The possibility that clear images could be obtained of certain objects in the human body such as tumors, aneurysms, stroke volumes, degenerative diseases of the brain such as Alzheimer's, Parkinson's and MID has been moving rapidly ahead during the past half dozen years. These advances are, on the one hand, better understood by the theory of photon migration, photon density, and on the other hand, by technical developments which have made the difficult problem of time resolved spectroscopy and phase modulation spectroscopy not only simple but also economical. The face that the laser community has seen fit to regard this as an important part of laser developments and technology is gratifying and surely essential to the development of the technology and its biomedical applications.

Recent infrared spectroscopic studies of the ultrafast responses of optically triggered changes in proteins are discussed. Examples from research on ions in aqueous media, hemoglobin, bacteriorhodopsin, and reaction centers illustrate the potential of time resolved infrared spectroscopy in the field of protein dynamics.

This report discusses the effects of pulsed, spatially inhomogeneous deposition of laser light energy that causes local heating of microregions in the exposed biotissue. The local heating effects may be due to: (1) the quantum character of absorption; (2) spatially inhomogeneous absorptivity of the tissue; and (3) spatially inhomogeneous light intensity distribution. The discussion involves various biological organization levels: molecular chromophores, cells, and biotissues (exemplified by laser ablation). The inference drawn are that: (1) it is necessary to take account of the local heating effects in the interaction between laser light and biotissue and (2) it is potentially possible to use these effects in laser therapy (laser photothermotherapy).

A comparison of photochemical properties of three aromatic and five aliphatic amino acids as well as seven peptides in neutral liquid aqueous solution at five different wavelengths was carried out. The photolysis quantum yield and the quantum yield of peptide bond scission were determined for the first time. The experimental dependence of tryptophan photodecomposition efficiency versus the energy excess above the ionization threshold confirms recent theoretical predictions of photoionization properties of molecules in diluted aqueous solution below the bottom of the conduction band.

We present a summary of our work on wave packet motion on laser coupled molecular potential surfaces. These are described by a time dependent version of the Born-Oppenheimer theory. We look at pulse excitation, level crossings and interference phenomena. Our numerical results are compared with simple semi-classical models.

By femtosecond ground-state recovery measurements the lifetime of the lowest excited singlet (S₁) state in extra long carotenes, with 15 and 19 conjugated double bonds, has been determined to 850 and 470 fs, respectively. The nonradiative relaxation rates, due to internal conversion (k_ic), are interpreted together with the k_ic found for shorter carotenes homologous to trans-(beta) -carotene with 5, 7 and 11 ((beta) -carotene) conjugated double bonds. By using the energy gap law derived in the weak coupling limit the radiationless transitions were analyzed. The variations of k_ic depend mainly on the large variations in the coupling parameter, and the negative exponential energy gap dependence plays a minor role, due to the low value on the (gamma) parameter.

New results of application of laser saturation fluorometry, relying on the measurement and analysis of nonlinear characteristics of laser-induced chlorophyll-a fluorescence, for study of primary photosynthesis processes and diagnostics of unfavorable environmental influences (nutrients depletion; presence of herbicides, heavy metal ions, etc.) on alga photosynthetic apparatus are reported. Mechanisms of the changes in fluorescent characteristics under these influences as well as structural and functional modifications to photosynthetic apparatus, causing these changes, are analyzed. It is shown that the laser saturation fluorometry permits to selectively identify different environmental influences oppressing photosynthesis.

Our work is devoted to the investigation of some problems of energy migration among C- phycocyanin (C-PC) chromophores: (a) determination of energy migration rates between chromophores at various aggregation states of C-PC; (b) determination of the main routes of energy transfer in the rods consisting of 2 - 4 C-PC hexamers; (c) whether the position and orientation of C-PC chromophores are optimal for energy migration; and (d) disclosing of the probability function of statistic of exciton jumping times between chromophores. We developed two approaches, both being based on the statistical analysis of consecutive exciton interchromophore jumps (microscopic level) during its 'life'. The macroscopic parameters are obtained via statistical averaging. The first is the Monte-Carlo method and the second one implies direct calculation of energy migration rates. The latter method has yielded detailed information about the rates of energy migration among chromophores of C-phycocyanin at various aggregation states and conditions of chromophore orientations.

