Biomedical subjects

Q H Gibson

Publications and source records attributed to Q H Gibson.

At least 55 records · Page 3Linked to original sources

PubMed1989

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa.

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.

Carbon Monoxide↗

PubMed1989

Flash photolysis of the serum albumin-heme-CO complex.

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.

Binding Sites↗

PubMed1989

The kinetics of ligand binding to plant hemoglobins. Structural implications.

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.

Amino Acid Sequence↗

PubMed1988

A comparison of the geminate recombination kinetics of several monomeric heme proteins.

The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.

Algorithms↗

PubMed1987

Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin.

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.

Binding Sites↗

PubMed1987

Ligand recombination to the alpha and beta subunits of human hemoglobin.

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.

Carbon Monoxide↗

PubMed1987

Oxygen binding and subunit interaction of hemoglobin in relation to the two-state model.

Mills and Ackers (Mills, F.C., and Ackers, G.K. (1979) J. Biol. Chem. 254, 2881-2887) have reported the subunit interactions of hemoglobin to decrease on binding of the fourth molecule of oxygen to hemoglobin. This effect, which they called quaternary enhancement, is incompatible with the two-state Monod, Wyman, and Changeux allosteric model. Their free energy of binding of the fourth molecule (-9.3 kcal/mol) has been compared with independent kinetic estimates which give -8.6 kcal/mol. This smaller value is consistent with literature values and allows reasonable representation of the equilibrium curve using the two-state model without invoking quaternary enhancement.

Allosteric Regulation↗

PubMed1986

Testing the two-state model: anomalous effector binding to human hemoglobin.

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.

Allosteric Regulation↗

PubMed1986

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide.

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.

Carbon Monoxide↗

PubMed1986

A kinetic description of ligand binding to sperm whale myoglobin.

Nanosecond recombination time courses were measured by photolyzing O2, NO, CO, methyl, ethyl, n-propyl, n-butyl, and tert-butyl isocyanide complexes of sperm whale myoglobin with a 30-ns laser pulse at pH 7, 20 degrees C. Absorbance was measured both during and after the excitation pulse and as a function of laser light intensity. The results were analyzed quantitatively in terms of a three-step reaction scheme, MbX in equilibrium B in equilibrium C in equilibrium Mb + X, where Mb is myoglobin, B represents a geminate state in which the ligand is present in the distal pocket but not covalently bound to the iron atom, and C, a state in which the ligand is still embedded in the protein but further away from the heme group. The fitted rate parameters were required to be consistent with the observed overall quantum yield, Q, which had been measured independently using much longer (approximately 0.5 ms) xenon flash pulses. Three major conclusions were derived from these analyses. First, the overall quantum yield of the ligand complex is determined primarily by the competition between the rate of iron-ligand bond formation from the initial photoproduct, kB----MbX, and the rate of migration away from state B, kB----C. For example, kB----C approximately equal to 30-100 microseconds-1 for all three gaseous ligands, whereas both Q and kB----MbX vary over 3 orders of magnitude (i.e. NO, Q = 0.001, kB----MbX approximately equal to 16,000 microseconds-1; O2, Q = 0.1, kB----MbX approximately equal to 500 microseconds-1; CO, Q = 1.0, kB----MbX approximately equal to 2 microseconds-1). Second, for NO, O2, and the isonitriles, the rate-limiting step in the overall association reaction starting from ligand in solution is the formation of state B. The rate constant for this process varies from 2 X 10(7) M-1 s-1 for the gaseous ligands to 0.02-1.4 X 10(5) M-1 s-1 for the isonitriles. In contrast, the B to MbX transition is limiting for CO binding. Third, for all the ligands except CO, the overall rate of dissociation is limited significantly both by the rate of thermal bond disruption, kMbX----B, and the competition between geminate recombination and migration away from the distal pocket (i.e. kB----C/(kB----MbX + kB----C]. In the case of CO, the rate of bond disruption is equal to the observed dissociation rate constant.

Animals↗

PubMed1986

Protoheme-carbon monoxide geminate kinetics.

