dipolar-decoupled spectrum is proportional to the amount of total hemoglobin present, free and aggregated). Curves b through e are spectra of the deoxygenated sample. Curve b is the 1H scalar-decoupled spectrum for the deoxyhemoglobin S sample. The intensity is diminished compared to the oxygenated sample spectrum (curve a) due to the aggregation or polymerization of some of the hemoglobin S in the gel state. Curve c is the 1H dipolar-decoupled spectrum of the deoxyhemoglobin S sample. Curve d is the difference spectrum between the 1H dipolardecoupled spectrum and the 1H scalar-decoupled spectrum, which is a measure of the amount of aggregated or polymerized deoxyhemoglobin S. Curve e is the proton-enhanced spectrum which is also proportional to the amount of polymerized or aggregated hemoglobin. However, this spectrum is about 2.5 times greater in intensity than curve d. Spectroscopic parameters are the same as given in ref. 9; chemical shift is based on CS2.
d) because of the intensity enhancement provided by crosspolarization. This enhancement factor was measured by nulling the proton-enhanced spectrum with the difference spectrum. The data in Fig. 3 can be extrapolated to zero polymer to obtain an estimate of deoxyhemoglobin S solubility. A linear regression analysis of the data gives zero polymer at a hemoglobin concentration of 17.3 b 2 g/dl. The fraction of free deoxyhemoglobin S is taken to be the ratio of the integrated intensity of the aliphatic region of the deoxyhemoglobin S scalar-decoupled spectrum to the integrated intensity of the oxyhemoglobin S scalar-decoupled
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Proc. Natl. Acad. Sci. USA, 76 (1979)
Biochemistry: Noguchi et al.
C
CD
0.8
26 Hemoglobin, g/dl
22
30
FIG. 2. Integrated intensity of the1H scalar-decoupled spectrum for the oxygenated control as a function of hemoglobin S concentration. The integrated intensity was obtained by numerical integration of the aliphatic region with the Nicolet software. The results are normalized to the maximum obtained (31.2 g/dl, oxygenated sample). Scaling each spectrum to this maximum until a null difference spectrum is obtained gives similar results. The straight line represents a linear regression analysis of the data (relative intensity = 0.081 + 0.030[HbS]; r2 = 0.949).
spectrum.t The polymer fraction is 1 minus the free fraction (Fig. 4). The polymer fraction increased with increasing deoxyhemoglobin S concentration, from 0.22 at 20.7 g/dl to 0.52 at 31.7 g/dl. The value of the fraction of free deoxyhemoglobin S was also calculated from the two scalar-decoupled spectra by using the Nicolet software for directly obtaining null difference spectra. The results of these methods fall within 10% of each other. Lastly, the fraction of polymerized deoxyhemoglobin S was obtained by using ultracentrifugation sedimentation which separates the gel into a sol phase and a pellet (3). The pellet is assumed to be composed of polymerized hemoglobin as well as free hemoglobin equal in concentration to the sol phase. By using a polymer concentration of 70 g/dl [obtained from studies of deoxyhemoglobin A and S mixtures (12)] and the sol phase concentration, the fraction of polymerized deoxyhemoglobin S was determined by assuming a two-phase model. When the NMR spectral experiments were completed, the sol phase concentration was determined after ultracentrifugation sedimentation, and the fraction of polymerized deoxyhemoglobin S was calculated. The calculated fraction of polymerized hemoglobin varied from 0.31 at 20.7 g/dl to 0.68 at 31.7 g/dl by this method. These uncorrected values have an absolute average 0.15 greater than the values obtained from the NMR spectroscopy, well beyond the uncertainties of the measurements. The apparent discrepancy between the two methods can be accounted for, in part, by considering the concentration gradient introduced during ultracentrifugation sedimentation of concentrated hemoglobin solutions. For example, for the sedimentation tubes with a sample volume of 350 Al and an oxyhemoglobin S solution of 12 g/dl, spinning under conditions t These methods are equivalent to method A in ref. 9. As was indicated in that paper,
we no
longer
use
method B [ratio of
integrated aliphatic
intensity in the difference spectrum (Fig. 1, curve d) to that in the oxygenated control (Fig. 1, curve a)l because incomplete spin-lattice relaxation and paramagnetic broadening cause underestimation of
polymer in that method.