Spectrally-resolved pico- and nanosecond fluorescence kinetics of photosystem II reaction center (RC) preparations and RC together with CP47 proximal antenna have been studied at room temperature and at 77 K. The following conclusions have been made. (1) At room temperature the average trapping time proportional to the charge separation time and the number of cooperating pigments is observed. This time is equal to 13 +/- 3 ps in RCs and 25 +/- 10 ps in RC + CP47 complexes. (2) All other decays at room temperature are of a recombination origin and reflect complex relaxation of the metastable radical pair state. (3) At low temperatures an energetically directed excitation transfer, qualitatively very similar to the one observed in the core antenna of some purple bacteria, is observed. This energy transfer is relatively slow with an apparent transfer time 10 - 20 ps at 77 K.

Molecular structures of chlorophyll a aggregates have been studied. Spectroscopic properties of these aggregates have been studied by absorption and fluorescence spectroscopy in hydrocarbon solution at various temperatures. Observed spectroscopic shifts were interpreted in terms of simple exciton theory. Exciton splittings were estimated from computer optimized models of previously suggested Chl a aggregate structures.

The primary photochemical events of photosynthesis occur in specialized, integral membrane proteins. The lightharvesting (LH) proteins collect photons and convey the resulting excitation energy to the reaction center (RC) proteins where charge separation occurs. Both LH and RC proteins generally contain several cofactors. Many of these cofactors are chiorophylls and carotenoids, which can be very selectively probed using resonance Raman (RR) spectroscopy at selected laser excitation wavelengths. The functional properties of these cofactors largely depend on their specific interactions within their protein host sites. RR spectroscopy uniquely permits a diagnosis of their conformations and molecular interactions, both in the resting and intermediate functional states of the protein

A model that assumes exciton interactions in the circular molecular aggregate is proposed for B875 (890) light-harvesting complex of purple bacteria. Picosecond absorbance difference spectra and induced absorption anisotropy are calculated.

The very first step in photosynthesis of green plants is the excitation of the accessory pigments in the antenna system. Then a chain of energy transfer steps funnels the excitation energy to the reaction center where its capture leads to creation of chemical energy. Of special interest is the antenna system of photosystem II, LHC II, which contains chlorophyll cM, and chlorophyll cM. In LHC II cM and chl are equally abundant and thus, this system constitutes a suitable candidate for studying the cM to cM energy transfer in detail. Furthermore, some investigations have already been carried out on this system. In an early study by Gillbro et a! [1] the energy transfer time between chl and cM was estimated to be in the range 240 ps. Later studies have, however, indicated that this was an underestimate of the transfer rate between cM and chl . From a study on a bacterial system, Eads et al [2] concluded from time resolved fluorescence experiments that the transfer time between chi and cM is in the order of 0.5 ps. Returning to the antenna systems of higher plants, Kwa et a! [3] in a recent work reported that this energy transfer step must be completed within 1 ps. LHC II does also belong to an exclusive group of membrane bound photosynthetic protein complexes that have been crystalized and whose structure have been partially determined (Kuhlbrandt et a! [4]). The outcome of that study was that the chiorophylls in each monomer are organized in two different levels, with each lve1 close to the bilayer leaflets. The 6 A resolution of this crystal structure did not, however, permit Kuhlbrandt et al [4] to distinguish between the chlorophyll residues of a or b type. In the same study Kuhlbrandt et at [4] also estimated the rates of energy transfer within one monomeric unit. This estimate is, however, rather crude since all the interacting chiorophylls were of only one type, namely cM and were interacting according to the exciton mechanism. With this assumption the transfer time was calculated to be as short as 10- 100 fs within the monomer unit, even with the Qy transition dipoles randomly orientated. With regard to the energy transfer step chi k to chl , thisis most likely an overestimate of the transfer rate. Furthermore, a more appropriate interaction mechanism could in fact be the Förster mechanism especially when chi and chl are interacting. We have recently shown that the Förster mechanism is valid for as short distances as ca 20 A in the PEC and other trimers of phycobilisomes [5]. The aim of this study was accordingly to re-examine the energy transfer dynamics within the LHC II complex. Previous studies showed that the need for better time resolution is obvious [1,3]. With a more accurate mesurement of the transfer time it should also be possible to see how well the distances calculated with the Förster model fit those obtained from crystal data

