Tuning the local solvent composition at a drug carrier surface: the effect of dimethyl sulfoxide/water mixture on the photofunctional properties of hypericin-ß-lactoglobulin complexes.

Aggregation is a major problem for the anti-microbial photodynamic applications of hydrophobic photosensitizers since it strongly reduces the amount of singlet oxygen generated in aqueous solutions. Binding of hypericin (Hyp) to the milk whey protein ß-lactoglobulin (ßLG), occurring at the two hydrophobic cavities located at the interface of the protein homodimer, can be exploited to confer water-solubility and biocompatibility to the photosensitizer. The introduction of a small amount of the organic cosolvent dimethyl sulfoxide (DMSO) leads to a remarkable improvement of the photophysical properties of the complex Hyp-ßLG by increasing its fluorescence emission and singlet oxygen photosensitization quantum yields. Surprisingly, the ability of the complex to photo-inactivate bacteria of the strain Staphylococcus aureus is strongly reduced in the presence of DMSO, despite the higher yield of photosensitization. The reasons for this apparently contradictory behavior are investigated, providing new insights into the use of carrier systems for hydrophobic photosensitizers.

High and low oxygen affinity conformations of T state hemoglobin.

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.

Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique.

We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.

Functional characterization of heme proteins encapsulated in wet nanoporous silica gels.

Myoglobin and hemoglobin were encapsulated in wet, nanoporous silica gels. A rigorous evaluation of the effect of the encapsulation on protein dynamics and function was carried out by measuring the kinetics of carbon monoxide rebinding after nanosecond laser flash-photolysis with transient absorption detection, and the oxygen affinity with absorption microspectrophotometry. The time course of carbon monoxide binding to myoglobin evidenced a strongly enhanced geminate recombination and a faster bimolecular rebinding with respect to solution, whereas T and R quaternary states of hemoglobin exhibited a geminate phase and a bimolecular binding rate very similar to those observed in solution. Oxygen affinity of T-state hemoglobin was found to be close to that observed for the binding of the first oxygen to T-state hemoglobin in solution. Results indicate that some conformational transitions are kinetically restricted, allowing to isolate distinct tertiary and quaternary states. This opens the way to their detailed functional characterization and application to biodevices.

Kinetics of local helix formation in poly-L-glutamic acid studied by time-resolved photoacoustics: neutralization reactions of carboxylates in aqueous solutions and their relevance to the problem of protein folding.

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.

Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump.

Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.

An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions.

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.

Spectroscopic and photoacoustic studies of hypericin embedded in liposomes as a photoreceptor model.

In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Lipopsomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-alpha-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

The interaction of melanin with 8-methoxypsoralen: effect on radiative and nonradiative transitions. A fluorescence and pulsed photoacoustic study.

The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8-MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen-melanin complexes, the photoacoustic parameters of 8-MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground-state level, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.

Porphyrin-melanin interaction: effect on fluorescence and non-radiative relaxations.

Optical techniques and pulsed-laser, time-resolved photoacoustics (PA) were employed to obtain information on the mechanism of interaction between cationic zinc tetrabenzilpyridilporphyrin (ZnTBzPyP) and synthetic L-Dopa melanins. Synthetic eumelanin and pheomelanin strongly quench the fluorescence of ZnTBzPyP, but Stern-Volmer plots suggest a mechanism of interaction quite different for the two pigments. This diversity was confirmed by PA: for eumelanin no thermal relaxation was observed other than prompt heat, whereas for the complexed form of ZnTBzPyP with pheomelanin we were able to detect a heat-emitting species with a non-radiative lifetime in the microsecond range. The involvement of oxygen in the photophysics of the complexes formed between the cationic porphyrin and the two pigments was demonstrated, but its role has yet to be described.

Application of photoacoustic phase angle spectroscopy (phi AS) to eumelanins and pheomelanins.

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment. Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.

Quenching of the excited states of 8-methoxypsoralen by synthetic Eumelanin.

The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluoresence and pulsed photoacoustic calorimetry. Spectroscopic data indicate the absence of interaction in the ground state, whereas the singlet excited state of 8-MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static mechanism. Photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.

Photoacoustic studies of non-radiative relaxation of excited states in melanin.

Photoacoustic measurements made at various chopping frequencies on dense acqueous melanin suspensions have revealed a significant dependence upon pH and redox state. It is shown that such behaviour, differing from the simple predictions of the Rosencwaig-Gersho theory, can be explained in terms of finite carrier diffusion and recombination times. The implications of these findings with respect to the amorphous semiconductor model proposed to describe the dynamic role of epidermal melanin are discussed. From the experimental data, values of physical parameters were calculated which allow a qualitative correlation between chemical states and electronic behaviour and, consequently, some aspects of the molecular biology of the melanosome, founded on a rigorous base.