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Picosecond to millisecond laser time-resolved transient absorption spectroscopy was used to study molecular oxygen (O2) rebinding and conformational relaxation following O2 photodissociation in the α and β subunits within human hemoglobin in the quaternary R-like structure. Oxy-cyanomet valency hybrids, α2(Fe2+–O2)β2(Fe3+–CN) and α2(Fe3+–CN)β2(Fe2+–O2), were used as models for oxygenated R-state hemoglobin. An extended kinetic model for geminate O2 rebinding in the ferrous hemoglobin subunits, ligand migration between the primary and secondary docking site(s), and nonexponential tertiary relaxation within the R quaternary structure, was introduced and discussed. Significant functional non-equivalence of the α and β subunits in both the geminate O2 rebinding and concomitant structural relaxation was revealed. For the β subunits, the rate constant for the geminate O2 rebinding to the unrelaxed tertiary structure and the tertiary transition rate were found to be greater than the corresponding values for the α subunits. The conformational relaxation following the O2 photodissociation in the α and β subunits was found to decrease the rate constant for the geminate O2 rebinding, this effect being more than one order of magnitude greater for the β subunits than for the α subunits. Evidence was provided for the modulation of the O2 rebinding to the individual α and β subunits within human hemoglobin in the R-state structure by the intrinsic heme reactivity through a change in proximal constraints upon the relaxation of the tertiary structure on a picosecond to microsecond time scale. Our results demonstrate that, for native R-state oxyhemoglobin, O2 rebinding properties and spectral changes following the O2 photodissociation can be adequately described as the sum of those for the α and β subunits within the valency hybrids. The isolated β chains (hemoglobin H) show similar behavior to the β subunits within the valency hybrids and can be used as a model for the β subunits within the R-state oxyhemoglobin. At the same time, the isolated α chains behave differently to the α subunits within the valency hybrids., O2 rebinding and conformational relaxation following O2 photodissociation were studied on picosecond to millisecond time scale in the α and β subunits within human hemoglobin in the quaternary R-like structure.

Excited electronic states and intraheme relaxation processes in the oxidized and reduced forms of mitochondrial cytochrome c oxidase extracted from a beef heart have been investigated by femtosecond absorption spectroscopy. The spectral and kinetic characteristics of short-lived intermediates have been measured from 80 fs to 20 ps after the photoexcitation. It is found that nonradiative electronic relaxation of the excitation energy in heme a, both in the oxidized (Fe(III)a) and reduced (Fe(II)a) forms, occurs successively as three processes, after the end of which heme a is in the ground state with a large store of vibrational energy. The subsequent vibrational relaxation (heme cooling) lasts for several picoseconds. It is found for reduced heme a3 (Fe(II)a3) that the electronic relaxation occurs as a result of two successive stages, which changes to vibrational relaxation in the ground state. The mechanism and dynamics of electronic excitation energy conversion in cytochrome c oxidase are analyzed.

Bimolecular recombination of molecular oxygen with human hemoglobin upon excitation in different spectral ranges is studied by nanosecond laser kinetic spectroscopy. The results obtained are fundamentally different; i.e., rather efficient photodissociation takes place in the case of excitation into the Q-band of the ππ* nature and is practically absent upon excitation into the near-IR band corresponding to the electron transfer from the porphyrin a2u orbital to a mixed orbital formed of the iron d orbital and the free π orbital of molecular oxygen. Analysis of these data, together with data obtained previously using the techniques of nano-, pico-, and femtosecond spectroscopy, allowed us to describe the mechanism and dynamics of the photodissociation reaction and intraheme relaxation processes.

The quantum yields and lifetimes of photosensitized luminescence of the 1Δ g state of singlet oxygen in an aquatic media with a controlled concentration of dielectric anisotropy centers (polyethylene glycol) have been measured using the methods of laser fluorometry. It is established that the quantum yield and the rate constant (k r ) of the a 1Δ g → X 3Σ - luminescence of 1O2 increase as the polymer concentration increases. The effect is analyzed within a general approach involving a relationship between kr and dielectric properties of the medium and is explained by the increased density of photon states and the local field factor in the space around O2(а 1Δ g ).