Analytical expressions for quantum yield of energy losses in light-harvesting antenna and quantum yield of charge separation in reaction center for bacterial photosynthesis were obtained. The dependence of these yields on spectral properties of 'minor' antenna component is discussed.

Substituting copper porphyrin (CuP) with pyridine substituents results in the formation of dimers with zinc porphyrins (ZnP). Different forms of self-assembling shorten the fluorescence lifetime of ZnP from 2 ns to 247 and 80 ps. This can be explained by electron transfer from ZnP to CuP.

Zn-Tetraphenylporphyrin-(SO^-₃)₄ forms two types of complexes with a covalently linked viologenanthraquinone (VA) compound both in H₂O and methanol. In both complexes fluorescence quenching occurs on a time scale < 10 ps and 0.5 ns, respectively, and its tentatively assigned to photo-induced electron transfer from porphyrin to VA.

Femtosecond processes in allophycocyanin, C-phycocyanin and phycoerythrocyanin trimers and monomers have been examined by means of polarization pump-probe technique. No femtosecond kinetics were observed in monomeric preparations. The isotropic absorption recovery kinetics with (tau) equals 440 +/- 50 fs which is not accompanied by anisotropy decay kinetics was obtained in allophycocyanin trimers at 612 nm. The conclusion about energy transfer between neighboring (alpha) 84 and (beta) 84 chromophores with different absorption spectra was made. The proposed model takes into account a stabilizing role of the linker peptide. Spectral and kinetic measurements were made in the 635 - 690 nm spectral region where the proposed acceptor should absorb. The bleaching of the 650-nm band occurs with a delay relative to the bleaching at 615 nm. Only a rise term was observed at 658 nm in consistence with the proposed model. Anisotropy values calculated around 650 nm at 3 ps after excitation are in the range 0.1 - 0.25 corresponding to an angle of 30 degree(s) - 45 degree(s) between the donor and acceptor transition dipole moments. A 500-fs absorption recovery and anisotropy decay process was obtained for C-phycocyanin trimers and explained by Forster energy transfer over 20.8 angstroms between neighboring (alpha) 84 and (beta) 84 chromophores of different monomeric subunits having similar absorption spectra and with a 65 degree(s) angle between their orientations. Energy transfer between violobilin ((alpha) 84) and phycocyanobilin ((beta) 84) chromophores was examined in donor and acceptor spectral regions of phycoerythrocyanin trimers, and was found to take 400 fs.

The transient microsecond absorption and photoconductivity in thin iodine(I)-doped (beta) - carotene (Car) films have been investigated. The laser nanosecond excitation ((lambda) equals 1064 and 512 nm) of the charge-transfer complex [Car⁺ (DOT) I_x^-] (where x equals 3,5,7) leads to the appearance of a new transient absorption spectrum at 1250 nm. This absorption decays by the hyperbolic low as a result of recombination of charged or neutral particles. The conductivity in doped films is possibly due to the flow of iodine anions in the external electric field. These charged particles arise in an unexcited state of the complex under the thermal influence of a laser pulse.

