Beta Subunits Of Hemoglobin Research Articles

Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry.

A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis.

Transfer of Human α- to β-Hemoglobin via Its Chaperone Protein: EVIDENCE FOR A NEW STATE

The alpha-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to alpha-hemoglobin (alpha-Hb) subunits acting as a chaperone protein, preventing the formation of alpha-hemoglobin-cytotoxic precipitates. We have engineered recombinant AHSP in a pGEX vector to study the functional consequence of interaction between AHSP and alpha-Hb. By in vitro binding assays, we have isolated the complexes glutathione S-transferase-AHSP.alpha-Hb and AHSP.alpha-Hb. The latter assembles as a heterodimer based on size-exclusion chromatography. These complexes exhibited monophasic CO binding kinetics, as observed for isolated alpha- and beta-subunits of hemoglobin. However, the rate of CO (or oxygen) binding to alpha-hemoglobin bound to its chaperone is three times slower than that observed for isolated alpha-hemoglobin, demonstrating a form that is intermediate to the R- and T-hemoglobin states. The physiologically relevant replacement of the chaperone by beta-hemoglobin chains could be detected by both ligand binding kinetics and tryptophan fluorescence quenching.

Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein.

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) <or= 2.25), denoted A and B. Averaged over many similar sites, the A-center has a rhombic EPR signal with a g-tensor, g(A) = [2.248(4), 2.146(1), 1.966(1)]; the B-center exhibits a less anisotropic EPR signal, g(B) = [2.216(3), 2.118(2), 1.966(1)]. Early measurement had suggested that, in the cryoreduced HbO(2) tetramer, the two centers corresponded to the two different chains [Symons, M. C. R., and Petersen, R. L. (1978) Proc. R. Soc. London, Ser. B 201, 285-300]. However, the present EPR and ENDOR results show that the two signals instead reflect the fact that the parent oxyhemes exist in two major conformational substates and that this is true for both alphaO(2) and betaO(2) subunits: alphaO(2)(A) (minor species) and alphaO(2)(B) (major species); betaO(2)(A)(major species) and betaO(2)(B) (minor species). Similar behavior is seen for MbO(2) [Kappl, R., Höhn-Berlage, M., Hüttermann, J., Bartlett, N., and Symons, M. C. R. (1985) Biochim. Biophys. Acta 827, 327-343]. The A/B g-tensors of alphaO(2) and betaO(2) chains vary little with the environment of the chains, while the relative populations of the substates depend greatly on glycols and IHP. These results suggest a quaternary influence on the oxyheme distal pocket of alpha chains and that the glycol-induced changes in the substate populations of the R-state HbO(2) tetramer are largely associated with the alphaO(2) subunit. (1)H ENDOR spectra from the distal histidine proton hydrogen-bonded to the peroxo ligand show very different isotropic coupling for the A- and B-centers. Analysis of the spectroscopic data suggests that the A- and B-centers represent different orientations of the oxyheme O(2) ligand relative to the distal histidine. It is likely that the A and B conformational substates in the alphaO(2) and betaO(2) subunits differ not only in their tertiary structures but in their affinities for O(2).

Doping control analysis of bovine hemoglobin-based oxygen therapeutics in human plasma by LC-electrospray ionization-MS/MS.

Since January 2000, hemoglobin-based oxygen carriers, such as Hemopure, belong to the list of prohibited substances of the International Olympic Committee. Hemopure is based on bovine hemoglobin, which is intra- and intermolecularly cross-linked by glutaraldehyde units causing an average molecular weight of approximately 250,000. Bovine and human hemoglobins differ by 15% in amino acid sequence; hence, tryptic digestion of these proteins generates species-common and -unique peptides. Those specific fragments originate from the alpha- and beta-subunits of hemoglobin, such as bovine Hb peptides alpha(69-90) (2367.2 Da) or beta(40-58) (2089.9 Da). By means of LC-MS/MS, peptides of human and bovine hemoglobin can be separated and identified, enabling the determination of compounds based on Hb of bovine origin and thus the administration of oxygen carriers such as Hemopure. Blank plasma samples were spiked with Hemopure or human or bovine hemoglobin, filtered, enzymatically digested, and analyzed on an Agilent 1100 Series HPLC interfaced to an Applied Biosystems API 2000 triple quadrupole mass spectrometer. In plasma aliquots of 50 microL containing 50 microg of Hemopure (1 mg/mL), peptides of bovine hemoglobin were confirmed, and blank plasma samples as well as 68 specimens of high-performance athletes were tested with the developed procedure.

