An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

Conformational consequences of the incorporation of arabinofuranosylcytidine in DNA. An NMR study of the DNA fragments d(CGCTAGCG) and d(CGaCTAGCG) in solution

The self-complementary octamers d(CGCTAGCG) and d(CGaCTAGCG) (aC, arabinofuranosylcytidine) were studied by means of NMR spectroscopy. It is shown that d(CGaCTAGCG), under suitable conditions of oligonucleotide concentration, ionic strength and temperature, exclusively adopts a hairpin structure. However, under the same experimental conditions (5 mM DNA, no added salt, 295 K) d(CGCTAGCG) mainly adopts a B-DNA-type duplex. At lower temperatures (less than or equal to 290 K) the hairpin form of d(CGaCTAGCG) occurs in slow exchange with an intact B-DNA-type duplex. When the DNA concentration of d(CGCTAGCG) is dramatically reduced (less than or equal to 0.5 mM) the hairpin form becomes highly favoured at the expense of the dimer. Moreover, proton-chemical-shift considerations indicate that the structural features of the hairpin structure of d(CGCTAGCG) mimic, in part, those of the modified octamer d(CGaCTAGCG), i.e. a loop comprising only the two central residues with the thymine located into the minor groove (Pieters, J. M. L., de Vroom, E., van der Marel, G. A., van Boom, J. H., Koning, T. M. G., Kaptein, R. and Altona, C. unpublished results). Thermodynamic analysis of d(CGCTAGCG) yields an average Tmd value of 342 K (1 M DNA) and a delta Hod value of -266 kJ/mol for the dimer/coil transition and an average Tmh value of 321 K and delta Hoh - 102 kJ/mol for the hairpin/coil equilibrium. For the duplex/coil equilibrium of d(CGaCTAGCG) an average Tmd value of 336 K (1 M DNA) and delta Hod value of -253 kJ/mol are deduced. The hairpin/coil transition of d(CGaCTAGCG) is characterized by a delta Hoh value of -104 kJ/mol and an average Tmh value of 331 K. It is concluded that incorporation of an arabinofuranosylcytidine in the octamer d(CGaCTAGCG) results in stabilization of the hairpin form, whereas the dimer is destablized by two aC.dG base pairs.     

Thermodynamics of the binding of Streptomyces subtilisin inhibitor to alpha-chymotrypsin

The binding of Streptomyces subtilisin inhibitor (SSI) to alpha-chymotrypsin (CT) (EC 3.4.21.1) was studied by isothermal and differential scanning calorimetry at pH 7.0. Thermodynamic quantities for the binding of SSI to the enzyme were derived as functions of temperature from binding constants (S. Matsumori, B. Tonomura, and K. Hiromi, private communication) and isothermal calorimetric experiments at 5-30 degrees C. At 25 degrees C, the values are delta G degrees b = -29.9 kJ mol-1, delta Hb = +18.7 (+/- 1.3) kJ mol-1, delta S degrees b = +0.16 kJ K-1 mol-1, and delta C p,b = -1.08 (+/- 0.11) kJ mol-1. The binding of SSI to CT is weak compared with its binding to subtilisin [Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202; Takahashi, K., & Fukada, H. (1985) Biochemistry 24, 297-300]. This difference is due primarily to a less favorable enthalpy change in the formation of the complex with CT. The hydrophobic effect is presumably the major source of the entropy and heat capacity changes which accompany the binding process. The unfolding temperature of the complex is about 7 degrees C higher than that of the free enzyme. The enthalpy and the heat capacity changes for the unfolding of CT were found to be 814 kJ mol-1 and 17.3 kJ K-1 mol-1 at 49 degrees C. The same quantities for the unfolding of the SSI-CT complex are 1183 kJ mol-1 and 39.2 kJ K-1 mol-1 at 57 degrees C.     