Earlier a comprehensive study of reaction substrate, product and their complexes with the enzyme has been carried out by means of stationary fluorescence spectroscopy. A hypothesis has been suggested according to which protein changes its conformation during the reaction. In this respect it is quite interesting to study both static and dynamic properties of the enzyme molecule. Luckily, according to genetic engineering data, luciferase molecule contains only one tryptophan residue, which makes it a very convenient object for the studies by means of time-resolved fluorescence spectroscopy. The scope of this paper is the study of luciferase tryptophan fluorescence. The methods applied were fluorescence spectrochronography and anisotropy decay analysis.

Recent photoselection measurements on bacteriorhodospin at high pH and high ionic strength showed a high change of the anisotropy factor in the ms region. If this is due to a conformation change in the moiety of retinal chromophore, one can expect that it is also reflected in the fluorescence decay of tryptophan residues in the close vicinity of the chromophore. Time resolved tryptophan fluorescence measurements were carried out at different stages of bacteriorhodospin photocycle by a pump and probe method. Early results indicate a correlation between the time constant of the faster phase of the fluorescence decay and the anisotropy factor.

The nature of the complex fluorescence of the indole chromophore of tryptophan is briefly reviewed. A recent molecular dynamics simulation of the relaxation of water surrounding the excited state of tryptophan demonstrates that this 'dielectric relaxation' aspect of tryptophan photophysics is well-understood. The relationship of this behavior in solutions to the photophysics of tryptophan in proteins is discussed. Another well-known feature of tryptophan fluorescence is the ability of a variety of 'collisional quenchers' to reduce the emission yield and lifetime. The relevance of this aspect of the photophysics of tryptophan to protein luminescence is also discussed.

Recombination intermediates of photodissociated CO of horse and human MbCO were investigated by using pump/probe time-resolved resonance Raman technique with 10 ns resolution. It became evident that the species with the (nu) _Fe-CO around 490 cm^-1 recombines CO much faster than that with (nu) _Fe-CO around 510 cm^-1, and that the 490 cm^-1 species is not a precursor of the 510 cm^-1 species, although the former and latter have been assumed to reflect the 'open' and 'closed' forms for the ligand entry. The faster recovery of the 490 cm^-1 species is attributed to the increased contribution of the recombination from the protein separated pair due to stabilization of CO under more hydrophobic protein environments.

Both experimental and theoretical results which indicate the presence of the triplet-excited region in retinoids and carotenoids are reviewed. The triplet- excited region is defined as a region where changes in the bond order take place, upon triplet excitation, toward its inversion, i.e., a double bond becomes more signal bond-like and a single bond becomes more double bond-like. (1) It has a span of approximately six conjugated double bonds, (2) it is localized in the central part of a conjugated chain, and (3) it triggers `cis' to `trans' isomerization in the T₁ state. The experimental and theoretical results include: (1) the T₁ Raman spectra of all-trans-retinal and its homologues; (2) the T₁-state isomerization of isomeric retinal; (3) the T₁-state isomerization of isomeric (beta) -carotene; (4) the PPP-SD-CI calculations of the bond orders of the carbon-carbon bonds in a set of model polyenes; and (5) the normal-coordinate analysis of the T₁ Raman lines of undeuterated and deuterated all-trans-retinal. Finally, (6) the biological implication of 'the triplet-excited region' is discussed in relation to the photo-protective function of a 15-cis carotenoid bound to the bacterial photoreaction center.

The application of resonance fluorescence energy transfer measurements for the analysis of conformational states of neuropeptide galanin revealed the coexistence of different structures in solution. This method is complementary to other spectroscopic structural methods like 2D NMR. The energy transfer between a fluorescence donor and acceptor gives direct information about the long distance separation of defined residues of a molecule in analogy to the nuclear Overhauser effect (NOE) which is limited to short distances. A special analysis of experimental results has been performed to get the distribution function of these distances instead of one averaged value for the investigated system. This approach enables the investigation of the coexistence of different sub-populations and the dynamic equilibrium between them. Due to its high time resolution of sub-nanoseconds, energy transfer measurements show the 'frozen' distribution of conformations which are not averaged during the time range characteristic for this process, in contrast to NMR experiments (milliseconds). Large mobility of the energy donor and acceptor leads to high precision of the distance determination. The distribution of conformations corresponds to the diversity of structures found for galanin with NMR spectroscopy.

