Laurent Chinsky

Excited state geometry of uracil from the resonant Raman overtone spectrum using a Kramers–Kronig technique

The Kramers–Kronig transform technique of Blazej and Peticolas is extended to permit the calculation of overtone and combination bands in a resonant Raman spectrum relative to the fundamentals. The method is applied to the ultraviolet (257 nm) resonant Raman spectrum of uracil which exhibits five fundamental bands and numerous overtone and combination bands. The method permits the determination of Δej the shift of the excited state potential energy minimum along the jth normal mode for each of the five resonant Raman active modes. The Δej values obtained are in reasonable agreement with those obtained earlier from the absorption spectrum and excitation profiles of the fundamentals. However, no internal standard for the experimental determination of Raman intensities is required nor is it necessary to assume any value for Γ the vibronic linewidth. The shape of the absorption band and relative intensities of the harmonics to the fundamental are sufficient to obtain the value of each Δej.

SUBCELLULAR DISTRIBUTION OF HYPERICIN IN HUMAN CANCER CELLS

Confocal laser microspectrofluorometric measurements on human T47D mammary tumor cells have been performed to assess the intracellular distribution of hypericin within the various cell compartments: cytoplasmic membrane, cytoplasm and nucleus. Confocal fluorescence measurements obtained from microvolumes (˜ 1 μm3) located within the three sites of interest show that, while being primarily located in the cell membrane and cytoplasm after a short‐term incubation in a 10−6M hypericin‐containing culture medium, hypericin actually reaches the inside of the cell nucleus after a long‐term incubation (210 min). Moreover, owing to the relative fluorescence quantum yields of hypericin determined in vitro when the molecule interacts with DNA, membrane and protein model systems, it is assumed that there is a significant accumulation of the drug into the cell nucleus. Consequently, the nucleus has to be considered as a possible target for the toxic action of hypericin.

TRIPLET STATES OF CAROTENOIDS BOUND TO REACTION CENTERS OF PHOTOSYNTHETIC BACTERIA: TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY

Time‐resolved, low‐temperature resonance Raman spectra of triplet states of the carotenoids specifically present in bacterial reaction centers in a strained cis conformation have been obtained, thus demonstrating the possibility of studying intermediate transient states of these structures using resonance Raman spectroscopy. Resonance Raman spectra of triplet cis spheroidene and cis methoxyneurosporene present in reaction centers of Rhodopseudomonas spheroides, (strains 2.4.1. and Ga, respectively) exhibit marked differences with those of triplet, all‐trans carotenoids previously studied in vitro. These differences, together with the frequency shifts measured for the v1 modes, indicate that triplet carotenoids bound to reaction centers retain a cis conformation, and that probably no isomerization occurs to all‐trans carotenoids upon T ← S0 excitation. Pi electron distributions along the polyene backbone are probably less regular in the triplet state than in the singlet ground state, although probably not to the extent suggested by previous theoretical calculations. The apparently anomalous behaviour of the v2 bands of all‐trans carotenoids upon T ← S0 excitation is shown to result largely from the actual complexity of this region of the Raman spectra, together with a weak participation of the vc—–c internal coordinate in the corresponding modes. Finally, the Raman scattering efficiency of triplet spheroidene bound to reaction centers is lower than that of the singlet, ground state form, under equivalent excitation conditions.

Interaction of Hypericin with Serum Albumins: Surface-enhanced Raman Spectroscopy, Resonance Raman Spectroscopy and Molecular Modeling Study¶

Abstract Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1–H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 Å between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.

Contribution of the Resonance Raman Spectroscopy to the Identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.

Amantadine–DNA interaction as studied by classical and resonance Raman spectroscopy

Abstract The interaction of the antiviral agent amantadine with calf thymus DNA was studied by classical and UV-resonance Raman spectroscopy. It was found that: (i) the drug interacts with purine bases adenine and guanine via hydrogen bonds formation between N7 positions of purines and amino group of amantadine and (ii) the interaction leads to partial DNA structure change, which is demonstrated by a deformation of the hydrogen bonds of the A–T base pairs and by a partial deformation of the sugar-phosphate backbone of DNA, which does not lead to the DNA conformation transition.

Fluorescence of actinomycin D and its DNA complex

In order to show that naturally occurring actinomycin D can be used as a fluorescent cytochemical probe, the fluorescence spectra of 50 micronM solution of actinomycin D in several solvents and in its DNA complex are reported. The excitation spectra of both bands observed are compared to the absorption spectrum, and the fluorescence quantum yields is estimated to be 0.65-10(-4), taking rhodamine B as a reference. Strong solvent-induced spectrum modifications are evidenced, interpreted as a solvent shell reorientation about the molecule chromophore in the excited state. The changes in the fluorescence spectrum of actinomycin due to an interaction with DNA (marked blue shift and decreased quantum yield) cannot be interpreted as a solvent effect; they express the properties of the DNA-chromophore eta-complex involving an orbital overlapping of actinomycin and guanine in DNA.

Interaction of antiviral and antitumor photoactive drug hypocrellin A with human serum albumin.

Absorption, resonance Raman, surface-enhanced Raman spectroscopy and differential scanning microcalorimetry were employed to study the interaction of hypocrellin A with human serum albumin. The identification of the binding place for hypocrellin A as well as the model for the albumin-hypocrellin A complex are proposed. In this model hypocrellin A interacts with albumin through more than one binding site placed on the protein surface. This model of non-specific interaction could explain why the absorption spectrum of hypocrellin A does not change in the presence of albumin and why the presence of the drug does not change significantly the thermodynamic parameters of the protein, while the Raman spectra show evident changes concerning both the protein and the drug structure. Even if hypocrellin A does not interact with an interior binding site, it can affect deeply the general albumin structure.

AZ conformational transition in poly(rArU) and structure marker bands in UV resonance Raman spectroscopy

Abstract Ultraviolet resonance Raman (RR) spectra of poly(rArU), poly(dAdU) and poly(rA)·poly(rU) in aqueous solutions, excited at 257 and 281 nm wavelengths, have been reported in the spectral region between 400 and 1800 cm −1 . In comparing the RR data of these various AU duplexes, conformational marker bands of the right-handed A structure have been determined. In addition, a conformational transition of poly(rArU) has been monitored in the presence of a high concentration of NH 4 F in aqueous solution. This transition has also been observed by CD spectroscopy (M. Vorlickova, J. Kypr and T.M. Jovin, Biopolymers, 27 (1988) 351) without any clearcut conclusion as to the final structure adopted by the polymer. The present RR data, compared with previous RR results obtained from other alternating purine—pyrimidine duplexes (polyribo- and polydeoxyribonucleotides), show that the final structure of the polyribonucleotide in highly concentrated NH 4 F solution is a left-handed Z helix. Moreover, the current study allowed RR structure marker bands of Z form duplexes to be determined in a general manner.