Photophysical properties of new tumor targeted photosensitizers based on cationic porphyrins and human transferrin were investigated. It was demonstrated that incorporation of the porphyrins into the protein causes slight changes of the photophysical properties of the photosensitizers. The kinetics of singlet oxygen luminescence photosensitized by porphyrin-transferrin complexes were studied.

Human hemoglobin reactivity towards ligands such as O 2 and CO as well as ligand migration via the protein matrix is modulated by internal cavities. In the present work, a timeresolved multiple-probe picosecond to millisecond infrared technique developed at Central Laser Facility (Didcot, UK) was applied to determine the dynamics of the ligand migration via the internal cavities of hemoglobin. We studied both native hemoglobin and its isolated α and β chains. We succeeded in following the evolution of photodissociated CO ligand inside the protein matrix during geminate recombination. Moreover, we managed to detect the photodissociated CO molecules escaped from the protein into external media.

A nanosecond laser near-infrared spectrometer was used to study singlet oxygen ((1)O2) emission in a protein matrix. Myoglobin in which the intact heme is substituted by Zn-protoporphyrin IX (ZnPP) was employed. Every collision of ground state molecular oxygen with ZnPP in the excited triplet state results in (1)O2 generation within the protein matrix. The quantum yield of (1)O2 generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 (1)O2 molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for (1)O2 generation within the protein matrix and for a subsequent (1)O2 deactivation was introduced and discussed. Rate constants for radiative and nonradiative (1)O2 deactivation within the protein were determined. The first-order radiative rate constant for (1)O2 deactivation within the protein was found to be 8.1 ± 1.3 times larger than the one in aqueous solutions, indicating the strong influence of the protein matrix on the radiative (1)O2 deactivation. Collisions of singlet oxygen with each protein amino acid and ZnPP were assumed to contribute independently to the observed radiative as well as nonradiative rate constants.

A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O 2 ) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1 atm of air and up to 25 atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O 2 escape by a factor of ~ 1.30 and 3.3, respectively. A kinetic model for O 2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O 2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O 2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites.

Bimolecular and germinate molecular oxygen (O(2)) rebinding to isolated α chains of human adult hemoglobin in solutions is analyzed. Multiple extended molecular dynamics (MD) simulations of the O(2) migration within the protein after dissociation are described. Computational modeling is exploited to identify hydrophobic pockets within the αchains and internal O(2) migration pathways associated with the experimentally observed ligand rebinding kinetics. To initiate dissociation, trajectories of the liganded protein are interrupted, the iron-dioxygen bond is broken, and the parameters of the iron-nitrogen bonds are simultaneously altered to produce a deoxyheme conformation. MD simulations provide 140 essentially independent trajectories (up to 25-ns long) of the O(2) migration in the protein. The time dependence of cavities occupancy, obtained by the MD simulations, and the kinetics of O(2) rebinding, measured by flash-photolysis techniques, allow us to obtain the kinetics of the entire O(2) migration process within the nanosecond time range and construct an explicit kinetic model of the O(2) migration and rebinding process. The amino acids that have the most pronounced effect on the ligand migration within the α chain matrix are predicted.

We have used luminescence and absorption spectroscopy to study the dynamics of photosensitized singlet oxygen generation and the photophysical characteristics of chlorin e 6 in Photolon ointment. We have shown that dimethylsulfoxide and polyethylene glycol 400, in concentrations comparable with their content in the ointment form of the drug Photolon, do not lead to appreciable changes in the spectral characteristics of chlorin e 6. Substantial changes in the photophysical characteristics of the photosensitizer are observed at significantly higher concentrations of the excipients in the ointment. We have shown that in buffer solution, chlorin e 6 forms a complex with polyethylene glycol 1500. For the first time, we have detected luminescence of singlet oxygen from the surface of biological tissue treated with Photolon ointment.

