A flash photolysis method to characterize hexacoordinate hemoglobin kinetics

A flash photolysis method is described for analyzing ligand binding to the new and growing group of hemoglobins which are hexacoordinate in the unligated, ferrous state. Simple analysis of a two exponential fit to time courses for CO rebinding at varying CO concentrations yields rate constants for formation and dissociation of the hexacoordinate complex, and the bimolecular rate constant for CO binding. This method was tested with a nonsymbiotic plant hemoglobin from rice for which these values had not previously been determined. For this protein, dissociation and rebinding of the hexacoordinating amino acid side chain, His(73), is rapid and similar to the rate of CO binding at high CO concentrations. These results indicate that hexacoordination must be taken into account when evaluating the affinity of hexacoordinate hemoglobins for ligands.     

Ultraviolet flash photolysis of gramicidin-doped lipid bilayers

We have examined the rate of gramicidin channel conductance inactivation by ultraviolet photolysis using 0.1 millisecond light flashes. The lower limit on the channel photolysis reaction rate has been reduced by four orders of magnitude over previous observations. Monoolein/hexadecane bilayers formed in 1.0 M KCl were doped with (1-3) x 10(6) gramicidin A' channels and exposed to a broad-spectrum light flash. The flash reduced membrane conductance abruptly by approx. 16%. Following the flash, a further slow reduction of approx. 3% was observed followed by a slow recovery of approx. 4%. The post-flash decay and recovery may be due to slow chemical reactions, conformational relaxations, or changes in the equilibrium between aqueous, lipid-bound, and channel-forming dimerized gramicidin. Under our experimental conditions, gramicidin M was insensitive to light flashes compared to gramicidin A', demonstrating that for gramicidin A' the photolysis mechanism depends specifically on the tryptophan side-chain. Flash photolysis of a membrane containing a small population of channels (approx. 30) indicated that the decay is due to the sudden inactivation of several channels. The recovery appears to result from insertion of normal channels into the membrane. Flash photolysis of single-channel membranes showed that the flash causes abrupt, complete channel inactivation.     

Laser flash photolysis studies of tris(2,2′-bipyridine)nickel(II) ion in aqueous solution

The photoreactions of tris(2,2′-bipyridine)nickel(II) complex, Ni(II)(bpy₃)²⁺ were studied and compared with that of the photoreactions of 2,2′-bipyridine ligand. Continuous photolysis of the complex shows that the photodecomposition corresponds to the absorption of light by the complex in the ligand centered excited state. Flash photolysis of the complex using a 248 nm excimer laser yields bipyridine cation radical and solvated electron as transients. Absorption spectra of the transient observed for the complex is found to be similar to that observed for the ligand on flash excitation using a 248 nm laser suggesting that the transients observed in the case of complex is due to the coordinated bipyridine in the complex. The formation of solvated electron is observed to be monophotonic and that of the bipyridine cation radical is found to be a biphotonic process. Significant change in Ni-N bond distance upon oxidation of Ni(bpy₃)²⁺ ion when compared to that observed in nickel(II)tetraazamacrocyclic complexes suggests that the formation of the trivalent complex by photolysis is not favored for the Ni(bpy₃)²⁺ ion and CTTS excited state in the complex is not observed in the present system.     

Activation parameters in flash photolysis studies of Mo(CO)6

Reported is a combined time-resolved optical (TRO) and infrared (TRIR) spectroscopic investigation of the flash photolysis of Mo(CO)₆ in cyclohexane solution. TRIR studies using 308 nm excitation led to transient bleaching of the strong ν_CO band at 1987 cm⁻¹ of Mo(CO)₆ and appearance of new bands at 1931 and 1964 cm⁻¹ attributed to Mo(CO)₅(Sol). Using a high pressure/variable temperature flow cell, the kinetics of back reaction with CO (k_CO) to regenerate the hexacarbonyl was studied over the P_CO range 1-20 atm and at five temperatures. These data gave k_CO=4.6±0.2×10⁶ M⁻¹ s⁻¹ (298 K) and the activation parameters kJ/mol and J mol⁻¹ K⁻¹ from which an interchange mechanism was proposed. The analogous species seen in the TRO experiment displayed a transient absorbance at 420 nm and analogous kinetics properties although at lower P_CO self-trapping with Mo(CO)₆ (to give Mo₂(CO)₁₁) is a competitive process. The Mo(CO)₅(Sol) transient could also be trapped by ⁿPrBr (k_RBr=5.3±0.7×10⁷ M⁻¹ s⁻¹).     

Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.     

