A Study of the Mechanisms of Slow Religation to Sickle Cell Hemoglobin Polymers Following Laser Photolysis

J. Mol. Biol. (1996) 259, 947–956

A Study of the Mechanisms of Slow Religation to Sickle Cell Hemoglobin Polymers Following Laser Photolysis Daniel B. Shapiro1, Raymond M. Esquerra1, Robert A. Goldbeck1 Samir K. Ballas2, Narla Mohandas3 and David S. Kliger1* 1

Department of Chemistry and Biochemistry, University of California, Santa Cruz CA 95064, USA 2 The Cardeza Foundation Department of Medicine Jefferson Medical College Philadelphia, PA 19107, USA 3

Life Sciences Division Lawrence Berkeley Laboratory, Berkeley CA 94720, USA

Time-resolved linear dichroism (TRLD) measurements are conducted on gels of sickle cell hemoglobin following laser photolysis of the carbonyl adduct to monitor religation kinetics to hemoglobin S polymers. The return of the polymer phase to its equilibrium ligation state has been found to be about 1000 times slower than that of the solution phase hemoglobin tetramers. Several mechanisms describing this slow religation to the polymer were proposed: (1) religation occurs through a biomolecular process involving all polymer hemes, (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate, and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase. To test these mechanisms, measurements are performed on gels having different domain sizes. The results show no relation between domain size and religation kinetics. The independence of religation kinetics and domain size is most consistent with the first of the three mechanisms described above (bimolecular recombination involving all polymer hemes). This result is discussed in terms of a model in which diffusion of the ligand is inhibited in the polymer phase. An understanding of the ligand binding kinetics of sickle hemoglobin polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation. 7 1996 Academic Press Limited

*Corresponding author

Keywords: sickle cell hemoglobin; time resolved linear dichroism; polymerization; kinetics; laser photolysis

Introduction Sickle cell anemia is a disease that affects approximately one out of 600 people of African descent born in the United States (Rucknagel, 1975). The disease is characterized by microvascular occlusions caused (at least in part) by abnormally shaped and rigid red blood cells formed under hypoxic conditions. The distortion of the cell is due to the polymerization of an abnormal hemoglobin: hemoglobin S (HbS; Pauling et al., 1949). This abnormality is the result of a single point mutation at the b6 (Glu : Val) position (Ingram, 1956). This substitution of a hydrophobic for a hydrophilic Abbreviations used: TRLD, time-resolved linear dichroism; CO, carbon monoxide; SVD, singular value decomposition; DIC, differential interference contrast; LD, linear dichroism; Hb, hemoglobin. 0022–2836/96/250947–10 $18.00/0

residue causes the polymerization reaction to occur when the hemoglobin S is in the T (deoxy) state. The microvascular occlusion characteristic of sickle cell disease results in a large degree of morbidity and mortality. The HbS polymer consists of a 21 nm diameter fiber containing seven intertwined double helical strands with the hemes of each hemoglobin molecule approximately perpendicular to the axis of the fiber (Eaton & Hofrichter, 1990). Under the conditions used in the present study, as well as in vivo, the fibers form a macroscopic gel. The hemoglobin molecules in the polymer (the polymer phase) are in dynamic equilibrium with those in solution (the solution phase). The organization of the fibers in the gel takes on many forms both in vivo and in vitro. Due to the birefringence of the hemes (and hence the fiber) the organization of the gel can be observed through crossed polarizers in 7 1996 Academic Press Limited

948

Figure 1. Optical micrograph of a sickle cell hemoglobin gel viewed between crossed polarizers. Light is transmitted when the polymers are oriented at 45° with respect to the crossed polarizers because in this orientation they both rotate and introduce ellipticity into the polarization state of the light. Areas of darkness correspond to regions where the polymers are oriented parallel or perpendicular to the crossed polarizers. The radial symmetry of the imaged domain is seen as a cross. The radial symmetry was confirmed by rotation of the sample without a change in the areas of light and dark in the image (data not shown). The image appears blue because the sample is most birefringent and linearly dichroic in the region from 400 to 550 nm. The image size is 2.1 mm × 1.8 mm. The sample thickness was 30 microns. A Leitz diaplan microscope was used with white light produced by a tungsten lamp.

a transmission microscope. Fibers oriented parallel or perpendicular to the polarizer axes do not transmit light whereas those oriented at 45° do. Fibers commonly organize into spheres radiating outward and produce a cross-image when viewed through crossed polarizers. An example of such an image is shown in Figure 1. The growth of polymers and gels is understood in terms of the double nucleation mechanism (Ferrone et al., 1985a,b; Dou & Ferrone, 1993). The process begins with homogeneous nucleation, the joining of a sufficient number of hemoglobin molecules to from a critical nucleus. After the critical nucleus has formed, the growth of a polymer from this nucleus becomes thermodynamically favorable. At this point heterogeneous nucleation may occur. Heterogeneous nucleation involves the formation of an additional polymer on the surface of an existing one. Homogeneous nucleation is the rate limiting step in polymer and

