RESUMEN
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 A 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.
Asunto(s)
Perileno/análogos & derivados , Perileno/química , Perileno/farmacocinética , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Animales , Antracenos , Bovinos , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Modelos Moleculares , Conformación Molecular , Fotoquímica , Ratas , Espectrometría RamanRESUMEN
The resonance Raman spectra of three oligonucleotides with different lengths containing a specific 5'AG3' target doublet for hypericin - a potent antiretroviral and anticancer photoactive agent, and their 1:1 and 1:2 (oligonucleotide: hypericin) complexes are reported. It is shown that the structural arrangement of the oligonucleotides, their structural stability and the local structural arrangement around the 5'AG3' hypericin target, are the factors which determine the formation of a stable, specifically bounded DNA-hypericin complexes.
Asunto(s)
ADN/química , ADN/metabolismo , Perileno/análogos & derivados , Fármacos Sensibilizantes a Radiaciones/metabolismo , Antracenos , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Perileno/química , Perileno/metabolismo , Fármacos Sensibilizantes a Radiaciones/química , Espectrometría Raman , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/metabolismo , Antivirales/metabolismo , Perileno/análogos & derivados , Fármacos Fotosensibilizantes/metabolismo , Quinonas/metabolismo , Albúmina Sérica/metabolismo , Antineoplásicos/química , Antivirales/química , Calorimetría , Medicamentos Herbarios Chinos , Humanos , Estructura Molecular , Perileno/química , Perileno/metabolismo , Fenol , Fármacos Fotosensibilizantes/química , Unión Proteica , Quinonas/química , Espectrometría RamanRESUMEN
The resonance Raman spectra of two oligonucleotides and their complexes with potent antiretrovirally and antineoplastic active photochemical drug hypericin are reported. The Raman spectra of two oligonucleotides containing twelve base pairs on addition of hypericin (one and two molecules per one oligonucleotide) were compared. The first one contains the first nine base pairs of the "rev" gene coming from HIV genome with three base pairs added to stabilize the duplex (5'ATGGCAGGATAT3') and the second one consists of the same content of the nucleotide bases but in changed sequence order which serves as a control sequence (5'ACGTGATGATGA3'). Differences in the spectra of the "rev" gene sequence and control sequence in interaction with the drug indicate that: i) the AG and GA nucleotide doublets are structurally specific targets for hypericin and ii) the hypericin interaction with 5'AG3' target is stronger than with 5'GA3' one.
Asunto(s)
Antineoplásicos/metabolismo , Antivirales/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Perileno/análogos & derivados , Antracenos , Perileno/metabolismo , Espectrometría RamanRESUMEN
In this paper we examine the interactions of Calf Thymus DNA and the model polynucleotides poly(dA).poly(dT), poly(dAdT)2 and poly(dG.dC)2 with a group of metalloporphyrins derived from the freebase porphyrin tetrakis(4-N-methylpyridyl)porphine, H2(TMpy-P4), by means of ultraviolet absorption spectroscopy, circular dichroism spectroscopy and microcalorimetry. We have studied the interactions of the copper, cobalt, nickel and zinc derivatives of H2(TMpy-P4) in addition to the free base porphyrin itself. We have found strong evidence for an external self-stacking interaction of the Cu(TMpy-P4) and Zn(TMpy-P4) derivatives with poly(dA).poly(dT) and poly(dAdT)2 even at low concentrations of porphyrin, and all of the porphyrin derivatives studied appear to display such a self-stacking in interaction with poly(dA.dT)2 at sufficiently high ratios of porphyrin to polynucleotide.
Asunto(s)
Calorimetría/métodos , Dicroismo Circular , Metaloporfirinas/metabolismo , Polinucleótidos/metabolismo , Animales , Bovinos , Poli dA-dT/metabolismo , Polidesoxirribonucleótidos/metabolismoRESUMEN
Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)2, poly(dA).poly(dT), poly(dG.dC)2 and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC). Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.
Asunto(s)
Metales/química , Polinucleótidos/química , Porfirinas/química , Espectrometría Raman , Adenina/química , Estructura Molecular , Timina/químicaRESUMEN
The first resonance Raman scattering observation of the low-frequency (LF) region (below 40 up to 12 cm-1) of DNA motions is presented. Since the concentration of the studied DNA solution was very low (1 mg/ml), the spectra features reflect internal vibrations of the macromolecule. The decomposition of the spectra into Lorentzians clearly indicate three intrahelical DNA modes: the corresponding peaks are located at the frequencies 16, 19, and 23 (+/- 1) cm-1. This result is in agreement with our quasi-continuity model of the LF B-form DNA dynamics (V. Lisy, P. Miskovsky and P. Schreiber, J. Biomol. Struct. Dyn. 13, 707 (1996)). The fit of the experimental frequencies to the theory, using the Genetic Algorithms approach, allowed us to make some conclusions about the model force constants which could be found by independent conformational energy calculations. Possible positions of five lowest-frequency DNA peaks, predicted by the model, are discussed.
