Human Deoxyhemoglobin Research Articles

High magnetic field effects on human deoxygenated hemoglobin light absorption

The effect of high static magnetic field on human deoxyhemoglobin spectrophotometric characteristics is studied. Five samples of partially deoxygenated hemoglobin in aqueous solution were tested. Magnetic field was applied in steps of 2 T (tesla), from 0 to 18 T. Spectral analysis showed a higher light absorption at wavelengths of 546 nm and at 581 nm. The applied magnetic field did not alter the wavelengths at which the absorption peaks occurred. On the other hand, the light absorption was found to increase with the applied magnetic field strength up to 14 T.

PDBtool: An interactive browser and geometry checker for protein structures

PDBtool version 1.0 is an interactive UNIX and X/Motif-based computer program for investigating the three-dimensional (3D) structure of proteins using data represented by the Protein Data Bank (PDB) format. Major components of PDBtool include a three-dimensional structure viewer, text browsers from which to investigate components of the structure under study and a comprehensive set of geometry-checking tools. The object-oriented software design provides a high level of interaction between display windows in an effort to provide a more intuitive level of interaction than is possible with existing tools. Examples of the use of PDBtool are provided to illustrate these capabilities. These include: analyzing the distribution of cysteines in a member of the family of short-chain neurotoxins; for the same protein, analyzing outliers in a Ramachandran plot and their associated isotropic temperature factors; and investigating the distribution of heme groups in human deoxyhemoglobin. A detailed description of PDBtool, including a tutorial, is available on the World Wide Web at the URL http:// www.sdsc.edu/CCMS/Packages/PDBtool.html.

A Doubly Cross-linked Human Hemoglobin: EFFECTS OF CROSS-LINKS BETWEEN DIFFERENT SUBUNITS

Human deoxyhemoglobin cross-linked with trimesyl tris(3,5-dibromosalicylate) produces the previously reported cross-linked hemoglobin in which the epsilon amino groups of the two beta chain 82 lysyl residues are joined by a trimesyl bridge. Further specific modification of this protein directed to the alpha subunits with bis(3,5-dibromosalicyl)fumarate gives a doubly cross-linked material in which the epsilon-amino groups of the two alpha chain 99 lysyl residues are now joined by a fumaryl bridge. The singly cross-linked beta chain species binds oxygen cooperatively with a high oxygen affinity (P50 = 4.8 torr at pH 7.4). The addition of the second cross-linking reduces the oxygen affinity to 15.9 torr, which compares with 13.0 torr for the singly cross-linked alpha chain species. The doubly cross-linked hemoglobin retains significant cooperativity with a Hill coefficient of 2.3 compared with 3.0 for unmodified hemoglobin. Because some of the groups responsible for the Bohr effect are acylated, this doubly cross-linked hemoglobin exhibits 25% of the normal Bohr effect and less than 20% of the normal chloride effect. The use of two distinct cross-links within the same tetramer provides a material for physical and structural analysis as well as for further modifications for specific applications. The results indicate that the cross-link introducing the lowest oxygen affinity in the two singly cross-linked species appears to control the overall affinity in this doubly cross-linked species.

Electrostatics of hemoglobins from measurements of the electric dichroism and computer simulations

Hemoglobins from normal human cells, from sickle cells, and from horse were investigated by electrooptical methods in their oxy and deoxy forms. The reduced linear dichroism measured as a function of the electric field strength demonstrates the existence of permanent dipole moments in the range of 250-400 Debye units. The reduced limiting dichroism is relatively small (< or = 0.1); it is negative for hemoglobin from sickle cells and positive for the hemoglobins from normal human cells and from horse. The dichroism decay time constants are in the range from about 55 to 90 ns. Calculations of the electrooptical data from available crystal structures are given according to models of various complexity, including Monte Carlo simulations of proton fluctuations with energies evaluated by a finite difference Poisson-Boltzmann procedure. The experimental dipole moments are shown to be consistent with the results of the calculations. In the case of human deoxyhemoglobin, the root mean square dipole is higher than the mean dipole by a factor of about 4.5, indicating a particularly large relative contribution due to proton fluctuations. The ratio of the root mean square dipole to the mean dipole is much smaller (approximately 1.1 to approximately 1.5) for the other hemoglobin molecules. The calculations demonstrate that the dichroism decay time constants are not simply determined by the size/shape of the proteins, but are strongly influenced by the orientation of the dipole vector with respect to the axis of maximal absorbance. The comparison of experimental and calculated electrooptical data provides a useful test for the accuracy of electrostatic calculations and/or for the equivalence of structures in crystals and in solutions.

