A novel conformational change of the anticodon region of tRNAPhe (yeast).

The temperature dependence of the fluorescence of the Y-base of tRNAPhe (yeast) was investigated kinetically by the temperature jump method. In the range between -15 degrees C and +30 degrees C A NOVEL CONFORMATIONAL TRANSITION OF THE TRNA could be characterized. This conformational change was found in the absence of any artificial label; it is a characteristic property of tRNAPhe in its native structure. This transition accounts for 30% of the total fluorescence change. Its activation enthalpy is 16 kcal/mole (67 kJ/mole), and the transition enthalpy is between -2 kcal/mole and +2 kcal/mole (+/-8 kJ/mole). A model is represented in which this transition can be explained by a a change in the stacking pattern of the anticodon loop. The experimental findings are discussed with respect to several hypotheses about the molecular mechanism of protein biosynthesis which postulate conformational rearrangements of the anticodon loop.     

Stoichiometry determination for carbon monoxide binding to Rhodospirillum molischianum cytochrome c'

The stoichiometry of CO ligation to the dimer heme protein Rhodospirillum molischianum cytochrome c' is determined. We have recently measured the enthalpy change of CO ligation to this molecule by the van't Hoff method and found the value of -10.7 +/- 1.2 kcal/mol CO (aqueous) (Doyle, M. L., Weber, P. C., and Gill, S. J. (1985) Biochemistry 24, 1987-1991). In the present paper the enthalpy change of CO ligation, measured directly by titration calorimetry, is found to be -9.5 +/- 0.2 kcal/mol heme. Since the van't Hoff method gives the heat value in units/mole of CO and the calorimetric method gives the heat value in units/mole of heme, the stoichiometry of the reaction is given by the ratio of the two values and found to be 0.9 +/- 0.1, or within experimental error, one CO molecule bound per heme.     

Denaturation of phycocyanin by urea and determination of the enthalpy of denaturation by microcalorimetry.

Denaturation of the protein phycocyanin in urea solution was investigated by microcalorimetry, ultraviolet and visible spectroscopy, circular dichroism and sedimentation equilibrium. The results consistently demonstrated that in the presence of 7 M urea this protein is completely denatured. By assumings a two-state mechanism, an apparent free energy of unfolding at zero denaturant concentration, (formula: see text) was found to be 4.4 kcal/mole at pH 6.0 and 25 degrees C. By microcalorimetry the enthalpy of denaturation of phycocyanin app was found to be -230 kcal/mole at 25 degrees C. The relatively large negative enthalpy change results from protein unfolding and changes in protein solvation.     

Heat of denaturation of lysozyme

The enthalpy of denaturation of lysozyme was determined by measuring the heat, capacity of an aqueous solution of this protein in the vicinity of the transition temperature, 46 °C at pH 1. Within experimental error the calorimetric, heat (56 ± 8 kcal/mole) was found to agree with the van't Hoff transition enthalpy (63 ± 6 kcal/mole) determined from optical rotation measurements as a function of temperature. This indicates that denaturation of this protein can be interpreted in terms of a two-state model. Successive measurements of the same sample showed, from several lines of evidence, that the transition was about 80% reversible for the particular environmental conditions and thermal history involved in the study.     

Energetic studies of lactose active transport in Escherichia coli membrane vesicles

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.     

A calorimetric study of the interaction of ATP with rabbit muscle phosphofructokinase.

The heat of interaction of ATP with phosphofructokinase from rabbit muscle was determined at 25 degrees C in 0.1 M potassium phosphate, pH 7.0 and 8.0. The limiting value of the enthalpy change at high ATP concentrations was found to be -11.5 kcal mol of enzyme polypeptide chains. Since phosphate and imidazole have very different heats of ionization (+0.8 and +7.5 kcal/mol, respectively), this suggests that the binding of at least two protons to the enzyme occurs concomitantly with the binding of ATP at the regulatory site.     

Calorimetric studies of oxyhemoglobin dissociation. I. Reaction of sodium dithionite with oxygen

Calorimetric studies of the reduction of free oxygen in solution by sodium dithionite are in agreement with a stoichiometry of 2 moles Na₂S₂O₄ per mole of oxygen. The reaction is biphasic with ΔH_t - 118±7 kcal mol^?1 (?494 ± 29 kJ mol^?1). The initial phase of the reaction proceeds with an enthalpy change of ca ?20 kcal (?84 kJ) and occurs when 0.5 moles of dithionite have been added per mole dioxygen present. This could be interpreted as the enthalpy change for the addition of a single electron to form the superoxide anion. Further reduction of the oxygen to water by one or more additional steps is accompanied by an enthalpy change of ca ?100 kcal (?418. 5 kJ). Neither of these reductive phases is consistent with the formation of hydrogen peroxide as an intermediate. The reduction of hydrogen peroxide by dithionite in 0.1 M phosphate buffer, pH 7.15, is a much slower process and with an enthalpy change of ca ? 74 kcal mol^?1 (?314 kJ mol^?1). Dissociation of oxyhemoglobin induced by the reduction of free oxygen tension with dithionite also shows a stoichiometry of 2 moles dithionite per mole oxygen present and an enthalpy change of ca. ?101 ±9 kcal mol^?1 (?423± 38 kJ mol^?1). The difference in the observed enthalpies (reduction of dioxygen vs. oxyhemoglobin) has been attributed to the dissociation of oxyhemoglobin, which is 17 kcal mol^?1 (71 kJ mol^?1).     

