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Resonance Raman and surface-enhanced Raman spectroscopy were employed to study the interaction of hypericin with human serum albumin. The identification of the binding place for hypericin as well as the model for albumin−hypericin complex are presented. In this model hypericin interacts with tryptophan placed in II A subdomain of albumin. This interaction reflects (i) a change of the hydrophobicity of the tryptophan environment, (ii) the formation of an H-bond between the carbonyl group of hypericin and N1−H group of tryptophan, leading to a protonated-like carbonyl in the drug, (iii) a decrease of the strength of H bonding at the N1−H site of tryptophan, and (iv) a change of the tryptophan side-chain conformation.

The dynamics and mechanism of the photoinduced reversible process of formation and decay of an exciplex species created between the water-soluble cationic metalloporphyrin copper 5,10,15,20-tetrakis[4-(Nmethylpyridy1)lporphyrin ) and the DNA model compound poly(dA-dT) have been studied in detail. Such a photoinduced process had been previously observed in transient resonance Raman (RR) spectra under high-power laser irradiation of complexes of Cu(TMpy-P4) with calf thymus DNA and some oligoand polynucleotides containing thymine (T) or uracile (U) residues. It was found that the interaction of excited Cu(TMpy-P4) with carbonyl groups of T or U involved in polymers having an appropriate secondary structure was responsible for the new transient species detected in high-power Raman spectra. In the present work, direct kinetic measurements of the exciplex formation between Cu(TMpy-P4) and poly(dA-dT) were carried out by using both picosecond transient absorption pump-probe technique (10-ps time resolution) and two-color time-resolved RR technique (100-ps time resolution). A comparative nanosecond Raman study of this exciplex and of the excited (d,d) state of copper meso-tetraphenylporphyrin (CuTPP) model compound dissolved in a number of oxygen-containing solvents has also been performed, to clarify the excited electronic state which is at the origin of this process. It has been found that the binding of one of the CO-groups of T or U to Cu(TMpy-P4) in its lowest excited triplet state results in a shortening of the triplet-state lifetime to 35 f 7 ps. In addition, a population of an excited 2[d,2,d+y2] state, Le., the most low-lying and long-lived excited state for the five-coordinated Cu(TMpy-P4) (exciplex state), occurs in the process of excitation relaxation. Large wavenumber shifts of structure-sensitive vibrational marker lines from the porphyrin skeleton reveal the promotion of one of the copper d electrons into the half-filled d$-?2 orbital and the expansion of the porphyrin core to accommodate the occupation of this d orbital. The exciplex deactivation process (excited (d,d) state decay) has a time constant of 3.2 f 0.5 ns and is accompanied by the CO-group deattachment with a disruption of the exciplex into initial components.

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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.

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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.