Max Perutz's achievements: how did he do it?

Protein ScienceVolume 3, Issue 10 p. 1625-1628 Invited TributeFree Access Max Perutz's achievements: How did he do it? David Eisenberg, Corresponding Author David Eisenberg [email protected] UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024–1570UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024–1570Search for more papers by this author David Eisenberg, Corresponding Author David Eisenberg [email protected] UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024–1570UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, and Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024–1570Search for more papers by this author First published: October 1994 https://doi.org/10.1002/pro.5560031001Citations: 14AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References Bragg WL, Perutz MF. 1954. The structure of haemoglobin: Fourier projections on the 010 plane. Proc R Soc Lond Ser A 225: 315– 329. Fermi G, Perutz MF, Shaanan B, Fourme R. 1984. The crystal structure of human deoxyhaemoglobin at 1.74 Å resolution. J Mol Biol 175: 159– 174. Haurowitz F. 1938. Das Gleichgewicht zwischen Hämoglobin und Sauerstoff. Hoppe-Seyler 254: 266– 274. Judson HF. 1979. The eighth day of creation: Makers of the revolution in biology. New York: Simon & Schuster. Kendrew JC, Dickerson RE, Strandberg BE, Hart RG, Davies DR, Phillips DC, Shore VC. 1960. Structure of myoglobin: A three-dimensional Fourier synthesis at 2 Å resolution. Nature 185: 422– 427. Perutz MF. 1970. Stereochemistry of cooperative effects in hemoglobin. Nature 373: 726– 739. Perutz MF. 1980. Origins of molecular biology. New Scientist 85 (1192): 326– 329. Perutz M. 1984. Review of Rutherford by David Wilson. London Review of Books 6 (7): 7– 8. [Also reprinted in Is Science Necessary?] Perutz M. 1985a. Early days of protein crystallography. Methods Enzymol 114: 3– 19. Perutz M. 1985b. That was the war: Enemy alien. The New Yorker Aug. 12 1985. pp 35– 54. [Also reprinted in Is Science Necessary?] Perutz M. 1989. Is science necessary? New York: E.P. Dutton. Perutz M, Johnson T, Suzuki M, Finch JT. 1994. Glutamine repeats as polar zippers: Their possible role in inherited neurodegenerative disease. Proc Natl Acad Sci USA 91: 5355– 5358. Perutz MF, Muirhead H, Cox JM, Goaman LC. 1968. Three-dimensional fourier synthesis of horse oxyhemoglobin at 2.8 Å resolution: The atomic model. Nature 219: 131– 139. Perutz MF, Rossmann MG, Cullis AF, Muirhead H, Will G, North ACT. 1960. Structure of haemoglobin: A three-dimensional Fourier synthesis at 5.5 Å resolution, obtained by X-ray analysis. Nature 185: 416– 422. Rossmann MG. 1960. The accurate determination of the position and shape of heavy-atom replacement groups in proteins. Acta Crystallogr 13: 221– 226. Citing Literature Volume3, Issue10October 1994Pages 1625-1628 ReferencesRelatedInformation

Consequences of chemical modifications on the free radical reactions of human hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA 0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA 0. As previously reported, exposure of oxyhemoglobin to H 2O 2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 μ m HbA (18.0 ± 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.

Allosteric properties of haemoglobin β41 (C7) Pheå Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli

In human deoxy haemoglobin, the α42(C7)Tyr-residue is hydrogen-bonded to β99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in β-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar K R and K T values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers.

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)

Site-directed mutagenesis in haemoglobin: Functional role of tyrosine-42(C7)α at the α1-β2 interface

To clarify the functional role of Tyr-42(C7)α, which forms a hydrogen bond with Asp-99(G1)β at the α1-β2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42α was replaced by Phe (Hb Phe-42α) or His (Hb His-42α), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99β mutants, Hb Phe-42α showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42α, the deoxy T quaternary structure is highly destabilized and the strain imposed on the FeN ε (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42α, Hb His-42α showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42α and Asp-99β. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42α and Asp-99β plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.

Free energy of sickling: A simulation analysis.