Religation Mechanism of Sickle Hemoglobin Polymers

gel formation. Polymer formation is therefore characterized by a long delay time during which no polymer is formed. Once the polymer begins to grow, more surface area becomes available for heterogeneous nucleation to occur so that polymer growth becomes exponential. Since homogeneous nucleation is a rare event, an entire network of polymers, known as a domain, results from a single nucleation. The number and size of the domains depends on how quickly the gelation occurs, and this depends on the ratio of the solubility of the hemoglobin (the concentration that will not polymerize) to the concentration of total hemoglobin. Many relatively small domains can be formed by rapidly increasing the temperature of a solution of HbS from a low temperature (where the solubility is high) to a temperature of lower solubility. Alternatively, fewer but larger domains can be formed by slowly heating the sample. Although much has been learned about the process and kinetics of polymerization, with few exceptions (Hofrichter et al., 1974; Messer et al., 1976; Harrington et al., 1977; Mozzareli et al., 1987; Gross et al., 1991; Briehl, 1995) little study has been devoted to depolymerization or melting. The kinetics of both polymerization and melting are important in the pathophysiology of sickle cell disease because they play a key role in determining whether polymerization will occur in some cells. A red blood cell typically spends about ten seconds under hypoxic conditions during the 14 seconds spent in one round of circulation (Mozarelli et al., 1988; Altman & Ditmer, 1971). If the delay time for polymerization is greater than ten seconds then there will be no polymerization. If, however, some polymerization occurs, then it is important to know if the polymers will melt completely after re-oxygenation at the lungs and before re-entering the microvasculature under hypoxic conditions. If melting is incomplete, then the presence of the existing polymer will allow relatively rapid heterogeneous nucleation to occur that could lead to occlusion. The physiologically relevant variable that is most important in determining whether polymerization will occur and how fast it will occur is ligation (Eaton & Hofrichter, 1990). The partial ligation of hemoglobin molecules in the polymer phase will also play a role in the kinetics of melting (Briehl, 1995). It has been shown that HbS molecules in the solution phase have the same equilibrium binding (Allen & Wyman, 1954) and the same kinetics involved in ligand binding as normal adult hemoglobin HbA (Pennelly & Noble, 1978; Deyoung & Noble, 1981; Shapiro et al., 1994). The equilibrium binding curve of HbS polymer for oxygen has been measured using linear dichroism spectroscopy (Sunshine et al., 1982), which measures the difference in absorption between horizontally and vertically polarized light. It was found that polymer HbS has an equilibrium binding of about 1/3 that of normal T-state HbA. A similar study was conducted using carbon

949

Religation Mechanism of Sickle Hemoglobin Polymers

monoxide as the ligand (Hofrichter, 1979). These studies take advantage of the fact that hemes are nearly planar absorbers (Eaton & Hochstrasser, 1967, 1968) so that light is absorbed more strongly when polarized parallel to the heme planes. Since the heme planes are oriented roughly perpendicular to the polymer long axis, light that is polarized perpendicularly to the long axis of the polymer is more strongly absorbed than when it is polarized along the direction parallel to the polymer axis (Hofrichter et al., 1973). A HbS gel is linearly dichroic because of the partial orientation of the polymers in the gel. The hemoglobin molecules in the solution phase do not contribute to the linear dichroism because they have no preferred orientation. Linear dichroism spectroscopy is thus able to distinguish absorption due to the HbS molecules in the polymer phase from that in the monomer phase and was used to obtain the equilibrium binding curve of oxygen to HbS polymers (Sunshine et al., 1982). Using time-resolved linear dichroism (TRLD) measurements, we have reported the religation kinetics of CO to HbS polymers following laser photolysis (Shapiro et al., 1995). In these experiments, the liner dichroism is measured at precise times after CO has been photolyzed by a polarized actinic beam. Due to photoselection, more hemes will be deligated that are parallel to the polarization of the actinic beam than those that are perpendicular. A linear dichroism measurement (absorption parallel to the laser polarization-absorption perpendicular) taken after photolysis (corrected for the static linear dichroism measured before photolysis) resembles a deoxy-carboxy difference absorption spectrum. Linear dichroism due to photoselection of hemoglobin molecules in the monomer phase disappears within hundreds of nanoseconds after photolysis because of rotational diffusion. The linear dichroism from the molecules in the polymer phase disappears with the kinetics of religation because the polymers do not rotate. We reported previously that religation of the polymer phase occurs about 1000 times slower than that of the monomer phase (Shapiro et al., 1995). We proposed several mechanisms for this slow religation: (1) religation occurs through a bimolecular process involving all polymer hemes (henceforth referred to as the bimolecular mechanism), (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate in polymer religation (ends mechanism), and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase (exchange mechanism). Another potential mechanism involves dissociation of CO from solution phase hemoglobin molecules followed by binding of these CO molecules to polymer phase hemoglobin. This mechanism involving CO dissociation was excluded as an explanation for polymer religation because the CO dissociation rate is too

