Biomedical subjects

S J Gill

Publications and source records attributed to S J Gill.

At least 19 recordsLinked to original sources

PubMed1992

Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.

Animals↗

PubMed1992

Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin.

The inability to localize and measure the free energy of protein structure and structure change severely limits protein structure-function investigations. The local unfolding model for protein hydrogen exchange quantitatively related the free energy of local structural stability with the hydrogen exchange rate of concerted sets of structurally related protons. In tests with a number of modified hemoglobin forms, the loss in structural free energy obtained locally from hydrogen exchange results matches the loss in allosteric free energy measured globally by oxygen-binding and subunit dissociation experiments.

Allosteric Regulation↗

PubMed1992

Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.

Animals↗

PubMed1991

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.

Cytochrome c Group↗

PubMed1991

A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris.

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.

Animals↗

PubMed1990

Nonlinear optical effects in oxygen-binding reactions of hemoglobin A0.

Optical spectra have been taken in the Soret band (440-400 nm) under different oxygen partial pressures for hemoglobin (Hb) A0 at pH 7.0, 15 degrees C, 2-3 mM heme, 30 mM inositol hexaphosphate, 0.1 Hepes and 0.1 M NaCl. Application of the matrix method of singular value decomposition (SVD) to the difference spectra for different oxygen pressures shows the presence of at least two distinct optical transitions. From this result one concludes that the optical response to oxygen binding is nonlinear in the Soret band. The degree of nonlinearity has been determined by fitting the data at different wavelengths to the four-step reaction Adair equation with the inclusion of optical parameters that describe the intermediate oxygenated species. It is found that the data are well-represented by two optical parameters at each wavelengths, one which represents the optical change for the addition of the first and second oxygen molecules and the other which corresponds to the change for the addition of the third and fourth oxygen molecules. The ratio of these optical parameters depends only moderately upon wavelength with an average value of 0.8 over the Soret band. Thus, there is an approx. 20% smaller optical response for the first two ligated species than that for the last two ligated species. The overall Adair equilibrium constants are evaluated as follows: beta 1 = 0.081 +/- 0.003 Torr-1, beta 2 = 2.53 x 10(-3) +/- 2.4 x 10(-4) Torr-2, beta 3 = 1.25 x 10(-5) +/- 1.0 x 10(-6) Torr-3, beta 4 = 1.77 x 10(-6) +/- 1.5 x 10(-7) Torr-4.

Heme↗

PubMed1990

Common features of protein unfolding and dissolution of hydrophobic compounds.

Protein unfolding and the dissolution of hydrophobic compounds (including solids, liquids, and gases) in water are characterized by a linear relation between entropy change and heat capacity change. The same slope is found for various classes of compounds, whereas the intercept depends on the particular class. The feature common to these processes is exposure of hydrophobic groups to water. These observations make possible the assignment of the heat capacity change to hydrophobic solvation and lead to the description of protein stability in terms of a hydrophobic and a nonhydrophobic contribution. A general representation of protein stability is given by the heat capacity change and the temperature.

Chemical Phenomena↗

PubMed1989

Stabilization of the T-state of hemoglobin.

The effect of inositol hexaphosphate and bezafibrate on binding of O2 and CO to HbAO at high concentrations (1 mM) has been evaluated using thin layer optical techniques. Data analysis shows 1) the occurrence of greatly reduced ligand dependent cooperativity (Hill slope of 2.23 for CO and 1.51 for O2), and 2) the presence of significant triply ligated species. The data fits a nested allosteric two-state MWC model in which the T state consists of two allosteric substrates, Tt and Tr, where Tt binds only to the alpha chains and Tr binds to both alpha and beta chains. The model indicates that the triply ligated species consists of a predominant amount of T form, agreeing with kinetic observations of CO ligated hemoglobin. The maximum amount of triply ligated R molecules (CO or O2) implicated is less than 1%, a result similar to that found previously for binding studies made in the absence of BZF and IHP.

Bezafibrate↗

PubMed1989

Information theory and the analysis of ligand-binding data.

The phenomenological principles of information theory are used in the analysis of ligand-binding phenomena in biological macromolecules. Information maps are constructed to visualize regions of ligand chemical potential with maximum amount of information and to devise suitable experimental strategies therefrom. Extensive simulation studies and analysis of experimental data also point out the properties of information used as a weighting procedure in nonlinear least-squares analyses.

Carbon Monoxide↗

PubMed1989

Binding of oxygen and carbon monoxide to the hemocyanin from the spiny lobster.

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O2 ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechanism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning "nested" allosteric interactions.

Animals↗

PubMed1989

Temperature- and pH-dependence of the oxygen-binding reaction of human fetal haemoglobin.

O2 binding to human haemoglobin F0 was studied at high haem concentrations (3 mM) in the temperature range 15-35 degrees C and in the pH range 6.8-8.7 at 25 degrees C. Comparison with O2 binding to human adult haemoglobin A0 under identical solution conditions reveals striking similarities in the Bohr effect and the enthalpy of oxygenation between the two haemoglobins.

Fetal Hemoglobin↗

PubMed1989

Carbon monoxide and oxygen binding to human hemoglobin F0.