In this work we have studied photon initiated processes of bacteriorhodopsin (BR) in Langmuir-Blodget (LB) monolayers and multilayers. As far as we know the present work is the first, where the photoresponse function and kinetics of BR is detected from a monolayer as well as the first report, where oriented monolayers have been used as a proton pump. The transient absorption spectra and the decay functions of multilayers were detected for films in water, in water vapor saturated air, and at room humidity. This had an influence on transient lifetimes. The method we have used to orient the bacteriorhodopsin molecules is based on the LB-technique and the fact that the protein orients during the coating process when a phospholipid binder is used. The photoelectric signals of the monolayers deposited by the different directions had opposite polarities. The amplitude due to five layers was five times as high as that due to a monolayer.

The picosecond evolution of the tertiary conformation of myoglobin following photodissociation of carbonmonoxymyoglobin was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13110 cm^-1 (763 nm) whose position is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin causing a blue shift of band III and a concomitant change in the protein conformation. The conformational relaxation reveals a viscosity dependence even at early times (< 2 ps), indicating that the primary motion of the protein involves a displacement of the surrounding solvent. This motion likely corresponds to a displacement of the F-helix. The ensuing relaxation is highly nonexponential, in agreement with recent molecular dynamics simulations. The conformational changes occurring near the heme likely affect the height of the barrier to ligand rebinding and may explain nonexponential rebinding of ligands at ambient temperatures.

The method of time-resolved polarization fluorescence spectroscopy combined with picosecond laser microscopy allows the development of adequate equipment for quantitative detection of porphyrin monomers, dimers and aggregates. In addition to steady state fluorescence microscopy which has been applied to study the intracellular distribution of photosensitizing porphyrins, time-resolved polarization microfluorometry provides more exact information about the size and shape of the porphyrins aggregates in different sites of the single living cell.

Optical Kerr effect (OKE) studies of myoglobin and water at wavelength of 627 nanometers with 45 femtosecond pulses have been performed. The nonresonant response of water consists of electronic, translational, librational and relaxational components. The low frequency components observed in the water OKE study are in qualitative agreement with previous depolarized light scattering (DLS) studies and molecular dynamics (MD) simulations. Operation on the edge of the Q-band of the myoglobin resulted in the pulse broadening to 100 fs. Two relaxational components, 195 fs and 2.4 +/- .2 ps were observed in both the carboxy and deoxy myoglobin samples studied. The 195 fs component is assigned to the OKE response of water while the 2.4 ps component is related to the transient birefringence of the myoglobin. A discussion of the origin of the transient signal as well as the calculation leading to the assignment to birefringence as opposed to dichroism is included. With this interpretation, the observed dynamics are related to the low frequency modes of the protein. Information on these modes is needed to understand the initial events that direct functionally important structural changes.

Multilayer LB films of chlorophyll a in L-(alpha) -phosphatidylcholine dipalmitoyl matrix have been prepared in order to study excitation energy relaxation processes. Chlorophyll a layers were deposited on a plate previously covered with 10 layers of stearic acid and separated from each other by three layers of stearic acid, and finally covered by 8 layers of stearic acid to avoid interlayer interaction and to protect the sample. Films were deposited on quartz plates with ends cut at an angle of 45 degree(s) to use the full inner reflection of the probing beam. Fluorescence of the samples were studied in the time range from 10 ps to 10 ns and transient absorption in time range from 100 ns to 100 microsecond(s) . Results are discussed from the point of two types of chlorophyll arrangements on the films.