Asunto(s)
ADN/química , Algoritmos , Animales , Bovinos , Modelos Moleculares , Espectrometría RamanRESUMEN
In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.
Asunto(s)
Fluorescencia , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Desoxirribonucleasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Fluoresceína , Fluoresceínas/química , Conformación de Ácido Nucleico , Oligonucleótidos/sangre , Rodaminas/química , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Espectrometría de Fluorescencia , Análisis Espectral/métodosRESUMEN
The four stranded form of polyriboinosinic acid, or poly(rl), formed under conditions of high ionic strength, has been studied principally by resonance Raman spectroscopy excited in the ultraviolet absorbent band of the hypoxanthine residues. UV Absorption and circular dichroism studies were made, principally in order to verify the presence of the quadruplex form at the low concentrations of poly(rl) used, and a trial experiment with the structural probe Tb3+ was also performed. Experimental evidence is found for highly stacked metastable forms present at low concentrations of polynucleotide, which are destroyed by heating in favor of the two well known forms.
Asunto(s)
Poli I/química , Polinucleótidos/química , Dicroismo Circular , Fluorescencia , Modelos Moleculares , Conformación de Ácido Nucleico , Poli I/metabolismo , Polinucleótidos/metabolismo , Espectrometría Raman , Terbio/química , Terbio/metabolismo , Rayos UltravioletaRESUMEN
Interactions of the antiretroviral hypericin molecule with polynucleotides, i.e. poly(dG-dC), poly(dA-dT), poly(rG) and poly(rC), have been studied in aqueous solutions by resonance Raman spectroscopy, using an UV excitation wavelength which induces a specific resonance enhancement of spectral band intensities corresponding to proper nucleic base modes of vibration. It is shown that: i) hypericin selectively interacts with the N7 sites of purines, ii) the strength of interaction depends on the polymer structure, and: iii) interaction with guanine is stronger than with adenine molecules.
Asunto(s)
Antivirales/química , Perileno/análogos & derivados , Polinucleótidos/metabolismo , Antracenos , Antivirales/metabolismo , Sitios de Unión , Guanina , Estructura Molecular , Perileno/química , Perileno/metabolismo , Poli C/metabolismo , Poli G/metabolismo , Poli dA-dT/metabolismo , Polidesoxirribonucleótidos/metabolismoRESUMEN
Resonance Raman spectra are reported for poly[dA.dT]2, poly[dA].poly[dT], poly[dG.dC]2, poly[dG].poly[dC], poly[dA.dC].poly[dG.dT] and Calf Thymus DNA in interaction with the porphyrin Cu(TMpy-P4). The spectra were obtained under conditions of low salinity and a ratio of porphyrin molecules to nucleotide base pairs of r = 0.05, with an excitation wavelength of 257 nm. The differences between the spectra in interaction with Cu(TMpy-P4) and those of the unmodified nucleic acids are interpreted in terms of the structural changes imposed by the porphyrin. Two different binding modes have been reported in previous studies, a 5'CG-3' specific intercalative mode and an A-T specific outside binding mode. Our results suggest that this latter mode may show a preference for A-A or T-T sites. Furthermore a transition in structure, while remaining within the B-form family of structures, is indicated for both poly[dA].poly[dT] and poly[dG.dC]2 upon addition of Cu(TMpy-P4).
Asunto(s)
Cobre , Sustancias Intercalantes/química , Metaloporfirinas/química , Nucleótidos/química , Polinucleótidos/química , Espectrometría Raman/métodos , Estructura Molecular , Conformación de Ácido Nucleico , Rayos UltravioletaRESUMEN
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 (approximately 1 micron3) 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(-6) M 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.
