[Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light].

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.

Direct observation of photolysis-induced tertiary structural changes in hemoglobin.

Human Hb, an alpha2beta2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the alpha subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.

Kinetics of nitric oxide binding to R-state hemoglobin.

Despite earlier work indicating otherwise, some recent reports have suggested that nitric oxide (NO) binds to hemoglobin cooperatively. In particular, it has been suggested that, under physiological conditions, NO binds to the high-affinity R-state hemoglobin as much as 100 times faster than to the low-affinity T-state hemoglobin. This rapid NO binding could provide a means of preserving NO bioactivity. However, using a flash-flow photolysis technique, we have determined that the rate of NO binding to normal adult R-state hemoglobin is (2.1 +/- 0.1) x 10(7) (s(-1) M(-1)), which is essentially the same as that reported for T-state NO binding. (c)2002 Elsevier Science (USA).

[An assessment of the degree of chromophore photo damage to the heme and globin components in UV-irradiated molecules and electrophoretic fractions of human carboxyhemoglobin]. / Otsenka stepeni fotopovrezhdeniia khromoforov gemovogo i globinovogo komponentov UF-obluchennykh molekul i élektroforeticheskikh fraktsii karboksigemoglobina cheloveka.

The contribution of hem and globin components of electrophoretic fractions of UV-irradiated human carboxyhemoglobin to photodestruction of the protein was studied. The changes observed are the result of summation of some processes unequal in intensity and direction that take place in microheterogeneous media of photomodified protein. Photosensitivity of hemoproteid in electrophoretic fraction depends on apoprotein condition, whereas the hem photoresistance cannot be the evidence of the photostability of the whole molecule.

Nanosecond optical rotatory dispersion spectroscopy: application to photolyzed hemoglobin-CO kinetics.

A standard technique for static optical rotatory dispersion (ORD) measurements is adapted to the measurement of ORD changes on a nanosecond (ns) time scale, giving approximately a million-fold improvement in time-resolution over conventional instrumentation. The technique described here is similar in principle to a technique recently developed for ns time-resolved circular dichroism (TRCD) spectroscopy, although the time-resolved optical rotatory dispersion (TRORD) technique requires fewer optical components. As with static ORD, TRORD measurements may be interpreted by empirical comparisons or may be transformed, via the Kramers-Kronig relations, to more easily interpreted TRCD spectra. TRORD can offer experimental advantages over TRCD in studying kinetic processes effecting changes in the chiral structures of biological molecules. In particular, the wider dispersion of ORD bands compared with the corresponding CD bands means that ORD information may often be obtained outside of absorption bands, a signal-to-noise advantage for multichannel measurements. Demonstration of the technique by its application to ns TRORD and the transform-calculated TRCD of carboxy-hemoglobin (Hb-CO) after laser photolysis is presented.

Heme-CO religation in photolyzed hemoglobin: a time-resolved Raman study of the Fe-CO stretching mode.

Time-resolved resonance Raman spectroscopy has been employed to monitor geminate heme-CO rebinding in photolyzed HbCO. The excitation frequency was tuned to enhance the scattering from rebound heme sites 20-500 ns subsequent to CO photolysis. The behavior of vFe-C during ligand rebinding has important ramifications concerning heme pocket dynamics of the distinct equilibrium configurations of the six-coordinate heme sites. During the geminate phase of recombination, the Fe-CO bond strengths and configurations of the rebound sites (inferred from the positions and line widths of vFe-C) were found to be the same as those of equilibrium configurations of HbCO within 500 ns of CO photolysis for all samples. No evidence was found for the existence of transient metastable configurations during geminate recombination. Spectra obtained at earlier times (100 ns) revealed small differences in the geminate rebinding rates of the two equilibrium configurations. Since there is little or no further CO rebinding between 100 and 500 ns after photolysis, some interconversion must occur between the dominant HbCO configurations on a submicrosecond time scale.

High-pressure laser photolysis study of hemoproteins. Effects of pressure on carbon monoxide binding dynamics for R- and T-state hemoglobins.

The bimolecular association reaction of carbon monoxide to human adult hemoglobin at pH 7, 20 degrees C, was examined as a function of pressure up to 1500 bar by means of high-pressure laser photolysis. The apparent quantum yield for a millisecond recombination reaction decreased with pressure, which was attributed to an increase in the fraction of nanosecond geminate recombination reaction. On the basis of the pressure dependence of the recombination rate, the activation volumes at normal pressure for the binding of carbon monoxide to the R- and T-state hemoglobins were determined as -9.0 +/- 0.7 and -31.7 +/- 2.4 cm3 mol-1, respectively. Since the activation volumes for the overall CO association reaction were negative, it seems that the iron-ligand bond formation process mainly contributes to the rate-limiting step for both quaternary structures. The characteristic pressure dependence of the activation volume was observed for the R-state Hb but not for the T-state Hb. At 1000 bar, the activation volume for the R-state Hb was reduced to nearly zero, probably resulting from the contribution of the ligand migration process to the rate-limiting step. The effect of pressure on the activation enthalpy and entropy was also extracted from the data.