Recombination kinetics of CO to protoheme after laser photolysis have been measured vs. temperature and viscosity. A 25-ns laser pulse was focused on the sample to produce an excitation rate of 10(9)/s per heme. This temporarily populates the heme-CO state of dissociated pairs which either separate or recombine on a picosecond time scale in viscous glycerol-water solutions. From the equilibrium amplitude of the fraction dissociated during the laser pulse, the geminate recombination rate constant is calculated to be 3 X 10(9)/s. This rate coefficient is only weakly dependent on temperature or viscosity. As previously observed the fraction that escapes depends on the solvent viscosity [Marden, M. C. (1983) Ph.D. Thesis, University of Illinois-Urbana]. A model consisting of a single barrier plus diffusive escape is used to simulate the kinetics during and just after the flash.

Carbon Monoxide↗

PubMed1985

Geminate reactions of oxygen and nitric oxide with the alpha and beta subunits of Fe-Co hybrid hemoglobins.

The alpha and beta subunits in Fe-Co hybrid hemoglobins differ in their rapid reactions with dioxygen and nitric oxide after dissociation by a 25-ns photoflash. The alpha subunits show little recombination on a scale of tens of nanoseconds, whereas the beta subunits show extensive recombination on this time sale. The alpha-beta difference is more marked with Fe than with Co and greater with dioxygen as ligand than with nitric oxide, but is clearly evident in all combinations of ligand and metal. Addition of inositol hexaphosphate slows ligand binding and reduces the proportion of rapid recombination of dioxygen and nitric oxide to beta-Fe subunits. The behavior of alpha-Fe subunits is unaffected by this compound. These results permit the beta subunit to be identified as the T-state species which equilibrates rapidly with oxygen in the T-state, i.e. the reverse of the identification suggested on structural grounds.

Carbon Monoxide↗

PubMed1984

Geminate combination of oxygen with iron-cobalt hybrid hemoglobins.

Photodissociation of oxygen from the ferrous subunits of hybrid hemoglobins in which the heme of either the alpha or the beta chain has been replaced by cobalt protoporphyrin IX shows large differences between the subunits. With a 25-ns light pulse, the apparent quantum yield at the end of the flash is greater for the beta-iron hybrid than for the alpha-iron hybrid. With the beta-iron hybrid, the yield is greater when solution conditions favor the T-state. After the flash, a part of the oxygen which has been dissociated recombines with a half-time of the order of tens of nanoseconds. The proportion is greatest in the R-state at low temperature and least in the T-state. With the alpha-iron hybrid, oxygen is much less readily removed, and the rapid recombination is slight or absent. It is seen most clearly at low temperatures in conditions which favor the T-state. The long term (greater than 100 ns) effect is that oxygen is much more readily removed from the beta-iron hybrid in the T-state than under any other condition. Analogous flash experiments performed with human hemoglobin A may be closely simulated by superposition of the results obtained with the two hybrid hemoglobins under the same conditions. Isolated human alpha and beta--SH chains show differences similar to, but less marked than, those of the iron-cobalt hybrids.

Hemoglobins↗

PubMed1984

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis.

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.

Animals↗

PubMed1984

The apparent quantum yield of T-state human hemoglobin. Contribution of protein and heme to rates of oxygen reactions.

The apparent quantum yield for dissociation of oxygen from T-state human hemoglobin has been determined using pulses of light 350 ns long at 540 nm. Two quantum yields were found. One was the same as for the R-state, and, like it, strongly temperature- and viscosity-dependent. The other, only slightly influenced by temperature and viscosity, was 10 times larger at 20 degrees C. Previous work (Sawicki, C. A., and Gibson, Q. H. (1977) J. Biol. Chem. 252, 7538-7547) has shown two distinct phases in binding of oxygen by T-state human hemoglobin at pH 7, 20 degrees C. When the apparent quantum yield was followed with time, the species with high quantum yield correlated with the rapidly reacting T-state species. The hemoglobin chains have different quantum yields in the T-state. Quantum yield data may serve as a measure of population of the liganded T-state in human hemoglobin, supplementing absorbance and circular dichroism data, and permit calculation of the rates of reaction at the heme in both R- and T-states.