Vibrational spectra of the double-stranded DNA genome of an icosahedral virus (P22) in packaged and unpackaged states have been accurately compared by digital difference Raman spectroscopy. The difference Raman spectrum, which is sensitive to structural changes at the level of < 2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking and the electrostatic environment of DNA phosphate groups. At the experimental conditions employed, the B form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid. However, the electrostatic environment of DNA phosphates is dramatically altered with packaging. The present results suggest a simple model for organization of the condensed dsDNA chromosomes of icosahedral viruses.

Time-resolved resonance Raman spectroscopy has been used to probe the lowest excited singlet and triplet states of free-base meso-tetraarylporphyrins. Both single-color and two-color time-resolved techniques have been used to probe the excited singlet states, while the triplet spectra were obtained in two-color experiments with a time-delay between pump and probe lasers of 30 ns. Marker bands for each of these three states have been identified. The information which qualitative analysis of the Raman spectra can give on the structural differences between these excited states is discussed.

Polyamines do not interact with neutral phospholipids (phosphatidylcholines) but they do interact in the presence of bivalent and trivalent cations. The effect of polyvalent cations is explained in terms of dehydration of the bilayer surface. Polyamines interact strongly with negatively charged phospholipids; the presence of bivalent and trivalent cations do not change sensitively the type of interaction between polyamines and phosphatidic acids.

Phthalocyanine interaction with cultured cells was studied by means of flow-cytometry, laser scanning confocal microscopy, fluorescent and transmission pH-microphotometry. Irradiation of Pc-loaded cells caused the following order of events: increase of Pc intracellular fluorescenceyieldsmitochondria damageyieldschanges of cell surface and/or volumeyields-plasma membrane potential disintegrationyields-severe damage of the cell plasma membrane and nuclear envelope. For all these processes it takes a few hours to reach a stationary value. Pc inside the cells is found in granules surrounding the Golgi apparatus and in the peripheral cytoplasmic region, last ones partially coinciding with the acidic cellular compartments. A temporary decrease of cytoplasmic and lysosomal pH is caused by Pc uptake and irradiation. Artificial acidation of the cytoplasm of Pc-loaded cells resulted in the enhancement of the efficiency of the photodynamic inactivation.

Surface-enhanced Resonance Raman scattering (SERRS) spectra were obtained for the reaction center complexes of the photosynthetic bacterium Rhodobacter sphaeroides (RC) and from photosystem II (PSII) of spinach, adsorbed on Ag and Au surfaces. These preliminary results demonstrate the considerable potential of this technique for selectively exciting resonance Raman scattering from reaction center components within their distinct absorption bands. Because of the high sensitivity afforded by SERRS, spectra could be measured from a single monolayer of reaction centers adsorbed on a metal surface. The surface-sensitivity provides new information indicating the topology of the PSII reaction center 47 kD light-harvesting protein complex. The activity of the PSII reaction center complex adsorbed on metal surfaces was monitored by photochemical reduction of cyt b-559. Measurement of fluorescence emission was shown to be a new and sensitive method for monitoring the structural and functional integrity of the PSII reaction center complex on the metal surface.

FT-IR transmission and reflection-absorption (RA) spectra were measured for Langmuir- Blodgett (LB) films of arachidic acid and the following conclusion could be reached from the measurements: (1) The structure of arachidic acid monolayer films deposited on CaF₂ plates changed with the surface pressure. (2) The structure of the monolayer films deposited on Au-evaporated glass slides did not show a marked dependency on the surface pressure, but the fraction of the trans configuration for the Ca-Cb-CequalsO group increased significantly with the pressure. (3) The carboxylic acid groups probably form a hydrogen bonding between the neighboring molecules in the monolayer films deposited on both CaF₂ plates and Au- evaporated glass slides. (4) Subcell packing of hydrocarbon chains in arachidic acid LB films on CaF₂ plates changes from hexagonal to orthorhombic upon going from the first monolayer to the second monolayer.

