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Two chemotypes were examined in vitro with CYPs 3A4 and 2C19 by molecular docking, metabolic profiles, and intrinsic clearance deuterium isotope effects with specifically deuterated form to assess the potential for enhancement of pharmacokinetic parameters. The results show the complexity of deuteration as an approach for pharmacokinetic enhancement when CYP enzymes are involved in metabolic clearance. With CYP3A4 the rate limiting step was chemotype-dependent. With one chemotype no intrinsic clearance deuterium isotope effect was observed with any deuterated form, whereas with the other chemotype the rate limiting step was isotopically sensitive, and the magnitude of the intrinsic clearance isotope effect was dependent on the position(s) and extent of deuteration. Molecular docking and metabolic profiles aided in identifying sites for deuteration and predicted the possibility for metabolic switching. However, the potential for an isotope effect on the intrinsic clearance cannot be predicted and must be established by examining select deuterated versions of the chemotypes. The results show how in a deuteration strategy molecular docking, in-vitro metabolic profiles, and intrinsic clearance assessments with select deuterated versions of new chemical entities can be applied to determine the potential for pharmacokinetic enhancement in a discovery setting. They also help explain the substantial failures reported in the literature of deuterated versions of drugs to elicit a systemic enhancement on pharmacokinetic parameters., Competing Interests: All authors were paid employees of Pfizer Inc at the time this work was done. Pfizer Inc provided all funding for this research. Some of the authors are owners of PFE stocks or shares and are named inventors on Pfizer patents. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Italian salami were sonicated in different times (0, 3, 6 and 9min) using ultrasound bath (US, 25kHz). The effect of sonication on microbial growth (lactic acid bacteria and Micrococcaceae), lipid and protein oxidation, total heme pigments (THP), non heme iron (NHI) and metmyoglobin (MMb) was investigated during processing (0, 2, 15, and 28days) and storage (1, 30, and 120days). US enhanced growth of microorganisms (P<0.05), mainly for the treatment 9min of sonication. The lipid (peroxide value and TBARS) and protein (thiol group) oxidative reactions were accelerated by US (P<0.05) and they should be considered to maintain Italian salami quality. Sonication contributed to maintenance of THP (P<0.05), especially during storage. MMb pigment was not affected by sonication (P>0.05). This study presented some features of US application that could be explored in the manufacture of Italian salami., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (K d ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO 2 ), δ(ONO), and ν s (NO 2 ) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO} 6 and {FeNO} 7 states, enabling FTIR measurements to distinguish ν s (NO 2 ) and ν as (NO 2 ) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H 2 O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the ν(Fe-His) mode, arising from the proximal His77-iron stretch, does not shift until 50 μs after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 μs after the photodissociation of CO, suggesting that the ν(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon CO binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 μs after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.

Purpose: As the conformational change of a protein is intimately related with its function in vivo, the determination of its structure and the understanding of its conformational change occurring in physiological condition is of critical importance. In this regard, we have investigated conformational changes in heme moieties of both folded and unfolded myoglobin (Mb) induced by one-electron reduction., Material and Methods: The conformational changes of the heme moiety of folded/unfolded metmyoglobin (metMb) induced by one-electron reduction were investigated using the combination of pulse radiolysis and time-resolved resonance Raman (TR(3)) spectroscopy. Guanidine-HCl (GdHCl) is used as an electron donor and a denaturant for a protein. Mb solutions containing 0.5 and 2.5 M GdHCl (100 mM phosphate buffer, pH 7.0) were prepared for the measurement of transient absorption and TR(3) spectra., Results: Upon reduction, the folded metMb, which had a six-coordinated heme geometry linked with a water molecule as a distal ligand, was structurally relaxed to the deoxymyoblobin (deoxyMb) form with a five-coordination heme geometry without water ligand. Meanwhile, the Raman spectrum of an unfolded metMb was almost identical to those of the unfolded deoxyMb formed by the reduction, indicating that both unfolded metMb and deoxyMb had similar heme geometries., Conclusions: The results provided herein show that upon reduction, the folded metMb with a six-coordinated heme geometry was structurally relaxed to deoxyMb with a five-coordination heme geometry, while both unfolded metMb and deoxyMb had a six-coordinated heme geometry linked with water molecule or histidine as a distal ligand.

The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV-vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of "protective action" by the blood to control the oxidative stress generated by the gamma irradiation. The UV-vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV-vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.

