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T Brittain

Publications and source records attributed to T Brittain.

At least 73 records · Page 4Linked to original sources
PubMed1985

A kinetic and equilibrium study of ligand binding to a Root-effect haemoglobin.

The blood of the striped marlin (Tetrapturus audax) contains one major Root-effect haemoglobin. Titrations of this haemoglobin with CO show that at high pH the molecule is highly co-operative (Hill coefficient 2.8) whereas at low pH the titration data can best be described as the sum of contributions from non-co-operating subunits of different affinity. In terms of the two-state model the R-state affinity constant is much more sensitive to pH than is that of the T state. Flash-photolysis studies were used to characterize the kinetics of ligand binding to this haemoglobin. Both T and R states show kinetic heterogeneity in their recombination time courses, associated with the alpha- and beta-chains of the molecule. The rate constants for ligand binding to each chain, in each quaternary state, were determined, and in conjunction with the allosteric equilibrium parameters determined at pH8.0 were used in the two-state analysis of reaction curves, over a range of ligand concentration. The two-state model, extended to take account of chain difference, adequately fits the homotrophic effects observed for this haemoglobin. The two-state model is, however, inadequate in its description of the heterotropic effects produced by protons.

Animals↗
PubMed1985

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine.

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.

Binding Sites↗
PubMed1984

A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm.

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.

Allosteric Regulation↗
PubMed1984

Characterization and function of isolated hemoglobins from the marine teleost Girella tricuspidata.

Five isohemoglobins were characterized from the marine teleost fish, Girella tricuspidata, using polyacrylamide gel electrophoresis and isoelectric focusing. O2-equilibrium (OEC) studies of the five components separated by column chromatography showed functional heterogeneity with respect to Hill's coefficient n, the O2 affinity, P50, and the Bohr coefficient phi, = delta logp50/delta pH. Recombination and examination of the isolates gave an OEC similar to that of "stripped" hemolysate and similar to a curve synthesized from a model incorporating the OEC parameters of the isoHbs.

Animals↗
PubMed1983

Molecular aspects of embryonic mouse haemoglobin ontogeny.

Embryos from C57BL/6J mice between the gestational ages of 9 and 16 days possess three embryonic haemoglobins EI, EII and EIII, the proportions of which change as a function of gestational age. Component EI, originally present at approx. 65% at day 9, decreases to approx. 20% by day 16, while component EII increases in an inverse manner to that of component EI. During this period component EIII remains essentially constant at approx. 25%. Separation of these species by ion-exchange chromatography has allowed the characterization of the Hill coefficient, Bohr effect, heat of oxygenation and binding of allosterically active organic phosphates for each component. The three components show marked functional heterogeneity and also differ from maternal haemoglobin. Oxygenation curves for whole embryonic blood show distinct deviations from simple binding behaviour. The presence of a high-affinity component within the blood samples may be accounted for by the presence of haemoglobin EI. By using parameters obtained from the study of the isolated components it has been possible to synthesize mathematically the O2-binding curves, obtained experimentally, throughout the gestational period. The characteristics of the isolated haemoglobin components of embryonic mouse blood are discussed in terms of the changing demands for O2 likely to be encountered by the developing embryo.

2,3-Diphosphoglycerate↗
PubMed1983

Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase.

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.

Animals↗
PubMed1983

Non-cooperative oxygen binding in the erythrocytes of pre-implanted sheep embryos.

The whole-blood oxygen equilibrium curve in sheep embryos at 17 days gestation was essentially hyperbolic, indicating non-cooperative O2 binding with Hill's coefficient, n approximately equal to 1.2. O2 affinity was higher in embryonic blood (P50 = 7.1 mmHg at pH = 7.4 and 39 degrees C) than in maternal blood (P50 = 32.6 mmHg at pH = 7.4 and 39 degrees C). The Bohr effect was apparently smaller in the embryo (delta log P50/delta pH = --0.52) than in maternal blood (--0.36).

Animals↗
PubMed1983

A comparison between the ease of haemoglobin oxidation and modification by sulphide.

The haemoglobin purified from a wide range of species undergoes facile sulphaemoglobin formation as well as oxidation. Oxidation by either anions such as azide or organic electron transfer reagents shows similar facility. Facile oxidation of a particular haemoglobin is associated with protection against sulphaemoglobin formation. Oxidation and sulphaemoglobin formation in some instances occur via competing electron transfer reactions.

Animals↗
PubMed1982

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

The CO complex of sulphaemoglobin was found to be photolytically dissociable at pH 6.0 and pH 8.5. The recombination kinetics of the CO complex after flash photolysis show two phases, which differ in rate by approximately 20-fold. At both pH 6.0 and pH 8.5 the two phases show an identical linear dependence on CO concentration, with associated second-order rate constants of 6.0 x 10(3) M-1 . s-1 and 1.2 x 10(5) M-1 . s-1 respectively. The percentage contribution of each of the two phases to the total absorbance change is independent of the CO concentration employed, but is a function of the protein concentration used. The percentage of the faster phase increases with protein dilution. These results taken together with computer simulation suggest the existence of appreciable amounts of monomeric species in solution at both pH 6.0 and pH 8.5 at low concentration of liganded protein. Estimates of the dissociation constants for the dimer in equilibrium monomer equilibrium yield values of 8 microM and 3.5 microM at pH 6.0 and pH 8.5 respectively. Stopped-flow delayed-flash-photolysis studies show that the liganded dimer decays to the liganded monomer with a half-life of approx. 0.5 s. Constant-illumination experiments allow an estimation of the half-life for the combination of deoxy monomers to deoxy dimers of 22 s. A model explaining the range of CO recombination kinetics seen for sulphaemoglobin does not require the existence of a photolytically produced high-activity form, even at high pH, in contrast with the case for normal haemoglobin.