Molecular dynamics simulations were performed to calculate the difference between the dimerization free energies of normal human deoxyhemoglobin (HbA) and the mutant sickle-cell deoxyhemoglobin HbS (Glu-beta 6----Val) for one of the lateral contacts in the HbS x-ray structure. The simulations yield a value of--15 kcal/mol. Although there is no quantitative experimental value for comparison, this is in qualitative agreement with the experimental result that HbS self-assembles into multistranded fibers that are responsible for erythrocyte sickling, while HbA does not. The free-energy difference was decomposed into enthalpic and entropic terms, both of which are significant, and the contributions of individual protein residues and of the solvent were examined. Electrostatic effects play the dominant role in favoring dimerization of HbS compared with HbA; van der Waals interactions make a negligible contribution to the difference. Both differential solvation and protein-protein interactions are important. Interactions within the donor tetramer (i.e., that containing the Glu-beta 6 mutation site), as well as those with the acceptor tetramer, contribute to the preferential free energy of dimerization of HbS.

New effectors of human hemoglobin: structure and function

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and morphological stasis during molecular evolution

The evolutionary distance between two sets of proteins was estimated using the techniques of Miyata and Yasunaga (1980) and Kimura (1980). Human beta 2-microglobulin was compared with the homologous murine molecule, while human and equine alpha-globin were similarly treated. It was found that a large amount of molecular evolution has occurred in beta 2-microglobulin since its divergence from the common ancestor of mice and humans. Kimura's estimate of evolutionary distance, K, is 0.353, while those of Miyata and Yasunaga are KS = 0.708 and KA = 0.171. The respective values for human and equine alpha-globin are 0.152, 0.293, and 0.084. In spite of this molecular evolution, it is shown that murine beta 2-microglobulin can effect the expression of HLA class I antigens on the surface of human-mouse hybrid cells and that the tertiary structures of human and equine deoxyhemoglobin are nearly identical. These observations are discussed in the light of Kimura's theory of neutral allelic drift.

Structure and Function of Human Hemoglobin Covalently Labeled with Periodate-oxidized Adenosine Triphosphate

Periodate-oxidized adenosine triphosphate (o-ATP), a ribose ring-opened dialdehyde derivative of ATP, reacts specifically with human deoxyhemoglobin to give a single major covalently modified product after reduction with sodium borohydride. This product, designated di-ATP Hb, was isolated using ion-exchange chromatography and shown to have incorporated two molecules of o-ATP/tetramer. Peptide mapping and x-ray crystallography at 2.8-A resolution indicate that a covalent adduct is formed between the ligand and residues Lys-82 EF6 of each beta chain in the organic phosphate-binding site of the molecule. di-ATP Hb exhibits a significantly decreased oxygen affinity (P50 = 20.8 mm Hg versus 5.8 mm Hg control; 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol, pH 7.4, 0.1 M C, 20 degrees C). The subunit cooper-activity of di-ATP Hb is also reduced (nmax = 1.9 versus 2.7 control).

Consequences of magnetization transfer on the determination of solution structures of proteins

The complete two-dimensional nuclear Overhauser enhancement (NOESY) spectra of several proteins were theoretically simulated to analyze the effect that spin diffusion has on the cross-peak intensity-interproton distance relationship as a function of rotational time τ and mixing time t m. The NOESY cross-peak intensities were calculated by solving the generalized Bloch equations from the crystal coordinates of basic pancreatic trypsin inhibitor, cytochrome b562, domain 1 of phage lysozyme, and human deoxy hemoglobin (MW 6500, 11,459, 18,625, and 64,450, respectively). It was found that correlations between cross-peak intensity and internuclear distance, useful for obtaining solution structure of proteins from nuclear magnetic resonance data, can be defined for cross peaks observed at t m = 100 ms, for rotational correlation times τ < 2.3 ns (corresponding to MW < 11,459 at 68°C), and at t m = 50 ms, for τ < 5.9 ns (corresponding to MW < 30,000 at 68°C). Above these limits, the correlations start to break down, and two protons at any given distance may give rise to NOE cross peaks varying greatly in intensity, depending on the local structure of the protein.

Radiation-induced changes of structural and functional properties of human hemoglobin. I. Spectral characterization of irradiated deoxyhemoglobin.

Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.

Nitrogen binding to deoxyhemoglobin at high pressures and its relation to changes in hemoglobin-oxygen affinity.