The purpose of the present work is to study the vacuum ultraviolet radiation action on the optical and chromatographic characteristics of human hemoglobin molecules and their components - haem and globin. Using the methods of spectrophotometry and thin layer chromatography (TLC), we have investigated into the structural changes of molecules of human hemoglobin, haem and globin, induced by the influence of vacuum UV light (gamma = 118-134 nm, dose - 1.2 kJ/m2). It has been shown that vacuum ultraviolet radiation induces an infringement of the higher types of the spatial organization ofglobin molecules, thus leading to the changes in the structural state of the albuminous globule.

Time courses of mitochondrial responses to illumination-induced physiological loads and to hypoxia, were recorded optically from eyes of blowflies Calliphora vicina chalky. We isolated changes in redox states of haems a(3), a, c, and b. Two types of responses to light stimulation were observed. Haems b and a(3) responded with transient oxidation and haems a and c with reduction. The same two groups emerged in response to anoxic exposure. The onset of reduction of haems a and c had virtually no latency, while haems a(3) and b exhibited a transient oxidation followed by reduction only after 10-20 s. The dependence of the steady-state reduction level on [Formula: see text] produced the same groups. Haems a and c were significantly reduced at [Formula: see text] levels around 10 kPa while with haems b and a(3) load-induced oxidation was only replaced by reduction below 2 kPa. We propose haems respond to physiological loads in accordance with their steady-state reduction, which in turn depends largely on barriers for electron transport imposed by the mitochondrial membrane potential. We also propose it may be possible to assess the values of tissue [Formula: see text] and O(2) consumption by monitoring haems that are highly oxidized at rest such as haem a.

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T(m) values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.

We have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at approximately 695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790 cm-1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron-->heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.

Time-resolved infrared (TRIR) flash photolytic techniques have been employed to initiate and observe the efficient dissociation of CO from a synthetic heme-CO/copper complex, [((6)L)Fe(II)(CO)..Cu(I)](+) (2), in CH(3)CN and acetone at room temperature. In CH(3)CN, a significant fraction of the photodissociated CO molecules transiently bind to copper (nu(CO)(Cu) = 2091 cm(-)(1)) giving [((6)L)Fe(II)..Cu(I)(CO)](+) (4), with an observed rate constant, k(1) = 1.5 x 10(5) s(-)(1). That is followed by a slower direct transfer of CO from the copper moiety back to the heme (nu(CO)(Fe) = 1975 cm(-)(1)) with k(2) = 1600 s(-)(1). Additional transient absorption (TA) UV-vis spectroscopic experiments have been performed monitoring the CO-transfer reaction by following the Soret band. Eyring analysis of the temperature-dependent data yields DeltaH(double dagger) = 43.9 kJ mol(-)(1) for the 4-to-2 transformation, similar to that for CO dissociation from [Cu(I)(tmpa)(CO)](+) in CH(3)CN (DeltaH(double dagger) = 43.6 kJ mol(-)(1)), suggesting CO dissociation from copper regulates the binding of small molecules to the heme within [((6)L)Fe(II)..Cu(I)](+)(3). Our observations are analagous to those observed for the heme(a3)/Cu(B) active site of cytochrome c oxidase, where photodissociated CO from the heme(a3) site immediately (ps) transfers to Cu(B) followed by millisecond transfer back to the heme.

N-dodecyl-N'-(2-phosphonoethyl)-1,4,5,8-naphthalenetetracarboxylic diimide (DNDI) is a novel naphthalenic diimide with amphiphilic character. DNDI was synthesized through the sequential reaction of 1,4,5,8-naphthalenetetracarboxylic dianhydride, first with dodecylamine and then with 2-aminoethylphosphonic acid. Fluorescence measurements showed that DNDI forms excimers in water at sufficiently high concentrations. The fluorescence quantum yield of DNDI in diluted solutions is sensitive to the polarity of the microenvironment, decreasing as going from water to less polar solvents. This property allowed to monitor the incorporation of DNDI into cetyl trimethyl ammonium bromide (CTAB) micelles, with a binding constant of 1.2x10(4) M-1. UV irradiation (365 nm) of solutions containing DNDI and the redox protein cytochrome c (cyt c) resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) state, a reaction that was inhibited by the incorporation of DNDI into CTAB micelles. DNDI formed host-guest complexes with alpha-cyclodextrin (alpha-CD) through the inclusion of the dodecyl group, resulting in an increased aqueous solubility of the compound.

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.

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.

Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.

The paper deals with the present views of some chemical properties and biological effects of nitric oxide (NO) and chiefly its formation and primary conversions. The interaction of NO with superoxide radical is shown to one of the most important reactions of the former, which gives rise to peroxynitrite whose breakdown yields a hydroxy radical. Emphasis is laid on the substances that are a temporary depot or a possible transport form of NO, such as nitrosothiols and nitrosyl complexes of non-hemic iron. NO is well-known to release when these compounds are degraded. It is suggested that hemoglobin is another NO depot, which forms stable complexes with the latter. These hemoglobin complexes may be degraded on exposure to laser radiation to form free NO that ha a vasodilatory effect. Photolysis of nitrosyl complexes of hemoglobin may be responsible for the therapeutical effect of laser radiation.