Our interest in the compounds lies in their special optical properties which distinguish themselves from typical metalloporphyrins such as heme and alkyl- or aryl-substituted synthetic porphyrins, e.g. octaethylporphyrin (OEP) or tetraphenylporphyrins (TPP). The compounds studied show blue-shifted weak Soret absorption in the near UV, but red-shifted STRONG Q band in the visible region. Resonance Raman spectra with excitations near Q absorption band display quite different enhancement pattern from those observed for the typical heme and model compounds (MOEP and MTPP).

The Surface Enhanced Raman Scattering (SERS) spectra of holopyruvate decarboxylase (PDC) and thiamine kinase (ThK) adsorbed on silver electrode were obtained. In contrast to the Raman, the SERS spectrum of PDC contained no modes of tryptophan residues, it indicates a removal of this moiety from the surface. In the SERS spectrum of ThK the bands belonging to ligands bound to the protein were observed. A correlation between the SERS signal intensity and the enzymatic activity of the ThK separate fraction and found. The influence of amino acids on SERS spectra of thiamine (Th) was studied to determine the possible composition on microsurrounding of coenzyme.

Near-infrared (NIR) excited FT-Raman spectra were measured in vivo for whole living photosynthetic bacteria. Rhodobacter (Rb.) sphaeroides G1C and R26 and Chlorobium (C.) limicola f. thiosulfatophilum. The obtained spectra, which were preresonanced with the Q_y bands of bacteriochlorophyll-a (BChl-a) or bacteriochlorophyll-c (BChl-c), did not contain contributions from major components of the bacteria such as proteins and lipids, and therefore, provided selectively structural information about BChl-a in the light-harvesting (LH) complexes (Rb. sphaeroides) or BChl-c in the chlorosome (C. limicola f. thiosulfatophilum) in a totally nondestructive manner.

Near-infrared (NIR) excited FT-Raman spectra were measured for intact, modified, and denatured lysozyme in solid states. The following conclusions could be reached from the Raman measurements. (1) FT-Raman spectrum of lysozyme treated at 90 degree(s)C and 10000 kPa is very close to that of the intact protein; only a slight shift of amide I band is observed. (2) Heat denaturation is clearly detected in the Raman spectrum for the protein treated at 120 degree(s)C; the Raman spectral changes suggest that most (alpha) -helix structure change into random coil structure upon the heat denaturation at 120 degree(s)C. (3) FT-Raman spectrum of lysozyme modified by glucose is close to that of intact lysozyme, indicating that the modification does not cause a marked secondary structure change. (4) The FT-Raman spectrum of the modified lysozyme by glucose treated at 120 degree(s)C and 15000 kPa is somewhat different in the amide I and III regions from that of the denatured lysozyme, suggesting that secondary structural changes caused by the heat denaturation are different from each other between the two denatured proteins.

Although a great deal of work has been done lately on unusual conformations of DNA, it is not always clear how relevant these structures are to the conformations found in the genome of living organisms. It appears that it an unusual form of DNA, such as the Z form, exists in the genome, it must exist as short sections of a specific sequence such as an alternating CG Z form sequence in the midst of very long sections of B-form DNA. To examine this problem of DNA structure, we have determined the minimum length of a string of alternating CG base pairs that can go into the Z form in the middle of a long section of B-form. Rules have been developed for determining the sequence lengths required to generate Z form in the presence of B form. Although Raman spectroscopy has been very useful as a semiquantitative indication of the conformation of proteins and nucleic acids it has not been by itself capable of giving precise structural parameters such as torsional angles or Cartesian coordinates. Recently we have shown how it is possible by a combination of Raman spectroscopy, thermodynamic analysis, hydrogen-deuterium exchange and molecular modeling to generate Cartesian coordinates for oliognucleotides in solution or in the crystal. In this paper we review the principles that lead to the actual structure determination. The possible extension of these techniques to the determination of the Cartesian coordinates for bent DNA is also discussed.