V
30
26 22 Hemoglobin, g/dl
18
FIG. 3. Integrated intensity at 30'C of the "3C proton-enhanced spectrum as a function of deoxyhemoglobin S concentration. The total integrated intensity of the 13C proton-enhanced spectrum including the carbonyl carbons was obtained by using the Nicolet software. The results are normalized to the maximal value obtained (31.2 g/dl deoxygenated sample). The relative intensity is proportional to the amount of aggregated or polymerized material present. The straight line represents a linear regression analysis of the data (relative intensity = -1.16 + 0.067 X hemoglobin concentration (g/dl); r2 = 0.945).
used for solubility studies results in a noticeable concentration gradient such that the mean (+SEM) hemoglobin concentration is 8.9 i 0.29 g/dl for the top lOO1,l and 10.0 i 0.1 g/dl for the top 250 ,l, as determined by the cyanmethemoglobin method at 540 nm (13).
0.60
(U
E 0o4k
0.2
2 22
26 Hemoglobin, g/dl
30
FIG. 4. Fraction of polymerized deoxyhemoglobin S as a function of deoxyhemoglobin S concentration at 300C. 0, Results obtained from 13C NMR by determining first the fraction of free hemoglobin from comparison between the 'H scalar-decoupled spectra of the deoxy-sample and the matching oxygenated control; O, results obtained from ultracentrifugation sedimentation on the same samples after the NMR data were collected, assuming a polymer concentration of 70 g/dl. The solid line represents the predicted fraction of polymer, assuming this polymer concentration and a solubility of 16.4 g/dl. The dashed line, which best fits the NMR data, extrapolates to a solubility of 18.4 g/dl.
Biochemistry: Noguchi et al. DISCUSSION NMR spectroscopy has been used to study sickle hemoglobin and to follow the gelation process. 1H NMR has been used to demonstrate several surface histidine residues which differ between oxy- and deoxyhemoglobin S (14, 15). Water 1H NMR relaxation methods have been used to follow the process of polymerization, in hemoglobin solutions and in cells (16-19). In contrast to these earlier studies, the 13C/IH magnetic double-resonance spectroscopic methods discussed here can be used to quantitate directly the fraction of polymerized hemoglobin molecules and thus can give useful direct information about the thermodynamic and kinetic properties of the polymerization process. In order to show the usefulness of the NMR spectroscopy for such quantitation, the NMR data were compared to data from sedimentation within the framework of a two-phase model. The two-phase model has been used extensively in the analysis of the thermodynamics of gelation (3-6). Our own measurements of 13C NMR linewidths of deoxyhemoglobin S gels, indicating the coexistance of an isotropically mobile phase and a polymerized or crystalline-like phase, are also consistent with a two-phase model (9). The two-phase model assumes that the deoxyhemoglobin S gel is composed of free hemoglobin of concentration C., and of polymer of concentration Cp. The total concentration of deoxyhemoglobin S, CT, can then be separated into the condensed phase and the free phase (CT = CP VP + C. V, in which vP and v, are the volume fractions of polymerized and free deoxyhemoglobin S). The fraction of polymerized deoxyhemoglobin S, FP = CP VP/CT, and the fraction of free deoxyhemoglobin S, F, = C, V,/CT, can also be completely expressed in terms of CT, CP, and C, to yield FP = Cp (CT - C,)/[CT(Cp - C,,)] and F, = C, (Cp - CT)/[CT(CP - C,.)]. The concentration of deoxyhemoglobin S in the polymer state, C., has been determined to be 70 g/dl (12), which is close to the value of 69.6 g/dl, the hemoglobin concentration of deoxyhemoglobin S crystals (20). In ultracentrifugation sedimentation studies the supernatant concentration is assumed to be the deoxyhemoglobin S solubility, C., or free homoglobin concentration (3, 4). By using that value for C,,, the total deoxyhemoglobin S concentration, CT, which is measured directly, and the above value for CP, the fraction of polymerized and free hemoglobin (FP and F,J, respectively) can be determined. In NMR studies, the fraction of polymerized and free hemoglobin are measured and are used to determine C,. If the two-phase model is valid, the NMR and ultracentrifuge sedimentation experiments should give similar values for the amount of polymerized and free material for any deoxyhemoglobin S sample. We find that, in the concentration range 24-32 g/dl, the polymer fraction obtained by using uncorrected ultracentrifugation sedimentation data (Fig. 4) is on the average 0.15 higher than that obtained by using 13C NMR data. For example, at a total deoxyhemoglobin S concentration of 28 g/dl, 13C