Arnaldo Vecli

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.

Reaction volume of water formation detected by time-resolved photoacoustics: photoinduced proton transfer between o-nitrobenzaldehyde and hydroxyls in water

Abstract The structural volume changes accompanying light-induced proton transfer reactions in aqueous solutions of o -nitrobenzaldehyde have been investigated using time-resolved photoacoustics. The solvation of the newly formed ions is accopanied by a contraction of the medium of about −5.2 ± 0.2 ml/mol. At a pH above 9.5, the formation of water molecules leads to an expansion of 24.5 ± 0.4 ml/mol. The apparent rate constant for the protonation of hydroxyls is k = (4.9 ± 0.4) X 10 10 M − s − . This is the first direct time-resolved measurement of the extent of the structual volume changes accompanying water formation.

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL

Abstract— 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. Liposomes 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‐a‐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.

Time-resolved photoacoustic spectroscopy: New developments of an old idea

Abstract Acoustic waves generated by heat emission in radiationless transitions from photoexcited molecules can be detected by suitable transducers. Their study allows the investigation of thermal relaxations, thus providing thermodynamic and kinetic data on short-lived species produced by the absorption of pulses of light. In this field of research the best technique has proved to be the so-called pulsed-laser, time-resolved photoacoustic spectroscopy, which is based on piezoelectric detection of pressure waves in the time domain. Deconvolution processing of the transient signals gives both the lifetimes of excited states and the energy content of the transitions, provided that decay times are in the range 5 ns-5 μs. Moreover, when compared with proper theoretical models emphasizing the energy balance, the photoacoustic results can help to build a complete picture of the deactivation pathways, including photochemical events. The biophysical applications, although numerous and widespread both in basic and applied research, offer the real possibility of giving information on photobiological processes in conditions very close to the living state. Among the more significant contributions obtained in this area, the results on photosynthesis and photosensitivity of plants and photosynthetic micro-organisms, structural and functional dynamics of respiratory proteins, photocycles of rhodopsin and bacteriorhodopsin and photophysical properties of several natural pigments are particularly relevant, together with some medical and biotechnological applications. Another promising field of application of photoacoustics concerns photoactive drugs and the photophysics of fluorescent probes for conformational studies of proteins, nucleic acids and membranes. In general terms, time-resolved photoacoustic spectroscopy promises to become one of the most powerful techniques in photobiophysics, provided that some limitations in data analysis and time resolution are removed by technical improvements.

Artificial models of biological photoreceptors: effect of quenchers on the fluorescence properties of hypericin embedded in liposomes

Abstract Liposomes loaded with hypericin at different local concentrations were used as artificial models of biological light sensors. The steady state and time-resolved fluorescence of the chromophore were investigated in the presence of fluorescence quenchers which penetrate differently into and diffuse differently through lipid bilayers: iodide (I−), 9,10-anthraquinone-2,6-disulphonate (AQDS2−) and 9,10-anthraquinone-2-sulphonate (ACMS−). The results of the experiments with I− indicate that two of the three fluorescent species detected in our system, the long-lived and intermediate-lived species, are relatively close to the lipid-water interface. Anthraquinone compounds, which partially penetrate into the liposome, not only significantly quench the chromophore fluorescence emission, but also, apparently, affect the distribution of hypericin molecules in the vesicle, shifting the long-lived fluorescent molecules towards chemical-physical configurations characterized by shorter fluorescence lifetimes.

Sequential hydration-dehydration studies of lysozyme by the thermally stimulated depolarization currents (TSDC) technique

To investigate the physical state of water in hydrating biological macro-molecules, the dielectric properties of water in hen egg lysozyme pellets with various moisture contents were studied using the thermally stimulated depolarisation currents technique. The water dipoles appeared to be directly involved in the relaxation processes, such that, by increasing the content of water of sorption from ho = 0.075 to ho = 0.29, the current density recorded increased abruptly at moisture content above 0.075. At a fixed starting hydration level, the time evolution of water content was also studied by isothermal sample aging in dynamic vacuum: the TSDC spectra changed in both intensity and position of their main peaks (TM = 245 K, 190 K, 150 K) with moisture content and showed hysteresis. The complex behaviour of the TSDC response can be compared with the results obtained with the same technique on other biological macromolecules and suggests possible models for water configurations and rearrangements.

Water induced bioelectret state in lysozyme

The role of water in a bioelectret such as lysozyme at different hydration levels is studied by coupling TSDC and i.r. absorption measurements. In addition to the 312 K-band reported by Mascarenhas, our TSDC spectra exhibit new, weaker but anyway relevant bands related to water in different coordinations. At fixed starting hydration levels, the time evolution of water contents was studied by isothermal sample aging in dynamic vacuum: 1) the whole TSDC spectrum was reduced, the decrease being more relevant on the high temperature side and 2) the water related i.r. bands decreased as well.

Water induced TSDC in hemoglobin and myoglobin

The hydration of hemoglobin (Hb) and myoglobin (Mb) has been investigated by the Thermally Stimulated Depolarization Currents (TSDC) technique. The TSDC spectra as a function of the hydration degree (/spl delta/=0.01+0.3 grams of water/grams of protein) and of the annealing time in vacuum were recorded in the temperature range 100-300 K on pellet samples. The distribution of water dipoles for both heme-proteins shows four main peaks in the range 170-300 K, indicating the presence of at least four different classes of bound water. A fifth peak at very low temperatures (T/sub m//spl sim/150 K) appears only at low hydration levels and probably it is not related to water dipole relaxations. Possible environments of water dipoles responsible for the TSDC peaks are discussed.

Water induced bioelectret state in aminoacidic homopolymers

In order to attribute each peak of the complex TSDC (thermally stimulated depolarization current) lysozyme spectrum to a water-induced polarization process occurring at a given polypeptide chain segment, the authors have measured and compared the TSDC and infrared absorption spectra of several aminoacidic synthetic homopolymers at different hydration levels. The A-band (135-160 K), present in all the homopolymers investigated, is due to water molecule reorientation around the peptide bond. The B-band ( approximately 215 K) is related to the relaxation of the side chains, whose mobility and reorientation parameters are affected by the hydration level.<<ETX>>