Chemical Phenomena↗

PubMed1984

Binding characteristics of a fluorescent analogue of diphosphoglyceric acid to human, amphibian and fish hemoglobins.

An attempt was made to establish the binding of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, DIPANS, as an estimator of conformation in the carbonmonoxy (CO)-hemoglobins (Hbs) of several vertebrates. DIPANS failed to bind menhaden I, trout I or tuna Hbs which are ligand insensitive. Below a pH of 7.0, DIPANS bound menhaden II, Bufo, Xenopus, and human Hbs with a binding stoichiometry greater than one. The charge of the DIPANS molecule does not control its binding to these Hbs. The binding to human CO-Hb can not be due to Hb conformation. For Xenopus Hbs and menhaden II, conformation predominates DIPANS binding. The binding to CO-Hb of DIPANS, can not be unambiguously attributed to the Hb's quaternary conformation.

Animals↗

PubMed1983

Oxygen equilibrium studies on hemoglobin from the bluefin tuna (Thunnus thynnus).

Oxygen equilibrium curves of the purified hemoglobin component I from the Atlantic bluefin tuna (Thunnus thynnus) have been determined between pH 6.5 and 8.75 at 25 degrees C, and for five temperatures between 10 and 30 degrees C at pH 7.0 and 7.5. From the equilibrium data oxygen equilibrium constants for four oxygenation steps, Ki (i = 1 to 4) were estimated. The number of the Bohr protons released on the ith oxygenation (delta Hi+), and the enthalpy and entropy changes at each oxygenation step (delta Hi and delta Si) were calculated. The Hill plot for oxygenation below neutral pH is biphasic; the top asymptote lies to the right of the bottom one and the linking limb between them exhibits a slope less than unity, exhibiting apparent negative co-operativity. The values of K1 and K2 exhibit little pH dependence, while those of K3 and K4 increase by two orders of magnitudes as the pH is changed from 6.5 to 8.75. In consequence, oxygen equilibrium above neutral pH exhibits a normal positive co-operativity. The oxygen equilibrium at lower temperature is biphasic as is that below neutral pH. The shape of the Hill plot is temperature-dependent. The affinity at low saturation decreases, and that at high saturation increases upon raising the temperature from 10 to 30 degrees C, resulting in crossing of the middle portion of the equilibrium curves at different temperatures. The delta H1 and delta H2 values are negative as are those of most other hemoglobins, but the delta H3 and delta H4 values are positive. Consideration of these results in a framework of the allosteric model extended to take account of differences between subunits has indicated that the deoxy quaternary structure is stabilized at low pH or low temperature, and that subunit heterogeneity gives rise to the biphasic oxygen equilibrium curve. An analysis of delta Hi+ suggests that the large number of the Bohr groups is responsible for the biased allosteric equilibrium towards the deoxy quaternary structure. The positive delta H3 and delta H4 values are also considered to arise from the large endothermic contribution of the Bohr protons released at the third and fourth steps of oxygenation.

Animals↗

PubMed1983

Circular dichroism and conformation of fish hemoglobins.

The circular dichroism spectrum of fully liganded CO hemoglobin from the Atlantic bluefin tuna (Tunnus thynnus) shows a pH- and temperature-dependent feature at 416 nm. It is half-developed at pH 5.9 and 20 degrees C and its change with temperature corresponds to a heat of 34 kcal/mol (tetramer) for the transition. Correlation with studies on function (Morris, R. J., and Gibson, Q. H. (1982) J. Biol. Chem. 257, 4869-4874) shows that the dichroism feature changes at about 1 pH unit below the R-T transition. There is a close correlation between the 416 nm band and changes in circular dichroism at 287 nm. The new 416 nm band is seen in several fish hemoglobins, but not with human hemoglobin. With hemoglobin from Brevoortia tyrannus, which has been sufficiently studied to permit the comparison, there is a smaller gap between the change in dichroism spectrum and the functional R-T transition. So far, no change in function has been associated with the appearance of the 416 nm circular dichroism band.

Animals↗