Asunto(s)
Antivirales/farmacocinética , Neoplasias de la Mama/metabolismo , Perileno/análogos & derivados , Antracenos , Fluorometría/métodos , Humanos , Perileno/farmacocinética , Proteína Quinasa C/antagonistas & inhibidores , Espectrofotometría Ultravioleta , Fracciones Subcelulares/metabolismoRESUMEN
The structural distortions of the duplex dodecamer d(ATTAACGTTAAT)2 monofunctionally alkylated by mitomycin C have been studied by the use of chemical probes reactivity and resonance Raman spectroscopy. This sequence contains the 5'-ACGT sequence for which mitomycin C was determined to present the best affinity (S. Kumar, R. Lipman, and M. Tomasz, Biochemistry 31, 1399 (1992)). Raman spectroscopy as well as osmium tetroxyde reactivity indicate that the distortion of the double helix structure is located around the central CG bases. Mitomycin C reacts exclusively with the 2-amino group of guanine and this binding does not disrupt the inter bases H-bonds, as indicated by chloroacetaldehyde reactivity. Although resonance Raman spectroscopy does not allow the handedness of the monoalkylated CG/GC sequence to be determined, it indicates a similarity between the base stacking and that which would be observed for alternating purine/pyrimidine sequences at high salt concentration.
Asunto(s)
ADN/efectos de los fármacos , Mitomicina/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Acetaldehído/análogos & derivados , Acetaldehído/química , Alquilación , Secuencia de Bases , Sitios de Unión , Sondas de ADN/química , Guanina/química , Hidroxilamina , Hidroxilaminas/química , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Compuestos de Osmio/química , Espectrometría RamanRESUMEN
Poly(dI-dC) in aqueous solution can undergo different equilibrium geometries, which strongly depend on salt nature and concentrations. These equilibrium structures have been monitored by resonance Raman spectroscopy (RRS) measurements in the ultraviolet region, i.e. by using 257 and 281 nm laser excitation wavelengths which favor the resonance enhancement of the Raman contributions from inosine and cytosine residues of poly(dI-dC), respectively. Spectral changes depending on the NaCl concentration and on the presence of Ni2+ ions have been observed and interpreted in comparison with RRS results previously obtained for other alternating purine-pyrimidine polydeoxyribonucleotides, i.e. poly(dG-dC), poly(dA-dT) and poly(dA-dC).poly(dG-dT), which also showed B to Z conformational transitions in varying the salt concentrations. It is shown here that: i) the base stacking geometries are nearly the same in the high-salt form (5 M NaCl) of poly(dI-dC) as in the low-salt form (0.1 M NaCl) of the polymer, ii) however, the high-salt structure yields important differences from a B-helix (obtained in low-salt solution) as regards the nucleoside conformations (sugar puckering and base-sugar orientation), and: iii) the addition of 9 mM NiCl2 in the high-salt (5 M NaCl) solution of poly(dI-dC) induces the Z-conformation of the polymer.
Asunto(s)
Polidesoxirribonucleótidos/química , Espectrometría Raman , Níquel/química , Poli dA-dT/química , Conformación Proteica , Cloruro de Sodio/química , Espectrofotometría UltravioletaRESUMEN
The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed. The three daunorubicin molecules that bind to one Fe(III) are not chemically modified through complexation with iron. In the case of the Fe(III)-adriamycin and Fe(III)-4'-O-tetrahydropyranyladriamycin complexes, about one of the three anthracycline molecules is chemically modified, yielding a highly lipophilic derivative, the 7,8-dehydro-9,10-desacetyladriamycin. The others molecules remain unchanged, i.e., highly hydrophilic in the case of adriamycin. These two species have a different fluorescent spectrum and can be identified inside the cell, using microspectrofluorometry. In the case of the Fe(III)-adriamycin complex, the lipophilic derivative is more rapidly internalized in the cell than the hydrophilic one. Diffusion into the plasmic membrane is the limiting step for the uptake of anthracycline by cells; this means that the plasmic membrane speeds up the dissociation of the Fe(III)-anthracycline complex.
Asunto(s)
Membrana Celular/metabolismo , Daunorrubicina/análogos & derivados , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Hierro/metabolismo , Compuestos Organometálicos/metabolismo , Animales , Línea Celular , Daunorrubicina/química , Difusión , Doxorrubicina/química , Hierro/química , Compuestos Organometálicos/química , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The interaction of poly(dG-dC).poly(dG-dC) with mitomycin C, an antitumor antibiotic, has been studied by various spectroscopic methods: circular dichroism, Fourier transform infrared resonance Raman scattering and using fluorescence emission of terbium bound to unpaired guanines as local conformation probe. The results allowed us to confirm the lack of long range modification of the DNA secondary structure upon binding. They also brought first information concerning the modification of the local structure of the nucleic acid at the level of mono- or bifunctional adducts.