slow to account for the observed religation lifetimes (Shapiro et al., 1995). Here we report the results of experiments designed to distinguish between these mechanisms proposed for slow religation. Both the second and third mechanisms described above (the ends mechanism and the exchange mechanism) depend on the number of ends in the polymer domains. Smaller domains are expected to have smaller fibers and more ends. The ends mechanism thus predicts that smaller domains become religated faster than bigger ones because there are more ends. Since polymer growth and depolymerization occurs only at polymer ends (Briehl, 1995), exchange would also be expected to occur exclusively at polymer ends. That smaller domains have more ends allows exchange between the phases to occur more readily. Thus both the ends mechanism and the exchange mechanism predict that smaller domains will be religated faster following CO photolysis than larger ones. On the other hand, the bimolecular mechanism predicts no dependence of the religation kinetics on domain size. We have measured the kinetics of polymer religation for gels as a function of domain size. We find no effect of domain size on the religation kinetics, thus supporting the bimolecular mechanism.

Results Typical visible band linear dichroism spectra of a HbS gel arising from the static, partial orientation of the hemes in the polymer phase are shown in Figure 2. The steady state linear dichroism resembles a deoxy-hemoglobin absorption spectrum, which has a single peak at 555 nm. Deviations from the deoxy-hemoglobin absorption spectrum are due to stray light artifacts in detection or to a distortion of the signal by the birefringence of the gel. The steady state linear dichroism varies as a function of orientation of the sample cell with respect to the probe polarizers because the polymers rotate along with the cell. A rotation of 90° causes a reversal in sign of the linear dichroism. Rotation of 45° from a position of absolute maximum signal amplitude gives a minimum absolute signal amplitude because the preferred orientation of the polymer is now at 45° with respect to the polarizers. We did not find any dependence of the time-resolved linear dichroism difference spectra (transient minus steady state) on the size or sign of the equilibrium spectra. This is consistent with the transient linear dichroism arising from photoselection. TRLD difference spectra are shown in Figure 3 for gels containing relatively large (Figure 3(a)) and relatively small (Figure 3(b)) domains. Each curve is the difference of the transient linear dichroism of the sample taken at a given time after photolysis minus the ground state linear dichroism. The curves resemble a deoxy-carboxy absorption difference spectrum. The spectral curves approach zero as the system returns to equilibrium by religation

950

Religation Mechanism of Sickle Hemoglobin Polymers

Figure 2. Linear dichroism spectra of a sickle cell hemoglobin gel at different orientations. The static linear dichroism arises from partial orientation of the gel. Polymers absorb more light polarized perpendicular to their long axes than that polarized parallel. The curve with the negative dichroism values was taken with the sample rotated by 90° around the probe beam axis from the position exhibiting the large positive peak. These curves derive their shape primarily from the absorption curve of the (mostly deoxy) polymers. The relatively flat curve was measured with the sample oriented at 45° with respect to the positions giving maximum and minimum peaks. The LD signal shown here is scaled by 1/b = 38 so that 0.4 corresponds to 0.01 optical density units of LD.

of the sickle hemoglobin polymers. The TRLD data were analyzed using singular value decomposition (SVD) and fit to an exponential lifetime. The most significant basis vectors obtained by SVD are shown in Figure 4(a) and (b). SVD confirms that the most significant spectral component in the TRLD data is the deoxy-carboxy difference curve. The time courses of these major spectral components (the first basis vectors) are shown in Figure 4(c) and (d). TRLD data were collected for three sets of samples, each set containing one sample that was made with relatively large domains and another sample with relatively small domains. The only difference in each matched set of samples (large domain versus small domain) is the number and size of the domains. Micrographs taken through crossed polarizers of each of the samples are shown in Figure 5. The micrographs (Figure 5) show that for each preparation there is a difference of about a factor of 4 to 10 in the radius of the domains between the smaller and larger domain samples. The truncated data for each sample, obtained using SVD, were fit to exponential lifetimes given in Table 1. The fit to an exponential function is justified for pseudo-first order kinetics where the concentration of free polymer hemes are in excess. However, the actual rebinding kinetics may be more complicated if the religation involves diffusional barriers or geminate processes. Nevertheless,