Photoproducts generated from hematoporphyrin under high-intensity picosecond flash photolysis

Summary Hematoporphyrin (Hp) solutions were subjected to a wide range high intensity (0.2–10.0 GW/cm²) near-UV laser pulse radiation ( _exc=355 nm, pulse duration 30 ps). The formation of stable Hp photoproducts was followed by UV-VIS absorption spectroscopy and liquid-gel column chromatography. As judged by the influence of free radial scavengers, a significant part of the products is assigned to arise from the reaction of Hp with OH · (and H ·) radicals. Using nitroxide radicals (TEMPO and TEMPONE) and the spin trap DMPO the generation of primary transient photoproducts, hydrated electrons (e _aq ^– ), OH · and H · radicals, was studied varying the pulse intensity at a constant absorbed light energy. The results showed that bi-photonic processes are responsible for the observed product generation (different fore _aq ^– photoejection and OH · (OH ·) formation). A tentative diagram of the Hp excitation routes involved in the present high intensity laser flash photolysis is suggested. According to it, OH · and H · radicals are supposed to be generated in a resonance energy transfer reaction from highly excited Hp^** to water molecules (H₂O sensitization).     

Charge motion in MbCO crystals after flash photolysis.

Charge motion accompanying the dissociation and recombination of carbon monoxide to oriented myoglobin crystals has been observed. The magnitude of the electrical signals detected after photodissociation by electrodes on either side of MbCO crystals of type A is consistent with the x-ray data showing that the doubly charged iron ion lies in the mean heme plane when a ligand is bound and moves out of the plane when deligated. Beyond 10 ms, the time development of the electrical signal is consistent with the kinetics observed optically after flash photolysis on the same crystals.     

Oxidation-state-dependent protein docking between cytochrome c and cytochrome b(5): high-pressure laser flash photolysis study

To characterize the protein-protein interaction during electron transfer, we used Zn-substituted cytochrome c (ZnCytc) as a model of ferrous Cytc and determined the volume change, DeltaV(d)(Zn), for the dissociation of its complex with ferric cytochrome b(5) (Cytb(5)) by the pressure dependence of its photoinduced electron-transfer kinetics. Under ambient pressure, the dissociation constant, K(d)(Zn), of the ZnCytc/Cytb(5) complex was dependent on the buffer concentration, 1.5 and 12 microM in 2 and 10 mM Tris-HCl, pH 7.4, respectively, which was consistent with formation of salt bridges in its complexation. The dissociation of one salt bridge is usually associated with large volume changes of -10 to -30 cm(3) mol(-1), while pressure dependence of K(d)(Zn) resulted in smaller value of DeltaV(d)(Zn), -8.5 cm(3) mol(-1). Therefore, the interaction between ZnCytc and Cytb(5) cannot be explained only by salt bridge interaction, and the partial cancellation by the positive volume change due to the additional hydrophobic interaction is a plausible explanation for the observed DeltaV(d)(Zn). In addition, DeltaV(d)(Zn) of -8.5 cm(3) mol(-1) was considerably smaller than the previously reported volume change, DeltaV(d)(Fe), of -122 cm(3) mol(-1) in the ferric Cytc/Cytb(5) complex dissociation [Rodgers and Sligar (1991) J. Mol. Biol. 221, 1453-1460]. ZnCytc used here has been assumed to be a reliable model of ferrous Cytc, and thus the discrepancy between our present DeltaV(d)(Zn) and the previous DeltaV(d)(Fe) is discussed on the basis of the protein docking dependent on the oxidation states of heme iron in Cytc.     

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca²⁺ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.     

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments

Pronase treatment of aqueous suspensions of purple membrane fragments from H. halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000-21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme.Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated.Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M₄₁₂ and the regeneration of bR₅₇₀. Simultaneously the yield of O₆₆₀ is reduced. As with untreated purple membrane, the correlation between the rates of decay of M₄₁₂ and regeneration of bR₅₇₀ is greatest when the yield of O₆₆₀ is lowest.     

Low-temperature flash photolysis studies of cytochrome oxidase and its environment

The CO-binding kinetics of cytochrome a₃ in isolated, detergent-solubilized cytochrome oxidase have been studied by flash photolysis over wide ranges of CO concentration and temperature. The results strongly suggest that CO has an intermediate bound state in its path to the final bound state at the heme iron. In the temperature range 230–273 K in frozen aqueous solutions, the recombination rates depend upon CO concentration; at low CO concentrations the kinetics are biphasic. The rate of the faster process depends upon the detergent concentration, that of the slower process upon the salt concentration. In addition, the faster process depends upon the amount of CO photodissociated. It is concluded that the cytochrome oxidase molecules are aggregated in regions that contain detergent and possibly some lipids. These regions retain considerable fluid character well below the macroscopic freezing point of the solution. The faster phase of the recombination is interpreted as the rebinding of CO molecules that remain in the fluid region after photodissociation. The slower phase would then be due to the migration of some dissociated CO out into the surrounding frozen solvent. The non-Arrhenius behavior of both phases probably represents partial melting of the medium; preliminary NMR measurements of mobile protons support this hypothesis. Many of the kinetic features described here are also seen in mitochondria; thus the detergent-solubilized cytochrome oxidase may be a useful model system for the ligand-binding behavior of the enzyme in the mitochondrial membrane.     