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to alpha and beta subunits within oxygenated human adult hemoglobin in solutions and porous wet sol-gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O2 binding to the alpha and beta hemes within the R quaternary structure is studied. The individual kinetic properties of the alpha and beta subunits within the hemoglobin encapsulated in sol-gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol-gel-free mediums. Interestingly, the alpha and beta subunits within the encapsulated hemoglobin possess the O2 rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O2 rebinding to the alpha and beta subunits within the oxygenated R-state hemoglobin in both solutions and wet sol-gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O2 rebinding properties, are discussed.

The different effects of pH and NaCl on individual O2-binding properties of α and β subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59–64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the α and β subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the α and β subunits were revealed. Regulation of the O2-binding properties of the α and β subunits within the liganded tetramer is proposed to be attained in two quite different ways.

The kinetics of nanosecond geminal recombination and bimolar association of molecular oxygen with the horse-heart myoglobin has been investigated by laser flash photolysis. The influence of Zn(II) ions on the dioxygenation and rebonding of myoglobin to a ligand has been considered. The kinetics of the geminal recombination was analyzed within the framework of the model of four states with a side path of ligand motion in the protein matrix. It is shown that an increase in the affinity of myoglobin to O2 in the presence of Zn(II) ions is predominantly caused by an increase in the rate constant of recombination of molecular oxygen from the primary intraprotein site.

The effect of DOPA melanin on the spectral and kinetic properties of the photosensitizers chlorin e 6 and Photolon in buffer solutions with different pH (8.5, 7.0, and 6.0) is studied. The data obtained indicate that no complex formation between molecules of DOPA melanin and either photosensitizer in the ground or excited states occurs (at least, at melanin concentrations ≤0.1 mg/ml). The presence of melanin in the samples has no effect on the spectral-luminescent or kinetic characteristics of either chlorin e 6 or Photolon. At a small concentration of DOPA melanin (≤0.02 mg/ml), the quantum yield of generation of singlet oxygen by the photosensitizers does not change; however, the generation efficiency of singlet oxygen decreases due to the shielding action of melanin.

Different effects on individual properties of α- and β-subunits within oxygenated human hemoglobin (HbA) are revealed at the pyridoxal 5′-phosphate modification. The rate constant of bimolecular oxygenation of the α-subunits within triliganded HbA and the apparent quantum yield of photodissociation of the α-subunits within completely oxygenated HbA decrease from 18.8±1.3 ( μ M s) −1 to 13.2±1.6 ( μ M s) −1 and from 0.0114±0.0012 to 0.0087±0.0009, respectively. The association rate constant of the β-subunits does not vary distinctly. However, the apparent quantum yield of photodissociation of the β-subunits is found to increase from 0.041±0.004 to 0.067±0.006. A previously proposed approach [B.M. Dzhagarov, S.V. Lepeshkevich, Chem. Phys. Lett. 390 (2004) 59.] is used for determination of the extent of subunit dissociation rate constant difference and the extent of subunit affinity difference from single flash photolysis experiment. The dissociation rate constant for the α-subunits within the tetramer is lowered by a factor of 1.3 with pyridoxylation. Since the association rate constant for the α-subunits are also lowered by a factor of 1.4, the α-subunits affinity to oxygen remains practically unaltered. The dissociation rate constant for the β-subunits is increased by a factor of ∼1.6. Because the association rate constant for the β-subunits does not vary virtually, the effect of the pyridoxylation on the oxygenation of the β-subunits is a 1.7-fold decrease in the O 2 affinity. Therefore the net pyridoxylation effect on the oxygenation of the liganded HbA is a reduction in the O 2 affinity mainly due to the decrease in the β-subunits affinity for oxygen. The present data of the bimolecular oxygenation study are analyzed in conjunction with the data of a previous geminate oxygenation research [S.V. Lepeshkevich, J. Karpiuk, I.V. Sazanovich, B.M. Dzhagarov, Biochemistry 43 (2004) 1675.]. For explanation of the pyridoxal 5′-phosphate regulation mechanism of the HbA subunits oxygenation, a parallel pathway model of ligand movement in the protein is favoured.