NMR yields a polymer fraction of 0.44 whereas ultracentrifugation sedimentation yields a polymer fraction of 0.56. The differences between sedimentation and NMR measurements can be examined in terms of the differences in the free hemoglobin concentration C,. C, is taken to be the supernatant concentration in sedimentation and was found to be 16.4 g/dl at 30°C under these conditions (21, 22). From Fig. 3, we estimate a solubility (no polymer present) of 17.3 g/dl. Our own experiments confirm the observations by others that the sedimenation process underestimates solubility and overestimates polymer, either by packing free hemoglobin molecules into the pellet or by forming new polymer at the top of the pellet (23, 24). Behe and Englander (23) estimated this ef-
Proc. Natl. Acad. Sci. USA 76(1979)
4939
feet at 5% [as did B. Magdoff-Fairchild (personal communication)]. Although our measurements suggest an effect several times higher, nonideality (25) would decrease this effect at the higher concentrations in which the NMR experiments were done. Thus, the value of 17.3 g/dl would fit both our data from proton-enhanced spectra and corrected ultracentrifugation runs. The solid line in Fig. 4, representing the polymer fraction, was calculated by using the 16.4 g/dl value and a polymer concentration of 70 g/dl; the dashed line, which better fits the NMR data, requires a solubility of 18.4 g/dl. Use of the 17.3 g/dl solubility value would lower the ultracentrifugation data almost to within the error limits of the NMR data. Several errors could arise in the process of collecting the 13C NMR data (9). Paramagnetism of deoxyhemoglobin S or paramagnetism due to the presence of sodium dithionite could decrease the signal intensity via dipolar broadening. Another source of error would exist if the exchange rate between free and bound (polymerized) deoxyhemoglobin S molecules were sufficiently slow as to broaden the signal peaks. However, these sources of error would result in loss of intensity for the free hemoglobin fraction, resulting in an overestimate of the polymer fraction. If the polymer contributed to the scalar-decoupled spectrum, this would result in an underestimate of the polymer fraction. We believe this is unlikely, however, because one would then expect a greater intensity contribution from the mobile side-chain carbon atoms than from the backbone carbon atoms and we find no difference in their relative intensities in the oxy- and deoxyhemoglobin spectra (Fig. 1, curves a and
b). §
Another possible source of discrepancy between the two methods relates to the assumed value of the polymer concentration. However, it would take significantly higher values, to reconcile the two sets of data, and the value we have used (12) is close to the maximum, which seems reasonable (27). A failure of the approximations of the two-phase model (7) could also be envisioned to account for our results. In any case, the residual divergence of data of the two methods is small enough to justify further equilibrium and kinetic studies of sickle hemoglobin polymerization. The major significance of the use of '3C/'H magnetic double-resonance spectroscopy is likely to be in the study of the mechanism of cell sickling. Methods of studying intracellular gelation have relied upon techniques such as light and electron microscopy, birefringence, and filterability (1), as well as water proton relaxation methods (16-19). None of these methods" gives a simple, linear measurement of the polymerization process and thus have been of limited utility in studying the mechanism of sickling or the effects of sickling inhibitors (1). We have shown that these new 13C NMR methods can be used to study intracellular sickle hemoglobin polymerization (9), and measurements of the extent of polymerization as a function of PO2, pH, temperature, and concentration of various covalent and noncovalent inhibitors are now possible. § The intensity of the scalar-decoupled spectrum of deoxyhemoglobin S was independent of Y2H2 in the range 1-3 kHz, indicating that intensity from anisotropically mobile groups in the polymer was not visualized in the scalar-decoupled spectrum. The absence of scalardecoupled spectral intensity in collagen provides further evidence that such groups are not detected in scalar-decoupled spectra (26). Smaller values of polymer concentration used previously (3, 4) were based on the assumption that the pellet contained no free monomer. Only now are reliable methods for measuring the kinetics of polymerization of sickle hemoglobin in single cells being developed
(28).
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Biochemistry: Noguchi et al.
