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Tritium-exchange experiments were performed to assess the dynamics of the spectrin molecule. From the kinetics presented, spectrin appears as a fast-exchanging protein compared to globular proteins. This is easily explained by its known elongated structure. Spectrin tritium exchange is also shown to be highly temperature sensitive, even in buffer conditions that greatly reduce the temperature dependence of hydroxyl catalyst concentration. At high ionic strength, the exchange rates of specific hydrogens deduced from a power-law simulation are shown to exhibit an Arrhenius behavior, with enthalpies ranging from 70 to 140 kjmol−1. These results are discussed in relation to the known high α-helix content of spectrin. Finally, a molecular model of α-helix opening is proposed that provides satisfactory agreement with the totality of the spectrin behavior in tritium-exchange experiments.

Summary. Background: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme-binding proteins. Free heme has been proposed to affect coagulation and has been co-purified with the factor VIII (FVIII)-von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results: Heme bound to several sites on FVIII with high apparent affinity. Heme-binding inhibited FVIII procoagulant activity in a dose-dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme-mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme-binding protein known for its protective activity in hemolytic conditions. Conclusions: Our data identify FVIII as a novel heme-binding protein. Occupation of high affinity heme-binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme-mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR-4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 3.73 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.

In the stereochemical model proposed by Perutz [1], the Fe-His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO)4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy-like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the alpha 2 beta 2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R-state. However, a significant amount of deoxy (T-state) kinetics are observed after addition of external effectors such as IHP. The iron-histidine bond is apparently crucial for the heme-heme interaction, but the allosteric equilibrium may still be influenced by external constraints.

We have tried to find out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1 mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J., Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like Kloek et al., we found its oxygen affinity to be as high as that of native Ascaris hemoglobin. We thought that this high affinity might be due to the heme-bound oxygen molecule being stabilized by two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in place of the leucine generally found there in vertebrate myoglobins and hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr., & Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these replacements lowered the oxygen affinity about 100-fold, to the same level of that of human alpha-globin chains. These results are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.

We have studied the infrared spectra of the bound and photodissociated states of Mb-12CO and Mb-13CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-12CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-1. The low-temperature photodissociated state (Mb) shows three lines (B0, B1, B2) at 2144, 2131, and 2119 cm-1 for 12CO. The absorbances of the three lines depend on temperature. B0 is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb have near-infrared, EPR, and Mössbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.

— We study the effect of solvent viscosity on the kinetics of the photocycle of bacteriorhodopsin (bR) from Halobacterium halobium. Solvent viscosity is altered by changing the glycerol concentration from 20 to 80% glycerol by volume. The kinetics of the photocycle are observed after flash photolysis at four wavelengths at several temperatures between 240 and 315 K. Assuming a sequential model, bR K - L M O bR, Arrhenius plots of the rate coefficients determine the activation enthalpies and frequency factors for each step. Kinetic data from all solvents are considered together and studied as a function of temperature for fixed solvent viscosities. The early steps of the cycle are insensitive to solvent viscosity, ; the later steps are retarded with increasing viscosity. Activation enthalpies are independent of viscosity; the frequency factors are proportional to η−K, where the exponent k 0.25 for the transition K L, 0.0 for L M, 0.8 for M O and 0.5 for O bR.

The association of dioxygen and carbon monoxide to soybean leghemoglobin (Lb) has been studied by laser flash photolysis at temperatures from 10 to 320 K and times from 50 ns to 100 s. Infrared spectra of the bound and the photodissociated state were investigated between 10 and 20 K. The general features of the binding process in leghemoglobin are similar to the ones found in myoglobin. Below about 200 K, the photodissociated ligands stay in the heme pocket and rebinding is not exponential in time, implying a distributed enthalpy barrier between pocket and heme. At around 300 K, ligands migrate from the solvent through the protein to the heme pocket, and a steady state is set up between the ligands in the solvent and in the heme pocket. The association rate, lambda on, is mainly controlled by the final binding step at the heme, the bond formation with the heme iron. Differences between Lb and other heme proteins show up in the details of the various steps. The faster association rate in Lb compared to sperm whale myoglobin (Mb) is due to a faster bond formation. The migration from the solvent to the heme pocket is much faster in Lb than in Mb. The low-temperature binding (B----A) and the infrared spectra of CO in the bound state A and the photodissociated state B are essentially solvent-independent in Mb, but depend strongly on solvent in Lb. These features can be correlated with the x-ray structure.