Differential binding curve measurements of carbon monoxide and oxygen binding to human hemoglobin F0 under near-physiological conditions (0.1 M NaCl and 15 mM 2,3-diphosphoglyceric acid, pH 7.35, and 37 degrees C) have allowed a detailed description of the binding and linkage between these two gaseous ligands. Comparison with human hemoglobin A0 under identical solution conditions shows that fetal hemoglobin F0 binds oxygen and carbon monoxide with higher affinity than human hemoglobin A0, but with the same cooperativity. Construction of the partition coefficient surface for carbon monoxide and oxygen binding reveals a failure of Haldane's laws for both hemoglobins. Linkage graphs are used to explore the phenomenological properties of the system. The graphs provide a quantitative description of the mechanism of carbon monoxide toxicity on oxygen transport by hemoglobin in vivo and demonstrate striking similarities between the functional properties of fetal and adult hemoglobins.

Carbon Monoxide↗

PubMed1989

Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin.

Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of Octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were not indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously [Miller, K. (1985) Biochemistry 24, 4582-4586]. The analysis shows two types of binding sites in the ratio of 4:3.

Animals↗

PubMed1989

Allosteric formulation of thermal transitions in macromolecules, including effects of ligand binding and oligomerization.

We examine the effects of concentration (aggregation), buffers, and ligation, under conditions of either constant ligand activity or limited total amount of ligand, upon thermal denaturation of macromolecules as measured by scanning calorimetry. In doing so we utilize and extend an earlier generalized allosteric treatment [S. J. Gill, B. Richey, G. Bishop, and J. Wyman (1985) Biophys. Chem. 21, 1-14], applicable to ligand binding, enthalpy changes, and volume changes in a macromolecular system. The approach is contrasted with formulations based on the idea of structural domains. We show how information from the full scanning calorimetric curves can be utilized in arriving at and testing appropriate models for observed behavior in selected examples.

Allosteric Regulation↗

PubMed1988

Quantitative analysis of linkage in macromolecules when one ligand is present in limited total quantity.

We present a general framework for analysis of two closely related problems in biochemical studies: (1) The first is analysis of binding data obtained under conditions in which a second, linked ligand is present in limited total quantity. In such conditions the free activity of the second ligand varies throughout the primary ligand binding curve, and the resultant behavior can be quite complex. Analysis of such curves enables one to quantitatively extract detailed information regarding the linkage of the two ligands at intermediate stages of ligation. The treatment is applied in an accompanying paper to oxygen binding in human hemoglobin in the presence of organic phosphates [Robert, C.H., Fall, L., & Gill, S. J. (1988) Biochemistry (following paper in this issue)]. (2) The second treatment we outline regards the analogous problem of analyzing differential scanning calorimetry (DSC) data obtained for a macromolecule binding a ligand present in limited quantity. A simple model is presented that accounts for dual transitions like those already seen in DSC data for human serum albumin in the presence of nonsaturating amounts of fatty acids [Ross, P., & Shrake, A. (1987) Abstracts of the 42nd Calorimetry Conference, University of Colorado, Boulder, CO].

Binding Sites↗

PubMed1988

Linkage of organic phosphates to oxygen binding in human hemoglobin at high concentrations.

We have performed high-precision oxygen binding studies on human hemoglobin tetramers in the presence of a series of limited, subsaturating amounts of the effector compounds 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP). The use of thin-layer optical methods enabled the use of high hemoglobin concentrations, preventing complications arising from the dissociation of the tetramer into dimers. Model-independent, simultaneous analysis of all data for each effector demonstrated that the intrinsic oxygen binding characteristics of the molecule are in agreement with those determined in earlier high-precision studies [e.g., Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002] and that the affinity of the tetramer for the tightly binding effector IHP changes most markedly between the second and fourth oxygen binding steps, perhaps indicating a large conformational change. The data were then analyzed by using the truncated allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which is based on the hypothesis that a quaternary conformational change occurs in the hemoglobin tetramer before the third and fourth oxygen molecules bind.

2,3-Diphosphoglycerate↗

PubMed1988

Nested allosteric interaction in tarantula hemocyanin revealed through the binding of oxygen and carbon monoxide.

We have examined the competitive binding of oxygen and carbon monoxide to the multisubunit hemocyanin of the tarantula Eurypelma californicum. Employment of high-precision thin-layer methods has enabled detailed characterization of the pure oxygen and pure carbon monoxide binding curves, as well as binding curves performed under mixed-gas conditions. The pure oxygen binding curve and the displacement of oxygen by carbon monoxide at full ligand saturation are highly cooperative, but in the absence of oxygen, carbon monoxide binds noncooperatively. The results were analyzed globally within the framework of a nested allosteric model [Robert, C.H., Decker, H., Richey, B., Gill, S.J., & Wyman, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1891-1895] which takes into account the hierarchy of subunit structure present in the macromolecule. The use of two ligands enables one to recognize two distinct levels of allosteric interaction functioning in the protein assembly. The binding characteristics of the allosteric states demonstrated for Eurypelma follow a similar pattern as those found earlier for Homarus americanus.

Allosteric Site↗

PubMed1988

Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins.

Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity. The opposite effect, inhibition of ice formation, has been described for a group of glycoproteins found in different fish and insect species. This group of substances, termed antifreeze glycoproteins (AFGPs), promotes the supercooling of water with no appreciable effect on the equilibrium freezing point or melting temperature. Substantial evidence now indicates that AFGPs act by binding to a growing ice crystal and slowing crystal growth. As the ice-nucleating protein surface is believed to have a structure similar to an embryonic ice crystal, AFGPs might be predicted to interact directly with a bacterial ice-nucleating site. We report here that AFGPs from the antarctic fish Dissostichus mawsoni inhibit the ice-nucleating activity of membrane vesicles from the bacterium Erwinia herbicola. The inhibition effect shows saturation at high concentration of AFGP and conforms to a simple binding reaction between the AFGP and the nucleation centre.

Animals↗