C.L. Choy

Ultrasonic absorption in myoglobin and other globular proteins.

The ultrasonic absorption of myoglobin has been measured by the resonance and pulse-echo techniques as a function of pH. The absorption at a given frequency can be separated into pH-dependent and pH-independent contributions. Like other proteins, two peaks at pH 3 and 11.5 are observed which can be accounted for a proton-transfer reactions of the side-groups. In addition, the absorption undergoes a large increase within a small range of 0.2 pH unit at pH around 4, when denaturation of myoglobin occurs, indicating that the absorption is sensitive to the overall protein conformation. To elucidate the origin of the pH-independent component, the absorptions of several other globular proteins at neutral pH are also measured. The absorptions of these proteins exhibit a linear correlation with their isothermal compressibilities, suggesting that the pH-independent component is related to volume fluctuations of protein molecules. The activation energy of 4 kcal/mol found for this relaxation is consistent with such an interpretation.

Kinetics of the pretransition of synthetic phospholipids a calorimetric study

The pretransition in aqueous dispersions of two synthetic phospholipids (dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine) has been examined in detail by differential scanning calorimetry. The transition from the high-temperature state (state above pretransition) to the low-temperature state (state below pretransition) is complex and appears to occur via some metastable states. In contrast, the kinetics of the transition from the low- to the high-temperature state is consistent with an activated two-state model. The observed hysteresis is shown to arise mainly from the kinetic nature of the pretransition.

Measurement of the protein-ligand bond energy of carboxymyoglobin by pulsed photoacoustic calorimetry

Abstract Pulsed photoacoustic spectroscopy has been used to study two horse heart myoglobin derivatives, deoxymyoglobin and carboxymyoglobin, in the temperature range of 0–25° C. The myoglobin-CO bond energy has been measured to be 13.4±0.5 kcal/mole. The volume change associated with the photodissociation of carboxymyoglobin into deoxymyoglobin and CO has also been observed.

Adiabatic compressibility of myoglobin. Effect of axial ligand and denaturation

An ultrasonic technique has been employed to study the adiabatic compressibility of three metmyoglobin derivatives (aquomet-, fluoromet- and azidometmyoglobin) at neutral pH, and aquometmyoglobin as a function of pH in the frequency range of 1-10 MHz at 20 degrees C. No difference was observed in the adiabatic compressibility of the various derivatives. This indicates that the binding of different axial ligands to myoglobin does not affect significantly the conformational fluctuations of the protein. The finding is consistent with the results of the hydrogen exchange rate experiment, indicating that both types of measurements are useful for the study of protein dynamics. Upon acid-induced denaturation, the adiabatic compressibility of myoglobin drops from 5.3 X 10(-12) cm2/dyn to 0.5 X 10(-12) cm2/dyn. Plausible reasons for such a decrease are discussed.

Thermal stability of hemoglobin and myoglobin: Effect of spin states

The thermal stability of various methemoglobin and metmyoglobin derivatives at different ligand concentrations and pH were studied by the method of differential scanning calorimetry. Comparing to water as a ligand, cyanide and azide are most effective in protecting the protein; fluoride stabilizes the protein slightly whereas imidazole and thiocyanate have little effect. Assuming thermal denaturation can be described by an activated two-state process, the activation parameters of all the derivatives were determined. We have also found that the stability of hemoglobin and myoglobin does not correlate with the iron atom spin state but depends instead on steric interactions with the ligands. On the reasonable assumption that structure and stability are related, this implies a definite dependence of structure on steric interactions.

Electron transfer between cytochrome c and metal hexacyanide complexes effect of thermodynamic driving force on the electron transfer rate

The electron transfer reactions between ferrocytochrome c and three isomorphic hexacyanide complexes, [Fe(CN)6]3-, [Os(CN)6]3- and [Ru(CN)6]3-, have been studied using the method of photoexcitation. The transfer rates for [Os(CN)6]3- and [Ru(CN)6]3- are, respectively, about 45- and 200-times higher than that of [Fe(CN)6]3-. A reorganization energy of approx. 0.8 eV was found for the cytochrome c-hexacyanide system when the data were analyzed according to the theory of Marcus.

Kinetics of electron transfer between cytochrome c and iron hexacyanides. Evidence for two electron-transfer sites

The electron-transfer reaction between ferrocytochrome c and ferricyanide has been studied by the method of photoexcitation. The observed transfer rate shows saturation behaviour at high ferricyanide concentration. Data analysis indicates that there are two binding sites of vastly different affinities at which electron transfer occurs. The binding constant for the strong binding site decreases from 1600 M-1 to 80 M-1 as the ionic strength increases from 15 mM to 140 mM. At 20 degrees C, the intramolecular electron-transfer rate for this site is 4.65 X 10(4) s-1, which gives an electron-transfer distance of approx. 9.7 A according to Hopfields model.

Electron transfer between cytochrome c and metalloporphyrins at high exothermicities

Abstract The electron-transfer rates between cytochrome c and the anion radical of two metalloporphyrins ZnTPPS and ZnTPPC (ΔE for the reaction is 1.42eV) have been measured by laser flash spectroscopy. The anion radicals were produced by reaction of the porphyrins with hydrated electrons which resulted from the photoionization of ferrocyanide ions. Together with results obtained previously, a reorganization energy of ≈ 1.1 eV was deduced for the cytochrome c-porphyrin system.