Further studies on the temperature dependence of the binding of testosterone to human pregnancy plasma proteins

The binding of testosterone by pregnancy plasma proteins has been studied by a new equilibrium dialysis system. The temperature dependence on the association constant has been investigated and the enthalpy change ΔH and entropy change ΔS have been calculated.By a computer optimization program, the binding constant of the high affinity testosterone binding protein has been estimated from Scatchard plots. The binding reactions were carried out at 5°, 25° and 37° C. The corresponding values were 3.1.10 1.2.10⁹ and 7.2.10⁸ liter/mole. The resulting enthalpy and entropy changes were ?2.0 kcal/mole and 35.0 cal/(mole.degree) respectively.It can be concluded that the binding of testosterone to the specific binding protein is an exothermic reaction and is stabilized by hydrophobic binding forces.     

The thermodynamics of hapten and antigen binding by rabbit anti-dinitrophenyl antibody.

Rabbit anti-dinitrophenyl antibody from a serum pool was obtained as five fractions of purified specific antibody by a limiting antigen precipitation method. Each fraction had a different binding affinity for epsilon-N-2,4-dinitrophenyl-L-lysine. The free energy changes for hapten binding to the five antibody fractions varied from -8.35 to -10.0 kcal/mol. An average deltaH of -13.9 kcal/mol was measured for the fractions with a batch calorimeter. The results indicate no significant correlation between enthalpy changes and free energy changes. However, a statistically significant correlation between the free energy changes and the entropy changes was found. The enthalpy of the anti-dinitrophenyl antibody interaction with multivalent dinitrophenyl human serum albumin was determined. These are the first enthalpy measurements of an antibody antigen reaction in which the intrinsic binding enthalpy between the antibody and the determinant group is known. The deltaH for the antigen binding reaction was -10.1 kcal/mol which is 3.8 kcal/mol less exothermic than the deltaH for the hapten binding reaction. The interactions that could lead to such a difference in enthalpy are discussed.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

Cooperative and thermodynamic parameters for oligoinosinate-polycytidylate complexes

The cooperative nature of the binding between polycytidylate and the oligoinosinates I(pI)_5–10 has been determined. Using the data of Tazawa, Tazawa, and Ts'o, it is shown that knowledge of the slope of the adsorption isothern allows one to determine the oligomer-polymer binidng constant, the oligomer–oligomer interaction constant, and the average degree of association (cooperative clustering) of the oligomers on the polymer. Knowledge of the above equilibrium constants as a function of temperature yields the respective thermodynamic parameters; no assumptions need to be made about the nature of the equilibrium constants or the thermodynamic parameters. For very long chains of polycytidylate, simple, explicit relations are given for the determination of the equilibrium constants involved. For finite chains of polycytidylate, the calculation of a single graph for each oligomer and polymer size allows the equilibrium constants to be determined for all experimental conditions of temperature and concentration. We find that the enthalpy and entropy of binding an oligomer n, bases to be δH_n = ±13.7 ? n(6.65) and δS_n = +32.5 ? n(18.8) given, respectively, in kcal/mole and e.u.; these parameters predict a melting temperature of 81°C for the poly(I)·poly(C) complex compared with the experimental value of 75°C. If the enthalpy is interpreted as arising from a sum of hydrogen bonding and stacking interactions, then the enthalpy of stacking is ?13.7 kcal/mole while the enthalpy of hydrogen bonding is +7 ± 4 kcal/mole; the positive enthalpy of hydrogen bonding presumably is a result of the fact that in the inosine-cytosine base pair, only two of the three sites on cytosine can hydrogen bond, the third being blocked from hydrogen bonding with water. The enthalpy of interaction between neighboring bound oligomers is found to be ?10.4 kcal/mole while the corresponding entropy is ?26.1 e.u. The binding is bound to be cooperative, though the extent of clustering varies markedly with temperature; the average number of oligomers in a cluster on the polymer is found to about five at a melting temperature of 25°C. The approach and equations given have generally applicability to oligomer-polymer associations.     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.     

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.     

Thermotropic behavior of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles exerts a profound effect upon the DPPC gel to liquid-crystalline phase transition. The glycoprotein was reconstituted into DPPC vesicles by octyl glucoside dialysis. The gel to liquid-crystalline phase transition of these vesicles was monitored by differential scanning calorimetry. Vesicles formed in the absence of glycoprotein (600--2100-A diameter) underwent the phase transition at 41.0 degrees C and had an associated enthalpy change of 8.0 +/- 1.6 kcal/mol. Increasing the mole ratio of glycoprotein to DPPC in the vesicles to 0.15 mol % reduced both the transition temperature and the transition enthalpy change. The enthalpy change as a function of the mole percent glycoprotein could be fit to a straight line by a least-squares procedure. Extrapolation of the results to the glycoprotein concentration where the enthalpy change was zero indicated one glycoprotein molecule bound 270 +/- 150 molecules of DPPC.     

Cytochrome c interaction with membranes. The enthalpy of oxidation of cytochromes c, c2, and a1,2.

The enthalpy of oxidation of horse-heart cytochrome c bound to phospholipid vesicles was found to be 14.6 ± 0.3 kcal/mole at 25 °C, pH 7.0, equal to the value for oxidation of the free form of the cytochrome. The affinity constants for binding of the reduced and oxidized forms of cytochrome c were the same at 4 °C and 30 °C, indicating that ΔH ° of binding contributes negligibly to the overall enthalpy of oxidation of the bound cytochrome c. The free energy (ΔG °′) of oxidation of the bound cytochrome c was 1.3 kcal/mole smaller than that for the free form, the difference being due to the change in entropy favoring the oxidized state of the cytochrome in the bound state. Measurement of the ΔH °′ for the oxidation of cytochrome a relative to the ferri/ferrocyanide couple shows it to be the same, within the limits of experimental error to that for the oxidation of cytochrome c.