The photoexcited metastable triplet state of Mg(2+)-mesoporphyrin IX (MgMPIX) or Mg(2+)-protoporphyrin IX (MgPPIX) located in the heme pocket of horse myoglobin (Mb) was investigated by optical and electron paramagnetic resonance (EPR) spectroscopy, and its properties were compared with the model complexes, MgMPIX, MgPPIX, and Mg2+ etioporphyrin I (MgETIOI), in noncoordinating and coordinating organic glasses. Zero-field splitting parameters, line shape, and Jahn-Teller distortion in the temperature range of 3.8-110 K are discussed in terms of porphyrin-protein interactions. The triplet line shapes for MgMPIXMb and MGPPIXMb show no temperature-dependent spectral line shape changes suggestive of Jahn-Teller dynamics, and it is concluded that the energy splitting is >> 150 cm-1, suggesting symmetry breaking from the anisotropy of intermal electric fields of the protein, and consistent with previous predictions (Geissinger et al. 1995. J. Phys. Chem. 99:16527-16529). Both MgMPIXMb and MgPPIXMb demonstrate electron spin polarization at low temperature, and from the polarization pattern it can be concluded that intersystem crossing occurs predominantly into in-plane spin sublevels of the triplet state. The splitting in the Q0.0 absorption band and the temperature dependence and splitting of the photoexcited triplet state of myoglobin in which the iron was replaced by Mg2+ are interpreted in terms of effects produced by electric field asymmetry in the heme pocket.

The effects of L-cysteine (LC) and N-acetylcysteine (NAC) on porphyrin accumulation in a human dermal microvascular endothelial cell line (HMEC-1) and a human epidermoid carcinoma cell line (A431) loaded with 5-aminolevulinic acid (ALA) and exposed to ultraviolet A (UVA) and blue light radiation were determined. Porphyrin accumulation was decreased in the presence of 0.1-7.5 mM LC (24.8%-31.4% suppression in HMEC-1 cell; 35.8%-48.9% suppression in A431 cells), and in the presence of 0.1-10.0 mM NAC (30.9%-58.0% suppression in HMEC-1 cells; 8.5%-45.3% in A431 cells). The suppression occurred in a LC or NAC dose-dependent fashion. The above was associated with partial reversal of suppression of ferrochelatase (FeC) activity in HMEC-1 cells and in A431 cells. As compared to FeC activity in cells treated with ALA and irradiation, enzyme activity was higher (by 31.9%-62.1%) in the presence of LC (1.0 mM or 5.0 mM) and in the presence of NAC (1.0 mM or 5.0 mM). These data indicate that LC and NAC have protective effects on porphyrin- and irradiation-induced diminution of FeC activity in HMEC-1 cells and A341 cells in vitro.

Changes in metabolic paramagnetic centers of the liver, kidney, spleen, brain and heart tissues, and blood after sodium nitrite intraperitoneal administration irradiation, or combined action of sodium nitrite and irradiation on mice, were studied using the EPR method. It was shown that irradiation of mice after sodium nitrite administration enhanced the effects induced by sodium nitrite alone: the levels of nitrosyl complexes Heme-NO in the tissues, cytochrome P-450 inhibition, and MetHb were higher.

To study porphyrin biosynthesis in human microvascular endothelial cells, HMEC-1 cells, a transformed human microvascular endothelial cell line, were incubated with 5-aminolevulinic acid (ALA), the precursor of endogenous porphyrins, and porphyrin accumulation was measured spectro-fluorometrically. The HMEC-1 cells accumulated porphyrin in a concentration-related and a time-dependent fashion. Protoporphyrin was the predominant porphyrin accumulated in the cells. The effect of light on protoporphyrin accumulation was evaluated by exposing the ALA-loaded HMEC-1 cells to ultraviolet-A (UVA) and blue light, followed by another incubation with ALA for 2-24 h. Enhancement of protoporphyrin accumulation in irradiated HMEC-1 cells was observed 2-24 h after irradiation, which was associated with a decrease in ferrochelatase protein and activity. Porphyrin accumulation from ALA after irradiation was significantly decreased when catalase (750-3000 U/mL, 29.3-44.3% suppression) or superoxide dismutase (270 U/mL, 36.4% suppression) was present during irradiation. These data demonstrate that HMEC-1 cells were capable of porphyrin biosynthesis, and that exposure of protoporphyrin-containing HMEC-1 cells to UVA and blue light, which includes the Soret band spectrum, decreased the ferrochelatase activity and its protein. These changes were mediated, at least in part, by reactive oxygen species.