Figure 3. Time resolved linear dichroism difference spectra. Each curve represents the linear dichroism measured at a given time after photolysis minus the equilibrium linear dichroism. The curves are that of a deoxy-carboxy absorption difference curve because photoselection causes hemes that are parallel to the laser polarization to be relatively deoxy and hemes that are perpendicular to the laser polarization to be relatively carboxy. The difference curves approach zero as the system returns to equilibrium. The TRLD signals shown here are scaled by 1/b = 38 so that a TRLD signal of 0.01 corresponds to 0.0025 optical density units of LD. (a) TRLD data taken on a sample that was slowly heated over two days from 0 to 23°C. The LD was measured at 0.025, 0.525, 1.025, 1.525, 2.025, 3.025 and 3.525 seconds following photolysis. (b) TRLD data taken on a sample that was immediately temperature jumped from 0 to 30°C and then allowed to reach room temperature. The LD was measured at 0.025, 0.525, 1.025, 1.525, 2.025, 2.525 and 3.025 seconds following photolysis.

the use of an exponential function serves as a good empirical characterization of the kinetics. The lifetimes for the samples with small domains were 7.3, 5.4, and 4.7 seconds. The lifetimes for the corresponding samples with large domains were 3.6, 5.3, and 5.7 seconds, respectively. The average of the lifetimes for the small domain samples is 5.8 (21.3) seconds and that of the large domains is 4.9 (21.1) seconds. The only preparation in which there seems to be a significant difference between the kinetics of religation is preparation 1. However,

Religation Mechanism of Sickle Hemoglobin Polymers

951 Figure 4. Singular value decomposition of TRLD data from Figure 3. The principle component basis spectra are shown together with their time courses multiplied by the square root of their singular values. The data for the sample that was slowly heated is shown in (a) and (c) and that for the sample that was rapidly temperature jumped is shown in (b) and (d). The first and largest basis spectra (shown in (a) and (b) (—)) correspond to the deoxy-carboxy difference spectrum. The second basis spectrum (– – –) is mostly random noise but appears to contain some spectral structure resembling a deoxy spectrum that may be due to polymerization and melting of the polymer. Unfortunately, our signal to noise is insufficient to pursue this possibility. SVD shows that the major kinetic component of our data is religation of the polymer. The time courses of the first basis spectra are shown in (c) and (d) plotted as the log(V) versus time. The second basis spectrum had a random time course.

the difference in the lifetimes for religation in this preparation is opposite to the prediction of the ends and exchange mechanisms. That the difference in lifetimes seen in preparation 1 is not reproduced in preparations 2 and 3 indicates that this difference is due to relatively poor signal to noise in measurements on preparation 1 and is not significant.

Discussion The main results of this study are given in Table 1. There is no significant difference in religation lifetimes for matched sets of samples with small domains versus the corresponding samples with large domains. The micrographs (Figure 5) show that there is at least a factor of four difference in the diameter of domains for each matched set of samples. An estimate of a factor of 4 is very conservative. Assuming the length of the polymers scale with the domain diameter this would correspond to a difference of a factor of 4 in the number of polymer ends. Thus there should be at least a fourfold difference in the ends where exchange between phases could occur and where exposed polymer ends could rebind CO. Accordingly, the ends mechanism and the exchange mechanism would predict that religation should occur at least four times faster in the small domain samples than in the large domain samples. The results of kinetic measurements given in Table 1 do not support this prediction. We conclude that the bimolecular mechanism, where all free hemes in the

polymer can rebind CO, is most consistent with these data. In general, the smaller domains formed using quick temperature jumps did not exhibit as many pure cross patterns in the polarizing microscope as the larger domain samples formed by slow heating. This could be because the concentration of hemoglobin molecules in solution approaches the solubility before a completely radially symmetric domain has formed. In addition, the small domain samples were temperature jumped to 30°C and then allowed to melt at room temperature, thus possibly distorting the spherical symmetry of the domain. In any case, the asphericity of the small domains would not affect the fact that they have more polymer ends than the large domains. The large difference between the rebinding kinetics for hemoglobin molecules in the monomer and polymer phases is perhaps most simply accounted for by the exchange model. Using known CO on rates for R and T state hemoglobin one predicts that CO recombination is complete by 10 ms after photolysis (Bertini et al., 1994; Shapiro et al., 1995). It then seems impossible that any CO would be left to rebind the polymer a second after photolysis. Therefore a mechanism in which completion of monomer rebinding is followed by religation of polymer through exchange between the two phases is enticing. However, the results of the present study are inconsistent with the exchange mechanism. How can the polymer CO recombination have a lifetime of the order of seconds when all of the CO

952 should have recombined to solution phase molecules in milliseconds? One explanation is that CO diffuses very slowly through a hemoglobin molecule in the polymer phase. It is well known that an examination of the crystal structure of

Religation Mechanism of Sickle Hemoglobin Polymers

hemoglobin does not reveal an obvious path for CO to get to the heme (Brooks et al., 1988; Carver et al., 1990; Gibson et al., 1992). It has been proposed that CO diffusion in hemoglobin is facilitated by internal motions of the protein (Brooks et al., 1988).