Subpicosecond resonance Raman spectroscopy of carbonmonoxy- and oxyhemoglobin.

In this paper we present the resonance Raman spectrum of the carbonmonoxy- (HbCO) and oxyhemoglobin (HbO2) photointermediates on a 800-900 fs timescale. In the case of HbCO, the frequencies of the so-called core-size markers (1500-1650 cm-1) are characteristic of a deoxylike photoproduct in a high spin state (S = 2) with a partially domed heme. The spectrum of the HbO2 photointermediate, on the other hand, is different, and may be characteristic of an excited-state species. These results are discussed in terms of a reaction scheme previously presented by Petrich, J. W., C. Poyart, and J. L. Martin (1988. Biochemistry. 27:4049-4060) and compared with those obtained in the literature on a 30-40 ps timescale. In both molecules a distinct downshift of the v4 mode was observed with respect to the equilibrium value, which is indicative of an elevated temperature of the heme after photodissociation.

A multipass cuvette for laser photolysis studies and its uses in studying hemoglobin kinetics and equilibria.

A simple multipass cuvette was constructed by cementing small first-surface mirrors to opposite optical faces of a standard cuvette, eliminating the need for complex alignment devices. The multipass cuvette could then be positioned to provide optical path lengths of approximately 5 and 7 cm for the observing beam directed perpendicularly to the laser photolytic pulse. Internal reflection losses in the cuvette elevated the baseline by 0.36 in absorbance for the seven-pass alignment. Heme proteins can easily be studied at 100 nM in this cuvette. Analysis of the concentration dependence of the rapid recombination phase following photolysis of HbCO allows KTD to be determined. Precise determination of this constant, however, requires that a large range of concentrations be studied, allowing the fraction of rapid phase to vary from 20 to 80%. Human HbCO at pH 7 cannot be effectively studied over this concentration range in ordinary cuvettes owing to the low concentrations required. By employing the multipass cuvette, we have been able to make very precise determinations of this constant and find at pH 7, 21 degrees C, a value for KTD of 0.66 microM. We also determined that the quantum yield for photolysis of HbCO dimers and tetramers must be very nearly the same. For HbCO in Tris buffer, pH 7.4, the R----T conformation change is some six to seven times slower than that in phosphate. We have developed a simple equation that allows both the rate constant for the conformational change and the KTD to be determined under these conditions. The KTD obtained is in excellent agreement with a reported value obtained by large-zone gel filtration.

Ligand dynamics in the photodissociation of carboxyhemoglobin by subpicosecond transient infrared spectroscopy.

Time-resolved infrared spectroscopy with 0.5-ps resolution is used to track the evolution of the CO stretching vibration after visible photoexcitation of carboxyhemoglobin in water at room temperature. Polarization measurements determine that the iron-complexed CO is oriented nearly perpendicular to the porphyrin plane. The dissociation appears to proceed via a metastable excited state with 2 +/- 1 ps lifetime. The dissociated CO binds weakly in the heme pocket for at least 500 ps. This state correlates with the internally bound state observed by Frauenfelder et al. at low temperatures in myoglobin.

Changes in the apparent quantum efficiency for photolysis of Hb(CO)1.

Recent studies suggest that the allosteric state of the protein surrounding the hemes in hemoglobin affects both geminate recombination of CO and the apparent quantum efficiency (AQE) for photolysis (Rohlfs, R.J., J.S. Olson, and Q.H. Gibson, 1988, J. Biol. Chem. 263: 1803-1813. We report combined flow/flash experiments in which the AQE for photolysis of Hb(CO)1 was measured as a function of time delay after its formation. Experiments were carried out at 20 degrees C in 0.1 M phosphate buffer at pH 7.0 with CO saturations of 10% or less. The AQE was observed to decrease from a value close to 1.0 at short times to approximately 0.6 after 2 s. The fundamental photolysis step for carboxyhemoglobin is known to have a quantum efficiency of nearly 1.0, whereas the lower AQE values we observe result from competition between rapid geminate recombination and a rapid reaction step leading to escape of the CO to the solution phase. Changes in AQE values reflect changes in these rapid reaction steps which presumably result from conformational change in Hb(CO)1. The change in AQE is consistent with conversion of one or more hemes to an R-like state but these changes could not be even approximately described in terms of a simple two-state allosteric model.

Effects of buffer salt on the geminate recombination of photodissociated carboxyhemoglobin and its isolated subunits.