Substitution of the Heme Binding Module in Hemoglobin α- and β-Subunits: IMPLICATION FOR DIFFERENT REGULATION MECHANISMS OF THE HEME PROXIMAL STRUCTURE BETWEEN HEMOGLOBIN AND MYOGLOBIN

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.

Design, construction, crystallization, and preliminary X-ray studies of a fine-tuning mutant (F133V) of module-substituted chimera hemoglobin.

A chimera betaalpha-subunit of human hemoglobin was crystallized into a carbonmonoxy form. The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 A. The crystal belongs to the monoclinic space group P21, with cell dimensions of a = 62.9, b = 81.3, c = 55.1 A, and beta = 91.0 degrees . These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study.

Synergic effects of NO and oxygen free radicals in the injury of ischemia-reperfused myocardium--ESR studies on NO free radicals generated from ischemia-reperfused myocardium.

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to beta-subunit of hemoglobin (beta-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginine. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (< 1 mmol/L) could protect the heart form the ischemia-reperfusion injury but at higher concentration aggravate the injury. Addition of NAME to the reperfusion solution could also protect the myocardium. Addition of xanthine (X)/xanthine oxidase (XO) or Fe2+/H2O2 to the reperfusion solution increased the production of NO and oxygen free radicals and the ischemia-reperfused injury simultaneously. Addition of superoxide dismutase (SOD) and catalase decreased the production of NO and oxygen free radicals and the ischemia-reperfusion injury.

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.

“Module” substitution in hemoglobin subunits. Preparation and characterization of a “chimera beta alpha-subunit”.

In the genes of alpha- and beta-subunits of hemoglobin, Go showed that modules F1, F2 + F3, and F4 correspond to exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90). The analysis of the correlation of function with its exon pattern showed that the residues associated with the defined function are concentrated in the specific exons encoding the "module" (Eaton, W.A. (1980) Nature 284, 183). To investigate the functional and structural significance of the "modular structure," we engineered a "chimera" subunit, in which module F4 of the beta-subunit was replaced by that of the alpha-subunit by use of mutagenesis. The NMR and resonance Raman spectra of the isolated "chimera beta alpha-subunit" have revealed that it has a beta-subunit-like heme environmental structure. However, the gel chromatography and NMR spectra of mixtures of the chimera and native subunits clearly showed that the chimera beta alpha-subunit binds specifically to the beta-subunit to form a heterotetramer, not to the alpha-subunit. These results led us to conclude that the predominant role of the module F4 is the subunit association and suggest that the modules are structural and functional units that have advantages in producing stable functional proteins.

Determination of iron-carbon monoxide geometry in the subunits of carbonmonoxy hemoglobin M Boston using femtosecond infrared spectroscopy

We have undertaken ultrafast infrared (IR) spectroscopic studies in order to elucidate the geometry of bound CO in the alpha and beta subunits of hemoglobin (Hb) M Boston 13CO. Hb M Boston is a mutant human Hb in which the distal histidine in the alpha subunits is replaced by a tyrosine. The IR absorptions of bound 13CO fall at 1925 cm-1 for the alpha subunits and 1907 cm-1 for the beta subunits. Despite a difference of nearly 20 cm-1 in these peaks, the measured anisotropies of the bound 13CO depletions following 30% photolysis are nearly identical, with values of -0.142 +/- 0.002 obtained for the alpha subunits and -0.140 +/- 0.003 obtained for the beta subunits. These translate to values of 20 degrees +/- 1 degree and 21 degrees +/- 1 degree for the values of the average angles between the CO bond and the normal to the heme planes in the alpha and beta subunits, respectively. Our present results and the work of previous investigators [Nagai, M., Yoneyama, Y., & Kitagawa, T. (1991) Biochemistry 30, 6495-6503] suggest that a change in the polar interactions of the bound CO with the heme pocket environment upon substitution of tyrosine for the distal histidine and a less bent structure for the Fe-C-O unit in the alpha subunits are responsible for the difference in the bound CO absorption frequencies in the alpha and beta subunits. A spectrum of the depletion of the bound 13CO peaks following photolysis indicates that both subunits photodissociate CO with the same quantum yield and neither subunit exhibits significant recombination within 1 ns.

Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin beta subunit tetramers.

Isolated beta chains from human adult hemoglobin at millimolar concentration are mainly associated to form beta 4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr approximately 66,000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated beta(CO) chains in solution and in beta(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated beta chains. Comparison of the assigned resonances of conserved amino acids in alpha chains, beta chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side.