Figure 5. Micrographs of sickle cell hemoglobin gels viewed through crossed polarizers. Micrographs of gels formed by slow heating are shown on the left and the gels from the same preparation that were rapidly temperature jumped are shown to their right. (a) Preparation 1, rapidly temperature jumped, total heme concentration 15.2 mM, total CO saturation 43%, concentration of polymer hemes 2.8 mM, the image size is 3.5 mm × 2.5 mm. (b) Preparation 1, slowly heated, total heme concentration 15.2 mM, total CO saturation 43%, concentration of polymer hemes 2.8 mM, the image size is 3.5 mm × 2.5 mm. (c) Preparation 2, rapidly temperature jumped, total heme concentration 14.9 mM, total CO saturation 35%, concentration of polymer hemes 4.7 mM, the image size is 1.4 mm × 0.98 mm. (d) Preparation 2, slowly heated, total heme concentration 14.9 mM, total CO saturation 35%, concentration of polymer hemes 4.7 mM, the image size is 1.4 mm × 0.98 mm. (e) Preparation 3 (preparation 2 samples were melted at 0°C over several days and reformed), rapidly temperature jumped, total heme concentration 14.9 mM, total CO saturation 35%, concentration of polymer hemes 4.7 mM, the image size is 2.7 mm × 2.0 mm. (f) Preparation 3, slowly heated, total heme concentration 14.9 mM, total CO saturation 35%, concentration of polymer hemes 4.7 mM, the image size is 2.7 mm × 2.0 mm. The samples were 30 microns thick. A Leica DMXRP microscope was used with white light produced by a tungsten lamp.

Religation Mechanism of Sickle Hemoglobin Polymers

Table 1. Exponential lifetimes for CO rebinding to sickle hemoglobin polymers Preparation 1 2 3

Lifetime (s)–small domain sample

Lifetime (s)–large domain sample

7.3 5.4 4.7

3.6 5.3 5.7

Thus in very highly viscous media, such as in a crystal or a glass, CO diffusion is greatly inhibited. A similar situation may arise in HbS polymers. The hemoglobin molecules in the polymer may have constrained internal motion like that of hemoglobin molecules in a crystal. When a CO molecule is photolyzed it would take longer for it to escape from its parent molecule. When it does escape it is more likely to become trapped in a neighboring molecule than to escape into solution. Limited diffusion within the polymer predicts that both the CO on and off rates would be slower. Thus the finding that the equilibrium binding of CO to HbS polymer is 1/3 that of T-state hemoglobin (Sunshine et al., 1982) is not inconsistent with the finding that the CO on rate to HbS polymer is 1000 times slower than to T-state hemoglobin (Shapiro et al., 1995). Diffusion limited rebinding for HbS polymers would not depend on the bulk concentration of polymer hemes in the gel. One might expect the rebinding to be exponential but to depend on the concentration of hemes within the polymer (0.69 g/ml) or on the concentration of hemes in a domain, but not on the concentration of polymers in the sample as a whole. We have not observed any obvious dependence of CO polymer rebinding kinetics on the concentration of polymer. It is possible that the rebinding of CO involves the CO molecule encountering diffusional barriers so that a stretched exponential would be a more appropriate function to describe the observed religation kinetics. However, the use of this function is not warranted due to our signal to noise and the number of available parameters in a stretched exponential function. Fitting the kinetics to exponential functions is thus a reasonable way to empirically characterize the data. Although the proposal that religation of the polymer occurs through direct rebinding to the polymer is currently the best description of our data, it is not the only explanation available. Another explanation that remains consistent with our data is that polymer religation is occurring through exchange between relatively ligated monomer phase hemoglobin molecules and relatively unligated polymer phase hemoglobin molecules and that this exchange can occur anywhere on the polymer. That exchange can occur within the polymer is inconsistent with observations that polymer growth and melting occur at the polymer ends (Briehl, 1995). Growth or melting can be viewed as resulting from a shift in the dynamic