Using the methods of steady-state absorption and fluorescence spectroscopy and flash-photolysis, the new photosensitizer “Photolon” (Stock Company “Belmedpreparaty”) and its basic chromophore part — the substance chlorin e6 — have been studied under model conditions. It has been shown that there are no considerable differences between the spectral-luminescent characteristics of Photolon and chlorin e6 at a concentration not exceeding 2.5·10−5 M in ethanol, phosphate-saline buffer (pH 8.5), and isotonic solution. That is, the polyvinyl-pyrrolidone (PVP) entering into the composition of Photolon does not influence the chromophore characteristics being investigated. It has been found that an increase in the mass fraction of PVP in the buffer solution of chlorin e6 leads to an increase in the degree of polarization of chlorin e6 fluorescence and to a change in the spectral-luminescent and kinetic characteristics, pointing to the fact that the pigment is built into the hydrophobic portions of the polymer. Using the method of multiple scattering of light, it has been shown that both Photolon and chlorin e6 are present in the whole blood of donors and oncological patients mainly in the monomeric form; Photolon is quickly removed from the blood of patients (after 24 h, C ≤ 10−3 mg/ml).

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k α = 27.2 ± 1.3 (μM·sec)-1 and k β = 62.9 ± 1.6 (μM·sec)-1. Both the observed processes have been assigned to the bimolecular oxygenation of α- and β-subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are γ α = 0.0120 ± 0.0017 and γ β = 0.044 ± 0.005 for α- and β-subunits, respectively. The oxygenation reactions of isolated αPCMB- and βPCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k α = 44 ± 2 (μM·sec)-1 and k β = 51 ± 1 (μM·sec)-1, respectively. The quantum yields of photodissociation of isolated αPCMB- and βPCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.

Time-resolved luminescence measurements in the near-infrared region indicate that photodissociation of molecular oxygen from myoglobin and hemoglobin does not produce detectable quantities of singlet oxygen. A simple and highly sensitive method of luminescence quantification is developed and used to determine the upper limit for the quantum yield of singlet oxygen production. The proposed method was preliminarily evaluated using model data sets and confirmed with experimental data for aqueous solutions of 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin. A general procedure for error estimation is suggested. The method is shown to provide a determination of the integral luminescence intensity in a wide range of values even for kinetics with extremely low signal-to-noise ratio. The present experimental data do not deny the possibility of singlet oxygen generation during the photodissociation of molecular oxygen from myoglobin and hemoglobin. However, the photodissociation is not efficient to yield singlet oxygen escaped from the proteins into the surrounding medium. The upper limits for the quantum yields of singlet oxygen production in the surrounding medium after the photodissociation for oxyhemoglobin and oxymyoglobin do not exceed 3.4×10(-3) and 2.3×10(-3), respectively. On the average, no more than one molecule of singlet oxygen from every hundred photodissociated oxygen molecules can succeed in escaping from the protein matrix.

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BMLepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.

Laser flash photolysis technique is used to study human hemoglobin (HbA) oxygenation. Monomolecular geminate oxygenation of triliganded R-state HbA molecules is described by a function of three exponentials. Geminate oxygenation of the alpha-subunit within R-state HbA is characterized by two components with time constants of 0.14 and 1 ns, while geminate oxygenation of the beta-subunit within HbA is characterized by two components with time constants of 1 and approximately 30 ns. Bimolecular oxygenation of triliganded R-state HbA molecules is described by a biexponential law. Two observed rate constants are assigned to oxygenation of the alpha- and beta-subunit within HbA. The bimolecular association rate constants for O(2) rebinding with the alpha- and beta-subunit within triliganded R-state HbA are k(alpha) = 18.8 +/- 1.3 (microM x s)(-1) and k(beta) = 52 +/- 4 (microM x s)(-1), respectively. The apparent quantum yields of photodissociation of the beta- and alpha-subunit within completely oxygenated R-state HbA differ from each other by a factor of 3.6 and are equal to 0.041 +/- 0.004 and 0.0114 +/- 0.0012, respectively. The apparent quantum yield of photodissociation of completely oxygenated R-state HbA is equal to 0.026 +/- 0.003.