To study porphyrin biosynthesis in human umbilical vein endothelial cells (EC), cultured EC were incubated with delta-aminolevulinic acid (ALA), the precursor of porphyrin synthesis, and porphyrins generated were measured spectrofluorometrically. EC accumulated porphyrins in an incubation time-dependent and ALA dose-dependent fashion. Porphyrin accumulation was enhanced by CaMg EDTA, a ferrochelatase inhibitor, suggesting the participation of ferrochelatase. The effect of porphyrin and irradiation on porphyrin accumulation was evaluated by exposing porphyrin-containing EC to the UVA and blue light, followed by incubation with ALA for 2-48 h. Decreased porphyrin accumulation 2-48 h after the irradiation was noted at all the time-points examined. However, no alteration of ferrochelatase activity was observed, suggesting that the decrease in porphyrin levels was due to suppression of the activity of the other enzymes in the earlier steps of the heme biosynthetic pathway.

In transient optical experiments the absorbed photon raises the vibrational temperature of the chromophore. In heme proteins at room temperature conversion of a 530-nm photon into vibrational energy is estimated to raise the temperature of the heme by 500-700 K. Cooling of the heme is expected to occur mainly by interacting with the surrounding protein. We report molecular dynamics simulations for myoglobin and cytochrome c in vacuo that predict that this cooling occurs on the ps time scale. The decay of the vibrational temperature is nonexponential with about 50% loss occurring in 1-4 ps and with the remainder in 20-40 ps. These results predict the presence of nonequilibrium vibrational populations that would introduce ambiguity into the interpretation of transient ps absorption and Raman spectra and influence the kinetics of sub-ns geminate recombination.

In male mice of the strain C57BL/10ScSnPh, both intact and X-irradiated with a whole-body sublethal dose, the distribution of the content of total and haeme iron was studied in individual bones, the spleen, liver, plasma and erythrocytes, using an original mineralization and extraction technique. In the erythropoietic organs as a whole and in the individual bones the haeme and non-haeme iron compartments were distinguished, and within these the iron accounted for by circulating plasma and by erythrocytes and erythrocyte precursors. In the radiation-depleted marrow there were quantitative and qualitative changes in the compartments, particularly the creation of an important compartment of non-circulating (fixed) erythrocytes.

Friend erythroleukaemia cells (FELC) were induced to a haem-producing state by X-rays. The percentage of haem positive cells was maximum for doses between 10 and 15 Gy. Heat treatment at 42.0 degrees C or 45.0 degrees C during or after irradiation inhibited haem induction whereas heating before irradiation enhanced it. Incubation at 37 degrees C between heating and irradiation resulted in a decline in induction levels, indicating repair of heat damage that interacts with X-ray damage. Incubation at 37 degrees C between irradiation and heating did not result in changed haem induction levels, indicating a lack of repair of radiation damage that could interact with subsequent damage produced by heating.

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 authors studied the effects of the whole-body x-irradiation on the activity of delta-aminolevulinate synthase and heme oxygenase in the liver of Wistar rats. The activity of delta-aminolevulinate synthase decreased to 81-49% of normal by the 1st-3d day after irradiation in a dose of 7 Gy followed by partial normalization of the enzyme activity by the 5th-7th day. The activity of heme oxygenase was over 2 times as increased by the 5th-7th day following irradiation in a dose of 7 Gy. Irradiation in a dose of 5 Gy did not alter the activity of heme oxygenase and caused a negligible reduction in the activity of delta-aminolevulinate synthase. During the most pronounced decrease in the rate of heme synthesis in the liver of irradiated rats, there was an elevation in the level of "free" heme (measured by the degree of tryptophane pyrrolase saturation with heme). This attests to a possible lowering of the rate of heme utilization in the synthesis of heme. A possible role of the effects described in the irradiation-induced decrease in the content of cytochrome P-450 in the animals' liver.

In order to verify the "radiation protection" effect of prosthetic groups in irradiated conjugated proteins and the influence of their electronic structure on intramolecular excitation transfer, two series of substances were examined. The first was composed of ferrihemoglobin and its constituents and the second was composed of similar hemoproteins with heme groups with major differences in electronic structure. The results point to a strong radiation protection effect of the heme groups and a dependence of radical yield of hemoproteins on the electronic structure of the heme. A possible interpretation of this latter effect is discussed.