Asunto(s)
ADN/efectos de los fármacos , Mitomicina/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Dicroismo Circular , ADN/química , Polidesoxirribonucleótidos/química , Análisis EspectralRESUMEN
The right to left helix structural transition in purine-pyrimidine alternating copolymers has been extensively studied by vibrational spectroscopies, amongst many other experimental approaches. Here, the use of resonance Raman spectroscopy in the ultraviolet region (223-, 257- and 281 nm excitation wavelengths) to monitor such structural changes is reviewed in the light of new results obtained on poly(dA-dC).poly(dG-dT) on one hand, and the previous results obtained on poly(dG-dC)2, poly(dA-dT)2 and natural DNA (Chicken erythrocytes) on the other. It is now possible to define B----Z transition marker bands involving the proper bases, which show a similar behaviour on structural transition whatever the composition of alternating purine-pyrimidine sequences: the 1580- and 1487 cm-1 lines of the purines, the 1486- and 1294 cm-1 lines of the pyrimidines are good markers in the vibrational spectra recorded at various UV excitation wavelengths.
Asunto(s)
ADN/química , Polidesoxirribonucleótidos/química , Espectrofotometría Ultravioleta , Espectrometría Raman , Conformación Molecular , Estructura Molecular , Conformación de Ácido NucleicoRESUMEN
The conformational changes of poly(d2NH2A-dT) in aqueous solution, induced by increasing the NaCl concentration from 0.1M to 4M, have been monitored by ultraviolet resonance Raman spectroscopy, in using the 222-, 257- and 281 nm excitation wavelengths. These changes have been interpreted in comparing the polymer spectra to those of the mononucleotide compounds on one hand, and to those of other alternating purine-pyrimidine polymers on the other hand, i.e. poly(dG-dC) and poly(dA-dT) which showed a B to Z transition in going from low- to high salt concentrations. The high salt poly(d2NH2A-dT) spectra do not show any Raman marker line of the Z conformation. The spectroscopic results indicate that most of the ribose puckering goes from C2'-endo/anti to C3'-endo/anti in increasing the salt concentration. In addition the base stacking interactions, to which the resonance Raman effect is very sensitive, are not drastically changed upon salt variations. Thus the high salt structure of poly(d2NH2A-dT) remains a right-handed helix, likely under a dominant A conformation.
Asunto(s)
Polidesoxirribonucleótidos/química , Cloruro de Sodio/farmacología , Espectrofotometría Ultravioleta , Espectrometría Raman , Conformación MolecularRESUMEN
The present work investigates the variations of electrostatic interactions within the myoglobin molecule associated with azide heme binding and pH variations. Far ultraviolet (223 nm) resonance Raman spectroscopy of the tryptophan and tyrosine residues, along with acid-base titration measurements, have been used to monitor variations in the protein matrix. With previously determined mode assignments, it is shown that the Trp and Tyr residues of the globin moiety are influenced by the charge spatial distribution. Upon ligand binding or under various pH conditions, the polar interactions inside the protein appear to be modulated by the electric field generated by the charge array. It is concluded that the binding site properties of myoglobin can be modulated by the charge spatial distribution within the protein, even in the absence of measurable conformational changes of the bulk.
Asunto(s)
Hierro/química , Mioglobina/química , Triptófano/química , Tirosina/química , Animales , Sitios de Unión , Electroquímica , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Espectrofotometría Ultravioleta , Espectrometría Raman , Ballenas , Difracción de Rayos XRESUMEN
The resonance Raman spectra of water-soluble porphyrins, Cu(TMpy-P4) and Ni(TMpy-P4), and their mixtures with DNA, Poly(dG-dC).Poly(dG-dC), and Poly(dA-dT).Poly(dA-dT) were measured using 426 nm pulsed laser excitation (and 556 nm for some applications). At high laser power, the solution of Cu(TMpy-P4) mixed with DNA or Poly(dA-dT).Poly(dA-dT) exhibits new bands at 1550 and 1349 cm-1 that are not observed for Cu(TMpy-P4) alone or for Cu(TMpy-P4) mixed with Poly(dG-dC).Poly(dG-dC). These extra bands do not appear when the resonance Raman spectra are measured by a cw laser or by a pulsed laser with low power. Similar mixtures of M(TMpy-P4) (where M = Ni, Zn, Co, Mn, and H2) with these nucleic acids exhibit no such bands even by high power pulsed laser excitation. We attribute the new resonance Raman bands to an electronically excited Cu(TMpy-P4), stabilized by forming an exciplex with the A-T site of the nucleic acid. The minimum lifetime value of such an exciplex was estimated to be on the order of 10 ps.