Carbon Monoxide↗
PubMed1982

An investigation of the allosteric functioning of the haemoglobin of the cane toad, Bufo marinus.

1. Cane toad haemoglobin shows co-operative equilibrium CO binding at pH 7.0 with n = 2.6. 2. Flash photolysis studies show that at high pH the R form of the protein is stabilised whilst low pH and the presence of IHP favours the T form. 3. The combination of stopped-flow and flash photolysis measurements allows estimation of the association and dissociation rates of CO binding for the two forms of the protein. 4. The protein functioning can be described by a modified two state model and mathematical modelling allows the estimation of the allosteric equilibrium constant for this haemoglobin.

Animals↗
PubMed1982

An investigation of the co-operative binding of carbon monoxide to the haemoglobin of the carpet shark Cephaloscyllium.

1. Carpet Shark haemoglobin shows co-operative equilibrium CO binding at pH 7.0 (n = 1.8). 2. Flash photolysis shows that the T form of the protein is stabilised at low pH and in the presence of Inositol Hexaphosphate (I.H.P.). 3. At high pH the R form of the protein is stabilised and exhibits kinetic heterogeneity, assigned to different chain reactivities. 4. Stopped flow and flash photolysis under various conditions allows estimates to be made of the association and dissociation rates of CO binding for the two forms of the protein. 5. The functionality of the protein, with regard to CO binding, can be described by a modified two state model.

Allosteric Regulation↗
PubMed1982

Limitations of the sodium fusion assay for fluorinated metabolites.

A method involving sodium fusion and analysis by fluoride electrode, which has been used for the determination of total fluorinated metabolites of fluorine-containing drugs in physiologic fluids, is shown to be inapplicable to volatile fluorinated metabolites of volatile fluorinated anesthetic agents. The yields of inorganic fluoride from the volatile metabolites 2,2,2-trifluoroethanol and trifluoroacetaldehyde were less than 2% of expected values, whether these metabolites were present in water, buffer, urine, or hepatic microsomes, although reliable results were obtained for the relatively nonvolatile sodium trifluoroacetate. It is proposed that the assay does not provide a valid method for determining total fluorinated metabolites of volatile or nonvolatile fluorine-containing drugs, but may be of some use in determining a single nonvolatile fluorinated metabolite of a volatile fluorinated drug.

Anesthetics↗
PubMed1981

Studies on ligand binding to sulphaemoglobin.

Equilibrium studies show that, at low protein concentrations, sulphaemoglobin, in the ferrous form, binds carbon monoxide at pH 6.0 in a non co-operative manner with a Hill coefficient of 1.15 and an affinity constant of 8 . 10(-7) M. At both pH 6.0 and pH 9.0 the kinetics of CO binding show the presence of a simple mono-exponential process with a second-order rate constant of 8 . 10(3) M-1 . s-1. The rate of dissociation of CO from sulphaemoglobin is approx. 0.01 s-1. The activation energy of the binding process is calculated as 40 kJ . mol-1. A comparison is presented between the CO binding properties of sulphaemoglobin, myoglobin and haemoglobin and a mechanism whereby the CO binding parameters of sulphaemoglobin are modified is proposed.

Carbon Monoxide↗
PubMed1981

The 20,000 molecular weight variant of human growth hormone. Preparation and some physical and chemical properties.

Extracts of frozen human pituitary glands contain, in addition to normal growth hormone (hGH (Mr = 22,005), a variant of lower molecular weight (approximately 20,000) (Singh, R. N. P., Seavey, B. K. and Lewis, U. J. (1974) Endocrinol. Res. Commun. 1, 449-464). This material, designated 20K hGH, occurs in various forms with the majority present as a heterologous dimer of Mr = 20,000 hGH and hGH. The purification scheme for obtaining the variant from these fractions is described. Analysis of Mr = 20,000 hGH in the sequenator has confirmed that residues 32-46 of hGH are absent in Mr = 20,000 hGH. The structures of hGH and Mr = 20,000 hGH have been compared by 1H nuclear magnetic resonance spectroscopy, circular dichroism, spectrophotometric titration, and nitration of the tyrosine residues by tetranitromethane. The results indicate that the folding of the polypeptide chain of the two proteins is similar but not identical.

Amino Acid Sequence↗
PubMed1981

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity.

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (k1 = 200 M-1 an K2 = 37 M-1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the alpha chains and the low affinity sites with the beta chains. Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k1 = 85 M-1 sec-1 k2 = 25 M-1 sec-1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the beta chains and the slow phase to the alpha chains. The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5-9.5 within which two ionization processes are discernable at pH 6.9 an 7.5.

Adult↗
PubMed1980

A stopped-flow study of the cupric ion oxidation of adult-human haemoglobin.

The addition of excess Cu2+ to adult human haemoglobin leads to the production of alpha 2(2+) beta 2(3+), in both the oxy and deoxy forms of the protein. Stopped-flow studies of the oxidation process yields apparent second-order rate constants of 196M-1 X S-1 and 41M-1 X S-1 for the deoxy and oxy forms respectively. The rate of the deoxy-form oxidation is linearly dependent on [Cu2+], whereas that of the oxy form is rate-limited above 2 mM to 0.11 S-1. Arrhenius activation energies of the two processes are almost identical at 91 kJ X mol-1, as are the activation enthalpies of 89 kJ X mol-1. The activation entropies show small differences, being 31 entropy units and 48 entropy units for the oxy and deoxy forms respectively. ATP and glycerate 2,3-bisphosphate at saturating concentrations do not affect the rate of oxidation of the oxy form, but halve the rate found for the deoxy form. These data are discussed in terms of the previously proposed mechanism of oxidation in which slow Cu2+ binding is followed by rapid electron transfer.

Adult↗