953 equilibrium between the concentration of monomer and polymer phase molecules. Thus, that growth and melting occur only at the ends implies that exchange occurs only at the ends. However, as far as we know, there is no direct evidence that shows that exchange between phases exclusively involves the polymer ends. Differential interference contrast (DIC) microscopy has been successful in showing that growth and melting occurs only at the polymer ends (Briehl, 1995), but the resolution of this technique cannot absolutely rule out some exchange occurring within the polymer as well. If exchange did involve HbS polymer molecules that were not at the polymer ends, then one might expect to see polymer breakage during melting. Although one could argue that exchange between polymer and monomer phase molecules occurs more slowly within the polymer than at the ends, and that DIC microscopy does not have the resolution to distinguish between a single polymer and a bundle of fibers, it seems very unlikely that exchange between phases can occur except at the polymer ends. Another possibility is that some other factor that we have not accounted for serves to slow down exchange between phases in relatively small domains thereby compensating for the fewer number of ends. For example, perhaps the concentration of monomers at the ends is different for small versus large domains, but this is unlikely. Although surprising, the mechanism in which polymer religation occurs through direct rebinding of CO to the hemoglobin molecules throughout the polymer remains the simplest mechanism that is consistent with all available data. It is important to emphasize that this result is not inconsistent with recent work on polymer melting that predicts faster melting in small domains (Briehl, 1995). In our experiments the solubility returns to its equilibrium value following photolysis through CO rebinding to monomer phase before any significant change is made in the total amount of polymer. Since rebinding to the monomer phase is complete within ten milliseconds after photolysis, we do not observe any significant transient polymerization or depolymerization. Moreover, in experiments using DIC microscopy to study polymer melting, whether CO binds to HbS molecules before or after they come off the polymer is not discernible. The work using DIC microscopy has focused on polymer melting and growth whereas this study has focused on the ligation state of the polymers. Polymer rebinding kinetics is related to melting kinetics, which in turn is related to the potential persistence of the polymer during circulation. Polymers that do not melt completely after reoxygenation at the lungs before entering the microcirculation provide seeds for heterogeneous nucleation with no delay times so that the kinetics of their growth would be governed by the rate of deoxygenation during circulation. The speed of melting will depend, in part, on the ligation of the polymer and hence the kinetics of oxygen

954

Figure 6. Time resolved linear dichroism apparatus. An actinic laser beam intersects the lamp probe beam at an arbitrary angle. The probe beam passes though a collimating lens (CL) and the probe polarizer (LP1) oriented along u = 45°2b before traversing the sample (S). A second probe polarizer (LP2) oriented along − 45°, analyzes the beam before detection. A laser polarizer (LP3) insures pure vertical polarization of the actinic beam. Vertical is defined as normal to the plane formed by the probe and actinic beams.

rebinding. Melting can occur upon reoxygenation by rapid rebinding to the molecules in the monomer phase, which disturbs the dynamic equilibrium between the polymer and monomer phases. Our results indicate that direct rebinding to the polymer phase plays a lesser role in melting than one would imagine if the CO-HbS polymer association rate were not found to be so slow.

Materials and Methods Hemoglobin S was obtained from excess blood originally donated by patients homozygous in HbS, following federal regulations and guidelines outlined by the National Institutes of Health. The hemolysate was prepared as described previously (Geraci et al., 1969). Cells were washed in 0.95% (w/v) NaCl and lysed by incubation in distilled water. Samples were then pelleted in liquid nitrogen for storage. HbS was separated from minor components (HbF and HbA2 ) by ionexchange chromatography using a DE-52 matrix (Whatman) developed with 0.05 M Tris-HCl (pH 8.3). After dialysis against 0.1 M sodium phosphate buffer (pH 7.3) the sample was concentrated using an Amicon stirred cell and centricon concentrators. All gas equilibrations were subsequently carried out with the samples on ice. The sample was equilibrated with one atmosphere argon after which a portion was ligated by exposure to CO. Approximately 0.05 M sodium dithionite was mixed into the sample. The CO bound portion was mixed with the deoxy portion prior to loading the 30 mm path-length sample cells (Hellma 121.000 QS) under anaerobic conditions. Two cells were loaded from the same preparation to ensure that each sample undergoing a different temperature jump would have the same concentration and CO saturation. The amount of polymer in the sample was calculated as described previously (Shapiro et al., 1995). One of the samples was immediately immersed into a water bath at 30° C and then brought to room temperature (about 23°C). The other was taken slowly to room temperature over a period of 48 hours using a temperature controlled bath. Static and time-resolved linear dichroism measurements were performed as described previously (Che

Religation Mechanism of Sickle Hemoglobin Polymers

et al., 1994; Shapiro et al., 1995). A schematic of the apparatus is given in Figure 6. A xenon flashlamp produced probe pulses at 0.25 to 2 Hz, which are incident on the sample with a diameter of about 3 mm. Actinic pulses of 15 mJ per pulse were produced by a Quanta Ray DCR-11 Nd:YAG laser, frequency doubled to 532 nm. The excitation pulses made an angle of about 30° with respect to the probe beam. The diameter of the laser beam was about 1 cm. A clean up Glan-Taylor polarizer was used to ensure the polarization purity (vertical) of the actinic beam. Two polarizers, oriented along directions 245 degrees with respect to the vertically polarized actinic beam, are placed before the sample and one after the sample, respectively. To conduct a linear dichroism measurement, the sample is probed at a given time following photolysis for two separate rotations by a small angle, b, of the initial polarizer. For a static measurement the sample is not photolyzed. The LD signal is calculated as the detected intensity when the initial polarizer is rotated +b (I+ ) minus the detected intensity when the polarizer is rotated by −b (I− ), divided by the sum of these intensities. As shown previously (Che et al., 1994) this gives LD signal0

