On the diffusion of hypericin in dimethylsulfoxide/water mixtures-the effect of aggregation.

Hypericin (Hyp) is a natural photosensitizing pigment with a possible application in the photodynamic therapy of cancer. Hyp is readily dissolved in dimethylsulfoxide (DMSO) but forms nonsoluble aggregates in an aqueous environment. Fluorescence spectroscopy and diffusion coefficient measurements are used to investigate the self-association of Hyp molecules in DMSO/water mixtures. Fluorescence measurements reveal that Hyp remains in its monomeric form in DMSO/water mixtures containing up to ∼20-30 wt % water. At higher water concentration, Hyp starts to form nonfluorescent aggregates. To determine the size of the aggregates, the diffusion coefficient of Hyp is determined for different DMSO/water mixtures both experimentally and theoretically. Our data indicate that the size of the aggregates increases as more water is added into DMSO. At 50 wt % water content, the effective diffusion coefficient is about 30% smaller than the calculated value for the stacked Hyp tetramer. The results indicate that in an aqueous environment, Hyp presumably produces large molecular weight stacked H-aggregates. We have also confirmed that in an aqueous environment at alkaline pH, molecules of Hyp remain in the monomeric state.

A novel surface shear viscometer.

A novel rotary viscometer--developed for the determination of rheologic properties of liquid/air interface layers--is presented. The instrument can be used to measure the shear viscosity and the shear elasticity of liquid surfaces. It contains a rotor floating on the liquid surface which is rotated by means of an electromagnetic torque. A torsion filament is used to calibrate the applied torque. The viscosity data are obtained on the basis of the Navier-Stokes equation solved for the rotation of a cylinder touching the surface of water and submerged into the water. The time behavior of the surface viscosity of films gradually formed from solutions of some proteins as well as their activation energy is presented.

How thick is the layer of thermal volume surrounding the protein?

Investigation on the volume properties of protein hydration layers is reported. Presented results are based on combination of Monte Carlo modeling and available experimental data. Six globular proteins with known data are chosen for analysis. Analyzing the model and the experimental results we found that water molecules bound to proteins by hydrogen bond are preferentially located at the places with local depressions on the protein surface. Consequently, the hydration level is not strictly proportional to the area of charged and polar surfaces, but also depends on the shape of the molecular surface. The thickness of the thermal volume layer as calculated in the framework of the scaled particle theory is 0.6-0.65 A for chosen proteins. The obtained value is significantly lower than that presented for proteins in earlier papers (where proportionality between the hydration level and the area of charged and polar surfaces was assumed), but is close to the value published for small solute molecules. Discussion including the influence of protein size and the thermal motion of the surface is presented.

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions.

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in neutral and acidic urea solutions. Spectroscopic and electrochemical studies.

The heme iron coordination of unfolded ferric and ferrous cytochrome c in the presence of 7-9 M urea at different pH values has been probed by several spectroscopic techniques including magnetic and natural circular dichroism (CD), electrochemistry, UV-visible (UV-vis) absorption and resonance Raman (RR). In 7-9 M urea at neutral pH, ferric cytochrome c is found to be predominantly a low spin bis-His-ligated heme center. In acidic 9 M urea solutions the UV-vis and near-infrared (NIR) magnetic circular dichroism (MCD) measurements have for the first time revealed the formation of a high spin His/H(2)O complex. The pK(a) for the neutral to acidic conversion is 5.2. In 9 M urea, ferrous cytochrome c is shown to retain its native ligation structure at pH 7. Formation of a five-coordinate high spin complex in equilibrium with the native form of ferrous cytochrome c takes place below the pK(a) 4.8. The formal redox potential of the His/H(2)O complex of cytochrome c in 9 M urea at pH 3 was estimated to be -0.13 V, ca. 100 mV more positive than E degrees ' estimated for the bis-His complex of cytochrome c in urea solution at pH 7.

Effect of temperature and guanidine hydrochloride on ferrocytochrome c at neutral pH.

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6+/-0.3 degrees C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by Delta G(D) at 25 degrees C, is 59+/-5 kJ mol(-1) (DSC) and 65+/-6 kJ mol(-1) (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 degrees C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.

Thermodynamics of a diffusional protein folding reaction.

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.