The stoichiometric and thermodynamic properties of nitrogen (N2) binding to human deoxyhemoglobin (Hb) at N2 saturation pressures up to 400 atm were derived from measured N2 solubilities in protein-free buffers (pH 7.1) and in corresponding buffer + Hb (6.5% w/w) solutions at 20.0, 25.0, and 37.0 degrees C. At each temperature, approximately 3 N2 molecules bind per Hb tetramer at N2 pressures of 100 atm, while about 7 N2 molecules bind per tetramer at 400 atm N2 pressure, where available binding sites are still not fully saturated. Calculated N2-Hb binding isotherms are well described by a simple binding model with 12 independent and equivalent binding sites/Hb tetramer. N2 binding at each of the sites is hydrophobic, exhibiting the typical increase of the dissociation enthalpy with temperature. Enthalpies of dissociation are slightly more negative, while corresponding unitary entropies are somewhat less negative than those for the transfer of N2 from olive oil to water. Calculated partial molar volumes of N2 in Hb are positive but less than the corresponding partial molar volumes of N2 in buffer. Results indicate that N2 binding to Hb is accompanied by only small protein conformational changes which entail slight structural destabilization and loss of free volume in the protein that partially accommodates the volume of the N2 ligand. Results are related to previously reported effects of high pressure and high-pressure N2 on HbO2 affinity, illuminating essential features of the molecular mechanisms for these effects.

Photolysis method for determination of the tetramer-dimer dissociation constant of deoxyhemoglobin.

A new method for determination of the tetramer-dimer dissociation constant Ku4.2 of deoxyhemoglobin is described. The method involves photolysis of hemoglobin solutions containing a few percent of bound CO (e.g. less than 3%). Under these conditions the nature of the observed CO rebinding is primarily determined by the properties of the dominant species, deoxyhemoglobin. The method makes use of the 30-fold difference in the rate constant describing CO binding to hemoglobin dimers and deoxyhemoglobin tetramers. Because of this large difference in rate constants CO rebinding is made significantly more rapid by the presence of even small concentrations of dimers. Treating this reaction as CO binding to a mixture of hemoglobin dimers and tetramers allows the determination of Ku4.2. Data is presented showing application of the method to human deoxyhemoglobin in the range from pH 9.5 to 11.2.

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes.

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.

X-Ray small-angle scattering from concentrated solutions of normal and sickle cell deoxyhemoglobin

X-ray small-angle scattering of human normal and sickle cell deoxyhemoglobin in aqueous solution was measured at various concentrations, including those approximating the intracellular. The calculated radius of gyration, determination of the overall shape, and Soule-Porod plots all indicate a concentration-dependent difference in strength of interparticle forces between normal and sickle cell samples at high concentration.

Modification of human hemoglobin by glutathione. III. Perturbations of hemoglobin conformation analyzed by computer modeling.

The perturbations of the conformation of human deoxyhemoglobin induced by the covalent attachment of glutathione at cysteine beta 93 have been investigated by computer simulation in conjunction with molecular graphics. In the first phase of the analysis, a systematic search was carried out of the conformational space of glutathione attached to deoxyhemoglobin. In this search, the conformation of the hemoglobin molecule was held constant, while the relative energies of a series of 186,624 glutathione conformations involving systematic variation of six dihedral angels were calculated. From this search, the most favorable conformation was selected as the starting conformation for energy minimization of the glutathionyl hemoglobin molecule as a function of all Cartesian coordinates. In order to provide a reference state, an independent minimization by the same procedures was carried out for deoxyhemoglobin in the absence of glutathione. Comparison of the minimized structures with and without glutathione attached revealed a number of significant differences. The most conspicuous difference in the protein moiety concerned the salt bridge between aspartate beta 94 and histidine beta 146 which is destabilized upon minimization of the glutathionyl-hemoglobin complex due to interactions of the aspartate residue with the glycyl NH group of glutathione. Other observed differences in the minimized structures are located at the alpha 1-beta 2 interface and include displacement of the carboxyl group of aspartate beta 99. In the minimized complex, the glutathione portion assumes a quasi-cyclic conformation stabilized through interactions between the free (gamma-glutamyl) amino and (glycyl) carboxyl ends of the tripeptide and between this carboxyl end and the epsilon amino group of lysine alpha 40. In a parallel conformational study of glutathione alone, a similar structure was found as the lowest energy form. These quasi-cyclic conformations contrast with the extended structures reported by Wright (Wright, W.B. (1955) Acta Crystallogr. 11, 632-642) for crystals of glutathione where interactions between molecules play a major role. The conclusions of our analysis are in agreement with the experimental investigations reported in the two preceding papers and permit, moreover, a coherent interpretation of the observed functional and structural changes in deoxyhemoglobin induced by glutathione.