1. Dean, J. & Schechter, A. N. (1978) N. Engl. J. Med. 299, 752-763; 804-811; 863-870. 2. Bertles, J. F., Rabinowitz, R. & D6bler, J. (1970) Science 169, 375-377. 3. Hofrichter, J., Ross, P. D., Eaton, W. A. (1976) in Proceedings of the Symposium on Molecular and Cellular Aspects of Sickle Cell Disease, DHEW Publication No. (NIH)76-1007, ed. Hercules, J. I., Cottam, G. L., Waterman, M. R. & Schechter, A. N. (GPO, Washington, DC), pp. 185-223. 4. Ross, P. D., Hofrichter, J. & Eaton, W. A. (1977) J. Mol. Biol. 115, 111-134. 5. Magdoff-Fairchild, B., Poillon, W. N., Li, T.-I & Bertles, J. F. (1976) Proc. Natl. Acad. Sci. USA 73,990-994. 6. Pumphrey, J. G. & Steinhardt, J. (1976) J. Mol. Biol. 112, 359-375. 7. Minton, A. P. (1974) J. Mol. Biol. 82,483-498. 8. Torchia, D. A. & VanderHart, D. C. (1979) in Topics in Carbon-13 NMR Spectroscopy, ed. Levy, G. C. (Wiley, New York), Vol. 3, pp. 325-360. 9. Sutherland, J. We H., Egan, W., Schechter, A. N. & Torchia, D. A. (1979) Biochemistry 18, 1797-1803. 10. Huisman, T. H. J. & Dozy, A. M. (1965) J. Chromatogr. 19, 160-169. 11. Hofrichter, J., Ross, P. D. & Eaton, W. A. (1976) Proc. Natl. Acad. Sci. USA 73,3035-3039. 12. Sunshine, H. R., Hofrichter, J. & Eaton, W. R. (1979) J. Mol. Biol., in press. 13. Van Assendelft, 0. W. (1970) Spectrophotometry of Haemoglobin Derivatives (Royal Vanogram, Assen, Netherlands), pp. 110-112.
Proc. Natl. Acad. Sci. USA 76 (1979) 14. Ho, C., & Russu, L. M. (1978) in Biochemical and Clinical Aspects of Hemoglobin Abnormalities, ed. Caughey, W. S. (Academic, New York), pp. 179-194. 15. Russu, I. M. & Ho, C. (1979) Blophys. J. 25, 127a. 16. Zipp, A., James, T. L., Kuntz, I. D. (1976) Biochim. Blophys. Acta 428,291-303. 17. Lindstrom, T. R., Koenig, S. H., Boussios, T. & Bertles, J. F. (1976) Biophys. J. 16,679-689. 18. Eaton, W. A., Hofrichter, J., Ross, P. D., Tschuden, R. G. & Becker, E. D. (1976) Biochem. Biophys. Res. Commun. 69, 538-547. 19. Cottam, G. L., Shibata, K. & Waterman, M. R. (1978) in Biochemical and Clinical Aspects of Hemoglobin Abnormalities, ed. Caughey, W. S. (Academic, New York), pp. 695-715. 20. Wishner, B. C., Ward, K. B., Lattman, E. E. & Love, W. E. (1975) J. Mol. Biol. 98, 161-178. 21. Noguchi, C. T. & Schechter, A. N. (1977) Biochem. Biophys. Res. Commun. 74, 637-642. 22. Noguchi, C. T. & Schechter, A. N. (1978) Biochemistry 17, 5455-5459. 23. Behe, M. J. & Englander, S. W. (1978) Biophys. J. 23,129-145. 24. Chung, L. L. & Magdoff-Fairchild, B. (1978) Arch. Biochem. Biophys. 189,535-539. 25. Ross, P. D., Briehl, R. W. & Minton, A. P. (1978) Biopolymers 17, 2285-2288. 26. Jelinski, L. W. & Torchia, D. A. (1979) J. Mol. Biol., in press. 27. Hofrichter, J. (1979) J. Mol. Biol. 128,335-370. 28. Sunshine, H. R., Ferrone, F. A., Hofrichter, J. H. & Eaton, W. A. (1979) in Development of Therapeutics Agents for Sickle Cell Disease, eds. Rosa, J., Beuzard, Y. & Hercules, J. (Elsevier/ North-Holland, Amsterdam), pp. 131-146.