Laser flash photolysis study of the triplet reactivity of beta-lapachones

The photochemical reactivity of beta-lapachone (1), nor-beta-lapachone (2) and beta-lapachone 3-sulfonic acid (3) has been examined by laser flash photolysis. Excitation (lambda = 266 nm) of degassed solutions of , in acetonitrile or dichloromethane, resulted in the formation of detectable transients with absorption maxima at 300, 380 and 650 nm. These transients, with lifetimes of 5.0 micros, were quenched by beta-carotene at a diffusion-controlled rate constant and assigned to the triplet excited states of 1-3. Addition of hydrogen donors, such as 2-propanol, 1,4-cyclohexadiene, 4-methoxyphenol or indole led to the formation of new transients, which were assigned to the corresponding ketyl radicals obtained from the hydrogen abstraction reaction by the triplets 1-3 . In the presence of triethylamine it was observed the formation of the long-lived anion radical derived from , which shows absorption maxima at 300 and 380 nm. The low values observed for the hydrogen abstraction rate constants for the beta-lapachones 1-3 using 2-propanol and 1,4-cyclohexadiene as quenchers led us to conclude that their triplet excited states show pi pi* character.     

Microtubule assembly and oscillations induced by flash photolysis of caged-GTP

Microtubule assembly and oscillations have been induced using the rapid liberation of GTP by UV flash photolysis of caged-GTP and monitored by time-resolved X-ray scattering. The flash photolysis method of achieving assembly conditions is much faster than the temperature jump method used earlier (msec vs. s range). However, the structural transitions and their rates are similar to those described previously. This means that the rates of the transitions in microtubule assembly observed before are determined by the protein itself, and not by the rate at which assembly conditions are induced. The advantages and limitations of using the photolysis of caged-GTP in microtubule assembly studies are compared with temperature jump methods. Caged-GTP itself reduces the rate of microtubule assembly and oscillations at mM concentrations, consistent with a weak interaction between the nucleotide analogue and the protein. X-rays are capable of slowly liberating GTP and other breakdown products from caged-GTP, even in the absence of UV flash photolysis, thus causing an apparent X-ray-induced microtubule assembly. This effect depends on the X-ray dose but is independent of the caged-GTP concentrations used here (mM range), suggesting that the breakdown of caged-GTP is caused not by the direct absorption of X-rays by the compound but by another intermediate reaction such as the generation of radicals by the X-rays.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-O,O-bis (2-amino ethyl ether)-N,N,N,N-tetraacetic acid - GDP guanosine-5-diphosphate - GTP guanosine-5-triphosphate - caged-GTP P³-1-(2-nitrophenyl) ethyl ester of GTP - HPLC high performance liquid chromatography - Mt-protein microtubule protein (=tubulin +MAPs) - MAP(s) microtubule-associated protein(s) - PC-tubulin phosphocellulose-purified tubulin - PIPES piperazine-1,4-bis(2-ethane sulfonic acid) - UV ultraviolet light Offprint requests to: E. Mandelkow     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.     

Nitric oxide binding properties of neuroglobin. A characterization by EPR and flash photolysis

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.     

Intermediates formed by laser flash photolysis of [PtCl(6)](2-) in aqueous solutions.

The stationary photolysis of [PtCl(6)](2-) in aqueous solutions (10(-5)-10(-4) M) at the region of 313 nm leads to its photoaquation with a quantum yield of 0.19. Laser flash photolysis experiments (308 nm) provided evidence of the formation of Pt(iii) intermediates, namely [PtCl(4)(OH)(H(2)O)](2-) and [PtCl(4)](-), and Cl(2) (-) radical anions. The Pt(iii) complexes formed as a result of an intrasphere electron transfer from Cl(-) ligands to the excited Pt(iv) ion. However, the main ( approximately 90%) photolysis channel was not accompanied by the transfer of Cl atoms to the solvent bulk. The photoaquation of [PtCl(6)](2-) results from the back electron transfer in the secondary geminate pair, [PtCl(5)(H(2)O)](2-)-Cl. The relative yield of Pt(iii) intermediates, recorded after the completion of all processes in the geminate pair, was less than 10% of the number of disappearing initial [PtCl(6)](2-) complexes.     

Further kinetics studies of intermediates formed by flash photolysis of Mo(CO)6

Kinetics studies of the behavior of Mo(CO)₅(CH) in the presence of radical initiators were conducted in cyclohexane (CH) solution by laser flash photolysis with time-resolved infrared detection. Activation parameters were determined for reactions of Mo(CO)₅(CH) with toluene and with photochemical radical generator dibenzylketone (DBK) in the presence of excess CO.