Salt, phosphate and the Bohr effect at the hemoglobin beta chain C terminus studied by hydrogen exchange

Hydrogen exchange experiments using functional labeling and fragment separation methods were performed to study interactions at the C terminus of the hemoglobin beta subunit that contribute to the phosphate effect and the Bohr effect. The results show that the H-exchange behavior of several peptide NH at the beta chain C terminus is determined by a transient, concerted unfolding reaction involving five or more residues, from the C-terminal His146β through at least Ala142β, and that H-exchange rate can be used to measure the stabilization free energy of interactions, both individually and collectively, at this locus. In deoxy hemoglobin at pH 7.4 and 0 °C, the removal of 2,3-diphosphoglycerate (DPG) or pyrophosphate (loss of a salt link to His143β) speeds the exchange of the beta chain C-terminal peptide NH protons by 2.5-fold (at high salt), indicating a destabilization of the C-terminal segment by 0.5 kcal of free energy. Loss of the His146 β 1 to Asp94 β 1 salt link speeds all these protons by 6.3-fold, indicating a bond stabilization free energy of 1.0 kcal. When both these salt links are removed together, the effect is found to be strictly additive; all the protons exchange faster by 16-fold indicating a loss of 1.5 kcal in stabilization free energy. Added salt is slightly destabilizing when DPG is present but provides some increased stability, in the 0.2 kcal range, when DPG is absent. The total allosteric stabilization energy at each beta chain C terminus in deoxy hemoglobin under these conditions is measured to be 3.8 kcal (pH 7.4, 0 °C, with DPG). In oxy hemoglobin at pH 7.4 and 0 °C, stability at the beta chain C terminus is essentially independent of salt concentration, and the NES modification, which in deoxy hemoglobin blocks the His146β to Asp94β salt link, has no destabilizing effect, either at high or low salt. These results appear to show that the His146β salt link, which participates importantly in the alkaline Bohr effect, does not reform to Asp94β or to any other salt link acceptor in a stable way in oxy hemoglobin at low or high salt conditions.

The Relationship Between in vivo Generated Hemoglobin Skeletal Protein Complex and Increased Red Cell Membrane Rigidity

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta-subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.

High pressure NMR studies of hemoproteins. The effect of pressure on the tertiary and quaternary structures of human adult ferrous hemoglobin.

The effect of pressure on the tertiary and quaternary structures of human oxy, carbonmonoxy, and deoxyhemoglobin was examined by high pressure NMR spectroscopy at 300 MHz. The increased pressure displaced the ring current-shifted gamma 1-methyl resonance of beta E11 valine for oxy- and carbonmonoxyhemoglobin to the upfield side, whereas that of the alpha subunit was insensitive to pressure. Such a preferential pressure-induced upfield shift for the beta E11 valine gamma 1-methyl signal was also encountered for the isolated carbonmonoxy beta chain. For deoxyhemoglobin, hyperfine shifted resonances of the heme peripheral proton groups and the proximal histidyl NH proton for the beta subunit were pressure-dependent, in contrast to the pressure-insensitive responses for these resonances of the alpha subunit. These results indicate the structural nonequivalence of the pressure-induced structural changes in the alpha and beta subunits of hemoglobin. The exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds which have been used as the oxy and deoxy quaternary structural probes were not changed upon pressurization. From all of above results, it was concluded that pressure induces the tertiary structural change preferentially at the beta heme pocket of the ferrous hemoglobin derivatives with the quaternary structure retained.

Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin.

A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.

Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits.

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts.

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.

Conformational relevance of the beta6Glu replaced by Val mutation in the beta subunits and in the beta(1-55) and beta(1-30) peptides of hemoglobin S.

The beta subunits of hemoglobin S showed a higher ellipticity than the beta subunits of the hemoglobin A, both in the Soret and near-ultraviolet regions. The apoderivatives of the beta subunits of hemoglobin S showed a lower helical content and a larger amount of beta conformation than the apoderivatives of the beta subunits of hemoglobin A. The beta(1-55) peptides and beta(1-30) peptides from the beta subunits of hemoglobin A and S have been separated and analyzed. The betaS(1-55) peptide showed a higher content of beta conformation and lower amount of alpha helix when compared to the betaA(1-55) peptide. This difference was present also in different concentrations of methanol. The apoderivative from the beta subunits of hemoglobin S and the betaS(1-55) peptide aggregated with increasing ionic strength. The CD measurement showed that their secondary structure did not change upon a 10-fold dilution of the sample. Very little secondary structure was present in the betaS(1-30) peptide, and the CD spectrum was very similar to that of the betaA(1-30) peptide. No significant difference in aggregation was found between the betaS(1-30) and betaA(1-30) peptides.