Delta H0 Research Articles

Calorimetric Study of the Reduction of the Disulfide Bonds in Insulin1

Calorimetric measurement was made on the reduction of the three disulfide bonds of insulin by dithiothreitol (DTT). The reaction was performed at 298 K in three different buffer solutions of pH 9.6. The observed heat changes were corrected for the enthalpy of proton release from the buffer components and the net heat of reaction of insulin with DTT was determined to be delta H0 = 51.5 +/- 1.4 kJ(mol insulin)-1. By subtracting the enthalpy of DTT oxidation (Fukada, H. & Takahashi, K. (1980) J. Biochem. 87, 1105--1110), the standard enthalpy of reduction of insulin was found to be delta H = 78.8 +/- 7.9 kJ(mol insulin)-1. Using the enthalpy change for reduction of random-coil proteins by dithioerythritol (Johnson, R.E., Adams, P., & Rupley, J.A. (1978) Biochemistry 17, 1479-1484), the enthalpy change associated with the conformational change of insulin alone was evaluated to be delta H = 74 +/- 2 kJ(mol insulin)-1 (or 13 J(g protein)-1, a value very similar to that observed for the denaturation of other globular proteins.

Calorimetric Study of the Oxidation of Dithiothreitol

The enthalpy changes of the eoxidation of dithiothreitol (DTT) by ferricyanide was measured calorimetrically in the pH range of 6 to 13 in various buffer solutions at 298 K. By combining the results of potentiometric titrations with the pH dependence of the observed enthalpy changes, the enthalpy changes of DTT oxidation and of the ionization of DTT sulfhydryls were evaluated. The enthalpy changes of oxidation of the non-ionized and the fully ionized DTT were found to be delta HDTT (non-ionized) = 32 +/- 3 kJ mol-1 and delta HDTT (fully ionized) = -38 +/- 3 kJ mol-1, respectively. The enthalpy changes of the first and the second ionizations of DTT sulfhydryls were found to be delta H1 = 48 +/- 2 kJ mol-1 and delta H2 = 21 +/- 3 kJ mol-1, respectively.

The thermodynamics of a negatively interacting allosteric effector system. The glutamate dehydrogenase . NADPH . ADP complexes.

The reaction catalyzed by bovine L-glutamate dehydrogenase is subjected to allosteric activation by ADP. We have measured the thermodynamic parameters (delta G0, delta H0, delta S0, and delta Cp) of the formation of the various possible binary and ternary complexes formed between the enzyme, NADPH, and either ADP or its analogs, adenosine, AMP, and ATP. delta H0 and delta Cp have been measured by flow calorimetry; delta G0 values obtained by calorimetry itself, differences spectroscopy, or gel filtration have been selected on the basis of accuracy under the conditions required for the formation of each complex. The data are interpreted in terms of "interaction parameters," the differences between the thermodynamic parameters of the formation of a ternary complex and the sum of those of the two related binary complexes. Both adnosine and ATP appear to loosen the binding of NADPH by simply preventing a subsite interaction of NADPH. AMP appears to have only minor and probably secondary effects. The negative effect of the binding of ADP on that of NADPH, however, involves the formation of a new interaction, which is exothermic, entropy compensated, has a moderately large negative delta Cp, and does not occur in either binary complex.

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide.

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.

The effects of chemical modification on the refolding transition of alpha-chymotrypsin.

The role of several active site residues of alpha-chymotrypsin in the prototypical refolding transition between active and inactive forms of this enzyme is examined using chemical modification. Oxidation of Met-192 to the sulfoxide results in a derivative which remains entirely in an active state from pH 6 to 9. The derivative becomes inactive only at high pH with pKa = 10.3, delta H0 = 9.5 kcal and delta S0 = -15 eu., indicating the sulfoxide group supplies about 2.1 kcal of active state stabilization relative to the unoxidized methionine side chain. The refolding transition of N-methyl-His-57-alpha-chymotrypsin, in which a nitrogen of the "charge relay" histidine is methylated, displays one ionization process with an apparent pKa of 9.45. The absence of an additional ionization process with a pKa near 7 provides evidence that one of the ionizations in the six state mechanism which describes this transition in alpha-chymotrypsin is linked to the charge relay system. We also demonstrate, using alpha-chymotrypsin, Met-192-sulfoxide-alpha-chymotrypsin and N-methyl-His-57-alpha-chymotrypsin, that the 230 nm circular dichroism band is a quantitative probe of the active-inactive equilibrium, although the chromophore or chromophores responsible for this and another very large negative band at 202 nm have not been identified. Circular dichroism was used to observe the active-inactive equilibrium in methan sulfonyl-alpha-chymotrypsin and phenylmethane sulfonyl-alpha-chymotrypsin. The enhanced stability of the active state of these derivatives relative to alpha-chymotrypsin can be rationalized in terms of steric effects in the substrate side chain binding site.

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction.

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.

Singularities of the dilute Ising ferromagnet

An analysis is undertaken for the case when the concentration p is less than the critical percolation concentration pc. It is found that delta 2M/ delta H2 undergoes a jump discontinuity at H=0. In discussing the critical behaviour of the dilute ferromagnet for p>pc, it is suggested that separate attention should be paid to the contribution of finite clusters, and of the infinite cluster. The former should give rise to a weak set of singularities with critical temperature Tc(1), the latter to standard Ising-type singularities with critical temperature Tc(p). An explanation is thus suggested for the non-analyticity discovered by Griffiths (1969).

Development of an expression for capillary depression of mercury

An equation has been developed for computing the capillary depression of mercury in tubes by fitting an equation to tabulated data. This empirical equation reads, for standard conditions of sea level and 70 degrees F, h=((0.2936r-0.42325) Delta h2+0.225r3- 0.325r2+0.009r+0.1306) Delta h/( Delta h2+r2) where h is the capillary depression, r the radius of the tube and Delta h is the height of the meniscus, all in centimetres. Capillary depressions can be computed by this equation within about 1 mu m.

Phase equilibria and kinetics of evolution of dilute solutions of hydrogen in niobium

Ultrahigh vacuum techniques have been used to measure the equilibrium pressure of hydrogen dissolved in niobium in dilute solutions over the pressure, concentration and temperature ranges, pe=10-7-10-2 Torr, H/Nb=0-0.035, and T=351-487 K. Sievert's law which indicates ideal solution behaviour was observed for hydrogen concentrations below about H/Nb=0.023; the solubility data are described by the expression square root pe=c(395.4+or-33.8)exp(-(4.345+or-0.161)*103/T) where c=H/Nb*100. The standard partial molar entropy and enthalpy of solution are Delta s0=49.6+or-2.3 J K-1 g-atom-1 and Delta h0=-36.12+or-1.34 kJ g-atom-1. The value of Delta s0 is compared with theoretical entropies calculated from configurational and vibrational contributions. The rate of evolution of hydrogen from a niobium filament has been studied over the pressure and temperature ranges 10-7-10-3 Torr and 465-553 K. The rate limiting process is identified as the recombination of hydrogen atoms on the metal surface rather than diffusion within the bulk provided the overall concentration is less than H/Nb approximately=0.02. The activation energy for the process is 67.56+or-1.26 kJ mol-1.