Calorimetric studies of the binding of Streptomyces subtilisin inhibitor to subtilisin of Bacillus subtilis strain N'

The binding of Streptomyces subtilisin inhibitor (SSI) to subtilisin of Bacillus subtilis strain N' (subtilisin BPN', EC 3.4.21.14) was studied by isothermal calorimetry at pH 7.0 and at various temperatures ranging from 5 to 30 degrees C. Thermodynamic quantities for the binding reaction were derived as a function of temperature by combining the data reported for the dissociation constant with the present calorimetric results. At 25 degrees C, the values are delta G degrees = -57.9 kJ mol-1, delta H = -19.8 kJ mol-1, delta S degree = 0.13 kJ K-1 mol-1, and delta Cp = -1.02 kJ K-1 mol-1. The entropy and the heat capacity changes are discussed in terms of the contributions from the changes in vibrational modes and in hydrophobic interactions.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Parallel-stranded duplex DNA containing blocks of trans purine-purine and purine-pyrimidine base pairs.

A 30 base pair parallel-stranded (ps) duplex ps-L1.L2 composed of two adjoined purine-purine and purine-pyrimidine sequence blocks has been characterized thermodynamically and spectroscopically. The 5'-terminal 15 residues in both strands ('left-half') consisted of the alternating d(GA)7G sequence that forms a ps homoduplex secondary structure stabilized by d(G.G) and d(A.A) base pairs. The 3'-terminal 15 positions of the sequence ('right-half') were combinations of A and T with complementary reverse Watson-Crick d(A.T) base pairing between the two strands. The characteristics of the full length duplex were compared to those of the constituent left and right halves in order to determine the compatibility of the two ps helical forms. The thermal denaturation curves and hyperchromicity profiles of all three duplexes determined by UV absorption spectroscopy were characteristic of ps-DNA, in accordance with previous studies. The thermodynamic properties of the 30 bp duplex corresponded within experimental error to the linear combination of the two 15-mers. Thus, the Tm and delta HvH of ps-L1.L2 in 10 mM MgCl2, derived from analyses according to a statistical mechanical formulation for the helix-coil transition, were 43 degrees C and 569 kJ mol-1, compared to 21 degrees C, 315 kJ mol-1 (ps-F5.F6) and 22 degrees C, 236 kJ mol-1 (ps-GA15). The UV absorption and CD spectra of ps-L1.L2 and the individual 15-mer ps motifs were also compared quantitatively. The sums of the two constituent native spectra (left+right halves) accurately matched that of the 30 bp duplex, with only small deviations in the 195-215 nm (CD) and 220-240 nm (absorption) regions. Based on analysis by native gel electrophoresis, the sequences studied formed duplex structures exclusively; there were no indications of higher order species. Chemical modification with diethyl pyrocarbonate showed no hyperreactivity of the junctional bases, indicating a smooth transition between the two parallel-stranded conformations. We conclude that under given salt conditions, oligonucleotides with normal primary chemical structures can readily form a parallel-stranded double helix based on blocks of very disparate non-canonical purine-purine and purine-pyrimidine base pairs and without perceptible destabilization at the junction. There are biological implications of these findings in relation to genetic structure and expression.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin.

Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.     

Thermodynamics of the Ca2+ binding to bovine alpha-lactalbumin

Bovine alpha-lactalbumin contains one strong Ca2+-binding site. The free energy (delta G0), enthalpy (delta H0), and entropy (delta S0) of binding of Ca2+ to this site have been calculated from microcalorimetric experiments. The enthalpy of binding was dependent on the metal-free bovine alpha-lactalbumin concentration. At 0.8 mg ml-1, metal-free bovine alpha-lactalbumin delta H0 was -110 +/- 6 kJ mol-1. At this concentration the binding constant was estimated from a mathematical analysis of the titration curves to be greater than 10(7) M-1. This means that delta G0 is smaller than -40 kJ mol-1 and delta S0 is less negative than -235 J.K-1 mol-1. The binding of Ca2+ is therefore enthalpy-driven. From binding experiments as a function of temperature, a delta Cp value of -4.1 kJ.K-1 mol-1 was calculated. This value is dependent on the protein concentration. A tentative explanation for this large value is given.     

Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies.

Dodecameric glutamine synthetase (GS) from Escherichia coli undergoes reversible, thermally induced partial unfolding without subunit dissociation. A single endotherm for Mn.GS (+/- active-site ligands) in the presence of 1 mM free Mn2+ and 100 mM KCl at pH 7 is observed by differential scanning calorimetry (DSC). Previous deconvolutions of DSC data for Mn.GS showed only two two-state transitions (with similar tm values; 51.6 +/- 2 degrees C), and indicated that cooperative interactions link partial unfolding reactions of all subunits within the Mn.enzyme dodecamer [Ginsburg, A., & Zolkiewski, M. (1991) Biochemistry 30, 9421]. A net uptake of 8.0 equiv of H+ by Mn.GS occurs during partial unfolding, as determined in the present DSC experiments conducted with four buffers having different heats of protonation at 50 degrees C. These data gave a value of 176 +/- 12 kcal (mol of dodecamer)-1 for delta Hcal corrected for buffer protonation. L-Glutamine and L-Met-(SR)-sulfoximine stabilize the Mn.GS dodecamer through the free energies of ligand binding, and these were shown to be partially and totally released, respectively, from the 12 active sites at high temperature. Ligand effects on Tm values from DSC were similar to those from spectral measurements of Trp and Tyr exposures in two subunit domains. Effects of varying [ADP] on DSC profiles of Mn.GS were complex; Tm is increased by low [ADP] and decreased by > 100 microM free ADP. This is due to the exposure of an additional low-affinity ADP binding site per GS subunit at high temperature with log K1' = 4.3 and log K2' = 3.6 at 60 degrees C relative to log K' = 5.5 for ADP binding at 30 degrees C, as determined by isothermal calorimetric and fluorescence titrations. Moreover, delta Hcal at > 27% saturation with ADP (corrected for ADP binding/dissociation) is approximately 80-100 kcal/mol more than in the absence of ligands. Changes in domain interactions could result from ADP bridging subunit contacts in the dodecamer. Each of the active-site ligands investigated here produces different effects on DSC profiles without uncoupling the extremely cooperative, partial unfolding reactions in the Mn.GS dodecamer.     

Temperature effects on the MgATP-induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii.

The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotobacter vinelandii has been investigated between 6 degrees C and 31 degrees C by stopped-flow spectrophotometry. Below 14 degrees C, the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14 degrees C) from about 1 s-1 to about 26 s-1. The rate constant for the reverse reaction was found to be approximately 4 s-1 and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6 degrees C and 12 degrees C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters delta H(0) ++ = 289 kJ.mol-1 and delta S(0) ++ = 792 J.K-1.mol-1. The Eyring plot of the stopped-flow data displays an inflection point around 14 degrees C. From the stopped-flow data obtained between 18 degrees and 27 degrees C the activation parameters delta H(0) ++ and delta S(0) ++ for the reduction of the MoFe protein by Fe protein are calculated to be 90 kJ.mol-1 and 99 J.K-1.mol-1 respectively. A second inflection point in the Eyring plot could exist around 28 degrees C.     

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.     

Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme

The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine alpha-lactalbumin.     