I+ − I− (b + LD/2)2 − (b − LD/2)2 LD = 1 , I+ + I− (b + LD/2)2 + (b − LD/2)2 b (1)

where we have assumed that LD is much smaller than b, which is a reasonable assumption for our measurements. A value of 0.033 radians was chosen for b in this work so that a signal to noise advantage of about 38 was obtained with respect to standard LD measurements probing vertical and horizontal direction separately. The probe beam was focused through a 250 mm slit into a Jarrel Ash spectrograph (150 grooves per mm, 800 nm blaze) and detected with an EG&G OMA II detector. A Stanford Instruments DG535 delay/pulse generator was used to control the timing of the detector gate (700 ns) and the firing of the flashlamp with respect to the laser. Approximately 150 measurements were taken and averaged for each transient linear dichroism spectrum. The average of equilibrium spectra taken before and after measurements of transient spectra was subtracted from each transient spectrum to yield TRLD difference spectra. The data were smoothed using a 15 point Savitzky-Golay algorithm (0.6 nm/point). The data were baseline offset at 579 nm. Data were analyzed using singular value decomposition (SVD; Golub & Reinsch, 1970; Henry & Hofrichter, 1992). SVD rewrites the data matrix of signals at each wavelength and time as the product of three matrices A = USV T.

(2)

U is an m × n matrix containing the optical density for n orthonormal basis spectra at m wavelengths. V T denotes the transpose of V, an n × n matrix giving the amplitude of each basis spectrum at n time delays. S is an n × n matrix containing the singular values of A, a determinant of the contribution of each basis spectrum to the measured spectrum at a given time. In practice, only the largest singular values and time amplitudes are retained. The smaller values and amplitudes are discarded as noise. Thus SVD provides a concise, noise-filtered representation of the data. This truncated representation was used to fit the TRLD data to an exponential lifetime using a (non-linear) least square global analysis fitting technique (Goldbeck & Kliger, 1993). The buildup of TRLD signal caused by photolysis of the sample before

Religation Mechanism of Sickle Hemoglobin Polymers

it was able to return to equilibrium was taken into account in the exponential fitting.

Acknowledgements We thank Dr James W. Lewis for helpful discussion and technical assistance. We also thank Drs Rhoda Elison Hirsch, Robert M. Bookchin, Frank A. Ferrone, and James Hofrichter for helpful discussions. We are grateful to Jonathan Krupp for help with the microscopy. D.B.S. thanks the NIH for support through NRSA no. F32HL08969. This work was also supported by NIH grants no. HL31579 (N.M.), GM35158 (D.S.K), and the NIH Comprehensive Sickle Cell Center grant no. HL38632 (S.K.B).

References Allen, D. W. & Wyman, J. (1954). Equilibre de l’he´moglobine de dre´panoctose avec l’oxyge`ne. Rev. Hematol. 9, 155–157. Altman, P. A. & Ditmer, D. S. (1971). Respiration and Circulation, pp. 417–422, 498, Federation of American Society of Experimental Biologists, Bethesda, MD. Bertini, I., Gray, H., Lippard, S. J. & Valentine, J. S. (1994) Biorganic Chemistry, pp. 186–187, University Science Books, Mill Valley, CA. Briehl, R. W. (1995). Nucleation, fiber growth and melting, and domain formation and structure in sickle cell hemoglobin gels. J. Mol. Biol. 245, 710–723. Brooks III, C. L., Karplus, M. & Pettitt, M. B. (1988). In Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics (Advances in Chemical Physics volume LXXI, Prigogne, I. & Rice, S. A., eds), pp. 111–116, Wiley, New York. Carver, T. E., Rohlfs, R. J., Olson, J. S., Gibson, Q. H., Blackmore, R. S., Springer, B. A. & Sligar, S. G. (1990). Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques. J. Biol. Chem. 265, 20007–20020. Che, D., Shapiro, D. B., Esquerra, R. M. & Kliger, D. S. (1994). Ultrasensitive time-resolved linear dichroism spectral measurements using near-crossed linear polarizers. Chem. Phys. Letters, 224, 145–154. Deyoung, A. & Noble, R. W. (1981). Oxygen binding to sickle cell hemoglobin. Methods Enzymol. 76, 792–805. Dou, Q. & Ferrone, F.A. (1993). Simulated formation of polymer domains in sickle hemoglobin. Biophys. J. 65, 2068–2077. Eaton, W. A. & Hochstrasser, R. M. (1967). Electronic spectrum of single crystals of ferricytochrome c. J. Chem. Phys. 46, 2533–2539. Eaton, W. A. & Hochstrasser, R. M. (1968). Single crystal spectra of ferrimyoglobin complexes in polarized light. J. Chem. Phys. 49, 985–995 Eaton, W. A. & Hofrichter, J. (1990). Sickle cell hemoglobin polymerization. Advan. Protein Chem. 40, 63–279. Ferrone, F. A., Hofrichter, J. & Eaton, W. A. (1985a). Kinetics of sickle hemoglobin polymerization. I. Studies using temperature jump and laser photolysis techniques. J. Mol. Biol. 183, 591–610. Ferrone, F. A., Hofrichter, J. & Eaton, W. A. (1985b). Kinetics of sickle hemoglobin polymerization. II. A