The intramolecular binding of carbon monoxide ($^{13}\mathrm{C}$$^{16}\mathrm{O}$/$^{12}\mathrm{C}$$^{16}\mathrm{O}$ and $^{12}\mathrm{C}$$^{18}\mathrm{O}$/$^{12}\mathrm{C}$$^{16}\mathrm{O}$ mixtures) to myoglobin at 20 and 60 K is observed with time-resolved Fourier-transform infrared spectroscopy. The binding rates (at \ensuremath{\approx} 1 ks) for the molecules $^{12}\mathrm{C}$$^{16}\mathrm{O}$, $^{12}\mathrm{C}$$^{18}\mathrm{O}$, and $^{13}\mathrm{C}$$^{16}\mathrm{O}$ are in the ratio 1:0.84:0.65 at 20 K, 1:0.87:0.84 at 60 K. The large isotope effect confirms earlier evidence for molecular tunneling. The fact that $^{12}\mathrm{C}$$^{18}\mathrm{O}$ binds faster than $^{13}\mathrm{C}$$^{16}\mathrm{O}$ implies that structure effects are significant in molecular tunneling.

Protoheme-CO in aqueous solution does not exhibit a geminate ligand recombination reaction. Addition of a protein, either globin or serum albumin, to which heme binds strongly, leads to an observable geminate reaction in aqueous solution. The bimolecular kinetic data for the albumin-heme-CO complex show two stable components, one heme-like in rate and difference spectrum, and one hemoglobin-like. The geminate reaction correlates spectrally with the hemoglobin-like component.

The association of dioxygen and carbon monoxide to soybean leghemoglobin (Lb) has been studied by laser flash photolysis at temperatures from 10 to 320 K and times from 50 ns to 100 s. Infrared spectra of the bound and the photodissociated state were investigated between 10 and 20 K. The general features of the binding process in leghemoglobin are similar to the ones found in myoglobin. Below about 200 K, the photodissociated ligands stay in the heme pocket and rebinding is not exponential in time, implying a distributed enthalpy barrier between pocket and heme. At around 300 K, ligands migrate from the solvent through the protein to the heme pocket, and a steady state is set up between the ligands in the solvent and in the heme pocket. The association rate, lambda on, is mainly controlled by the final binding step at the heme, the bond formation with the heme iron. Differences between Lb and other heme proteins show up in the details of the various steps. The faster association rate in Lb compared to sperm whale myoglobin (Mb) is due to a faster bond formation. The migration from the solvent to the heme pocket is much faster in Lb than in Mb. The low-temperature binding (B----A) and the infrared spectra of CO in the bound state A and the photodissociated state B are essentially solvent-independent in Mb, but depend strongly on solvent in Lb. These features can be correlated with the x-ray structure.

Proteins are dynamic systems. Recent evidence demonstrates that they exist in a large number of conformational substates and can continuously move from one substate to another; motion of a small ligand inside a protein may be possible only through these conformational fluctuations. To test this idea, we study with flash photolysis the binding of CO to protoheme and O2 and CO to myoglobin in many different solvents. The standard evaluation of such experiments yields information only about the protein-solvent system. A novel approach is presented which permits conclusions concerning the protein: Data from all solvents are considered together, and the rates for transitions of the ligand over various barriers are studied as a function of temperature for fixed solvent viscosities. Results show that over a wide range in viscosity the transition rates in heme-CO are inversely proportional to the solvent viscosity and can consequently be described by the Kramers equation. The rates of O2 and CO in myoglobin also depend on the solvent viscosity and are most sensitive to the solvent at the lowest viscosity. Viscosity influences protein reactions even in aqueous solutions. The data dan be interpreted by a dynamic model in which transitions into and inside myoglobin are governed by fluctuations between conformational substates corresponding to closed and open pathways. Ligand motion thus is mainly controlled by gates and not by static potential barriers. Some characteristic parameters for the substates are determined, and they agree approximately with similar parameters found in Mossbauer experiments. As expected, the barrier parameters evaluated in the novel approach deviate markedly from the ones obtained by the conventional procedure. Comparison with model calculations or basic theories will be meaningful only with the new evaluation, and the method may be essential for many or possibly all biochemical reactions.

The replacement reaction of myoglobin (Mb), MbCO + O2 leads to MbO2 + CO leads to MbCO + O2, has been studied with flash photolysis in the temperature range from 140 to 320 K and the time range from 2 mus to 200 s. In a fraction of the Mb, the photodissociated CO remains within the protein; rebinding is not affected by the presence of O2 and occurs with rates that are identical with the ones observed earlier in solvents containing only CO. In the remaining fraction CO migrates into the solvent and Mb combines preferentially with oxygen. The rate of the subsequent replacement of O2 by CO permits calculation of the oxygen dissociation rate ko2; ko2 has been determined from 260 to 320 K. The measurements support a multibarrier model.