The geminate recombination of photodissociated carboxyhemoglobin (human adult) and its isolated subunits was studied by nanosecond laser photolysis. The rebinding kinetics were found not to depend upon the kind of buffer used or on pH but changed appreciably with the buffer concentration. The results are compared to oxygenation properties under the same conditions.

Picosecond transient absorption study of photodissociated carboxy hemoglobin and myoglobin.

The optical transient absorption spectra at 30 ps and 6.5 ns after photolysis are compared for both carboxy hemoglobin (HbCO) and carboxy myoglobin (MbCO). Both 355- and 532-nm excitation pulses were used. In all cases the shapes of the optical difference spectra thus generated are stationary over the complete time-scale studied. The photolysis spectra for MbCO are not significantly different from the equilibrium difference spectra generated on the same picosecond spectrometer when measured to an accuracy of +/- 0.5 nm. In addition, spectral parameters for delegated HbCO generated on the same spectrometer but detected by two different techniques, either by a Vidicon detector or point by point with photomultiplier tubes, are reported; the results are different from some of the previously reported picosecond experiments.

Time-resolved Raman spectroscopy with subpicosecond resolution: vibrational cooling and delocalization of strain energy in photodissociated (carbonmonoxy)hemoglobin.

A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.

X-ray scattering study on hemoglobin solution with synchrotron radiation: a simple analysis of scattering profile at moderate angles in terms of arrangement of subunits.

X-ray scattering profiles in moderate-angle regions were recorded from carbon-monoxy-, oxy-, and deoxyhemoglobin solutions, using synchrotron radiation. They all display four distinct scattering peaks at R = 0.030, 0.055, 0.078, and 0.102 A-1 up to 2 theta approximately 10 degrees in addition to the main scattering around R approximately equal to 0. Contrast variation experiments, in which sucrose was used to change the electron density level of the solvent, revealed that the outer two scattering peaks are attributable to the variation of electron density within subunits in hemoglobin. The inner two were assigned as peaks due to the whole molecule and interpreted in terms of an interference function that is calculated from the inter-subunit distances in a molecule. This result is important in connection with evaluating the arrangement of constituent subunits in allosteric proteins and oligomeric proteins. The scattering profiles indicate that there is no difference in electron density variation within subunits between oxy- and deoxyhemoglobin. However, the arrangement of subunits is different between oxy- and deoxyhemoglobin molecules, as the scattering peaks at R = 0.030 and 0.055 A-1 shift toward smaller angles for deoxyhemoglobin.

Laser photolysis study of conformational change rates for hemoglobin in viscous solutions.

Rates for the R leads to T conformational change of deoxyhemoglobin formed by laser photolysis of carboxyhemoglobin were determined from CO rebinding observed in three solution systems with viscosities between 1 and 6 cP. Experiments were carried out at 20 degrees C and pH 8.3 in solutions consisting of borate buffer containing various amounts of sucrose, glycerol, or ethylene glycol. As in the case of earlier experiments in borate buffer (Sawicki and Gibson, 1976, J. Biol. Chem., 251:1533-1542), a simple two-state allosteric model which takes into account tetramer-dimer dissociation was found to give a good description of all experimental results. Using measured values for the R- and T-state CO-binding rate constants and the tetramer-dimer dissociation constant, values for the conformational change rate were determined by fitting this model to the experimental data. These rates were compared with Gavish's transient strain model (Gavish, 1978, Biophys. Struct. Mech., 4:37-52), which predicts an inverse dependence of conformational change rate on viscosity. Although fair agreement is found for hemoglobin in sucrose/borate solutions, in glycerol/borate and ethylene glycol/borate solutions, conformational change rate falls off much more rapidly with increasing viscosity than predicted by the model.

Geminate recombination of O2 and hemoglobin.

The photolysis of HbO2 and HbCO has been studied by measuring transient absorption spectra in the Soret region after excitation with picosecond pulses at 530 nm. Dissociation occurred promptly in both cases, followed (for HbO2) by geminate recombination of ca. 40% of the photodissociated O2 with a lifetime of 200 +/- 70 psec (25 degrees C). No recombination of Hb + CO was observed up to 1200 psec after photolysis. The HbO2 and HbCO photoproduct spectra were broader, weaker, and red-shifted in comparison to the spectrum of stable Hb and Gibson's fast-reacting form, Hb. For HbO2 the spectrum was initially much broader to longer wavelengths but relaxed to a constant shape within 90 psec, whereas for HbCO there was no spectral evolution. The photophysics is analyzed by considering the effect of spin constraints as well as spin--orbit coupling and orbital correlation among the various electronic states of liganded and deoxy hemoglobins. The small quantum yield of HbO2 dissociation is not primarily due to rebinding but rather to electronic relaxation to nonreactive states.