955 double nucleation mechanism. J. Mol. Biol. 183, 611–631. Geraci, G., Parkhurst, L. J. & Gibson, Q. H. (1969). Preparation and properties of a- and b-chains from human hemoglobin. J. Biol. Chem. 17, 4664–4667. Gibson, Q. H., Regan, R., Elber, R., Olson, J. S. & Carver, T. E. (1992). Distal pocket residues affect picosecond ligand recombination in myoglobin. An experimental and molecular dynamics study of position 29 mutants. J. Biol. Chem. 267, 20022–20034. Goldbeck, R. A. & Kliger, D. S. (1993). Nanosecond time-resolved absorption and polarization dichroism spectroscopies. Methods Enzymol. 226, 147–177. Golub, G. H. & Reinsch, C. (1970). Singular value decomposition and least squares solutions. Numer. Math. 14, 403–420. Gross, C. T., Salamon, H., Hunt, A. J., Macey, R. I., Orme, F. & Quintanilha, A. T. (1991). Hemoglobin polymerization in sickle cells studied by circular polarized light scattering. Biochim. Biophys. Acta, 1079, 152–160. Harrington, J. P., Elbaum, D., Bookchin, R. M., Wittenberg, J. B. & Nagel, R. L. (1977). Ligand kinetics of hemoglobin S containing erythrocytes. Proc. Natl Acad. Sci. USA, 74, 203–206. Henry, E. R. & Hofrichter, J. (1992). Singular value decomposition: Applications to experimental data. Methods Enzymol. 210, 129–192. Hofrichter, J. (1979). Ligand binding and the gelation of sickle cell hemoglobin. J. Mol. Biol. 128, 335–369. Hofrichter, J., Hendricker, D. G. & Eaton, W. A. (1973). Structure of the hemoglobin S fibers: Optical determination of the molecular orientation in sickled red blood cells. Proc. Natl Acad. Sci. USA, 70, 3604–3608. Hofrichter, J., Ross, P. D. & Eaton, W. A. (1974). Kinetics and mechanism of deoxyhemoglobin S gelation. A new approach to understanding sickle cell disease. Proc. Natl Acad. Sci. USA, 71, 4864–4868. Ingram, V. M. (1956). A specific chemical difference between the globins of normal human and sickle cell anemia haemoglobin. Nature, 178, 792–794. Messer, M. J., Hahn, J. A. & Bradley, T. B. (1976). The kinetics of sickling and unsickling of red cells under physiological conditions: rheologic and ultrastructural correlations. In Proceedings of the Symposium on Molecular and Cellular Aspects of Sickle Cell Disease (Hercules, J. I., Cottam, G. L., Waterman, M. R. & Schechter, A. N., eds), pp. 225–234, DHEW Publ. No. (NIH) 76–1007, Bethesda, Maryland. Mozarelli, A., Hofrichter, J. & Eaton, W. A. (1987). Delay time of hemoglobin S gelation prevents most cells from sickling in vivo. Science, 237, 500–506. Pauling, L., Itano, H. A., Singer, S. J. & Wells, I. C. (1949). Sickle cell anemia, a molecular disease. Science, 111, 543–548. Pennelly, R. R. & Noble, R. W. (1978). Functional identity of hemoglobin S and A in the absence of polymerization. In Biochemical and Chemical Aspects of Hemoglobin Abnormalities (Caughey, W. S., ed.), pp. 401–411, Academic Press, New York. Rucknagel, D. L. (1975). In Sickle Cell Anemia and Other Hemoglobinopathies (Levere, R. D., ed.), p. 1, Academic Press, New York. Shapiro, D. B., Paquette, S. J., Esquerra, R. M., Che, D., Goldbeck, R. A., Hirsch, R. E., Mohandas, N. & Kliger, D. S. (1994). Nanosecond absorption study of kinetics associated with carbon monoxide rebinding to hemoglobin S and hemoglobin C following laser

956

Religation Mechanism of Sickle Hemoglobin Polymers

photolysis. Biochem. Biophys. Res. Commun. 205, 154–160. Shapiro, D. B., Esquerra, R. M., Goldbeck, R. A., Ballas, S. K., Mohandas, N. & Kliger, D. S. (1995). Carbon monoxide religation kinetics to hemoglobin S

polymers following ligand photolysis. J. Biol. Chem. 270, 26078–26085. Sunshine, H. R., Hofrichter, J., Ferrone, F. A. & Eaton, W. A. (1982). Oxygen binding by sickle cell hemoglobin polymers. J. Mol. Biol. 158, 251–273.

Edited by F. E. Cohen (Received 30 November 1995; received in revised form 4 April 1996; accepted 15 April 1996)