Thermodynamics of hydrolysis of sugar phosphates

Thermodynamics of the enzyme-catalyzed (alkaline phosphatase, EC 3.1.3.1) hydrolysis of glucose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate, and ribulose 5-phosphate have been investigated using microcalorimetry and, for the hydrolysis of fructose 6-phosphate, chemical equilibrium measurements. Results of these measurements for the processes sugar phosphate2- (aqueous) + H2O (liquid) = sugar (aqueous) + HPO2++-(4) (aqueous) at 25 degrees C follow: delta Ho = 0.91 +/- 0.35 kJ.mol-1 and delta Cop = -48 +/- 18 J.mol-1.K-1 for glucose 6-phosphate; delta Ho = 1.40 +/- 0.31 kJ.mol-1 and delta Cop = -46 +/- 11 J.mol-1.dK-1 for mannose 6-phosphate; delta Go = -13.70 +/- 0.28 kJ.mol-1, delta Ho = -7.61 +/- 0.68 kJ.mol-1, and delta Cop = -28 +/- 42 J.mol-1.K-1 for fructose 6-phosphate; delta Ho = -5.69 +/- 0.52 kJ.mol-1 and delta Cop = -63 +/- 37 J.mol-1.K-1 for ribose 5-phosphate; and delta Ho = -12.43 +/- 0.45 kJ.mol-1 and delta Cop = -84 +/- 30 J.mol-1.K-1 for the hydrolysis of ribulose 5-phosphate. The standard state is the hypothetical ideal solution of unit molality. Estimates are made for the equilibrium constants for the hydrolysis of ribose and ribulose 5-phosphates. The effects of pH, magnesium ion concentration, and ionic strength on the thermodynamics of these reactions are considered.     

Reaction heats and heat capacity changes for intermediate steps of the ATP hydrolysis catalyzed by myosin subfragment 1

The interaction of myosin Subfragment 1 with ATP in 0.1 M KCl containing 0.01 M MgCl2 and 0.02 M Tris/HCl (pH 8.0) was studied by microcalorimetry at temperatures of 4, 12, and 23 degrees C so that values of the heat capacity change (delta Cp) could be obtained for intermediate steps of the ATPase cycle. The delta Cp values are large compared to the value for the overall cycle, indicating that large changes in the hydrophobic effect are involved in transitions between different intermediate states. However, the heat capacity changes themselves show peculiar temperature dependences. Thus bindings of ATP and ADP to Subfragment 1, both of which are strongly exothermic processes, take place with large negative delta Cp of about -3 kJK-1 mol-1 between 4 and 12 degrees C but with very small delta Cp of 0.3-0.4 kJ K-1 mol-1 between 12 and 23 degrees C. On the contrary, the delta Cp for the endothermic hydrolysis of ATP bound to Subfragment 1 is positive (congruent to kJK-1 mol-1) in the lower temperature range but strongly negative (congruent to -4 kJK-1 mol-1) in the higher temperature range. The magnitude of delta Cp for the slow Pi dissociation process is similar but its sign is just opposite to that for the hydrolysis. These anomalous changes in the heat capacity may be due to the temperature-induced changes in a balance between large opposing effects which result from distinct, local conformation changes within the Subfragment 1 molecule.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

The thermal stability of the tryptic fragment of bovine microsomal cytochrome b5 and a variant containing six additional residues.

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment of bovine microsomal cytochrome b5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (Tm) of approximately 67 degrees C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b5 shows a higher transition temperature of approximately 73 degrees C at pH 7.0 and this is reflected in the values of delta Hm, delta Sm and delta(delta G) of approximately 310kJ.mol-1, 900J.mol-1.K-1 and 5 kJ.mol-1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b5 do not differ significantly in structure with the results suggesting that the reorganisation energy (lambda) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b5 may result from the presence of additional N-terminal residues on the surface of the protein.     

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 1. Thermodynamic analysis.

We have investigated the spin-state equilibrium of adrenal mitochondrial P450scc (cholesterol-side-chain-cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 degrees C to -20 degrees C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are delta HA = 0.7 kJ.mol-1 and delta SA = 5J.K-1.mol-1 at low temperatures and delta HB = -42 kJ.mol-1 and delta SB = -152 J.K-1.mol-1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH. The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 +/- 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.     

An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C-G-m5C-G-A-G-m5C-G) in solution. The B-duplex and hairpin forms

By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.