The structural characterization of five oligomers from the carrageenan family and three oligomers of agarose, has been the aim of the present work. The compounds were chosen so as to study principally the effect of the substitution by a sulphate group on the two main vibrations due to the two glycosidic linkages (the (alpha) 1,3 and the (beta) -1,4). The C-O stretching modes of the two quoted glycosidic linkages have previously been identified in the spectral region between 1120 and 1160 cm^-1, whereas the C-O-C bending mode has been demonstrated to occur at about 730 cm^-1.

Two Raman spectroscopic methods are presented to investigate the structures of membrane- bound peptides. In the first method, water accessibility to each tryptophan side chain of gramicidin A incorporated into phospholipid liposomes has been measured by use of hydrogen-deuterium exchange, selective isotopic labeling, and visible Raman spectroscopy. The water accessibility data are best explained by a newly proposed ion channel structure of gramicidin A. In the second method, we utilize ultraviolet resonance Raman intensity as a probe of environments of aromatic residues. Application of this method to enkephalin has shown that the tyrosine side chain is buried in the hydrophobic region of the lipid membrane while the phenylalanine side chain is not.

The conformational changes of poly(dI-dC) in aqueous solution, induced by increasing the NaCl concentration from 0.1 to 5 M and in the presence of Ni²⁺ ions, have been studied by resonance Raman spectroscopy. It was shown that: (1) the high salt structure of the polymer is A/B with a predominant A conformation, (2) the addition of 9 mM NiCl₂ in the high salt solution induces the Z-conformation of the polymer, (3) the high salt structure is not a mere intermediate between the B and Z conformations of poly(dI-dC).

Complexes of Cu(TMpy-P4) with natural and synthetic DNAs have been investigated by using resonance Raman spectroscopy with both visible and UV laser excitations. Visible excitations in the Soret or the Q bands of the porphyrin provides important vibrational intensity enhancements from the Cu-porphyrin moiety, while UV excitations favors the Raman intensity enhancements from the DNA moieties of the complexes. In addition, high-power laser excitations in the visible region induces the formation of complexes between the porphyrin in one of its excited states and DNA thymine/uracil residues: this new species is called an exciplex. Here we focus on the structural conditions required for the exciplex formation, and attempts are made to determine the nature of this exciplex. On the other hand, the DNA conformational changes induced by the interaction with the porphyrin in its ground- and/or excited states are explored and discussed.

The absorption line shape at frequency-degenerate two-wave mixing in an ensemble of three- level quantum systems with the doublet structure of the upper state has been calculated. The effect has been predicted of weak signal amplification at the saturating pump frequency in the regime of stationary interaction at homogeneous broadening of resonance absorption lines.

Molecular dynamics simulation of bacterial ferredoxin (Peptococcus aerogenes) at constant temperature have been performed. There are two iron-sulfur Fe₄S₄ clusters in the protein molecule of ferredoxin, which can be in various charge states. After the protein was changed instantaneously from oxidized state in reduced one, the unexpected behavior of the distance between clusters had been observed. Strong coupling of negative charged clusters to the protein matrix provides the clusters come closer together by approximately 0.5 angstroms when the charge on one of them have been increased, even though this charge change leads to an increase in the intercluster repulsion. The molecular dynamics trajectory have been used to specify a quasi-harmonical model to describe a low frequency motions of the protein. In the model each of amino acids and each of iron-sulfur clusters appears as an elementary unit.

We present here the results of molecular dynamic simulation of the dynamics of ethylene molecule (C₂H₄) excited by picosecond IR laser pulse. The parameters of the potential energy surface described by the molecular mechanics method, were determined using fitting method. With the parameters, the molecule's dynamics, the temporal dependence of its total energy, and its power emission spectrum were calculated.

A computer simulation procedure is developed for modeling intramolecular dynamics in polyatomic molecules. Electronic-vibrational excitation by ultrashort laser pulses (20 fs divided by 1 ps) is treated explicitly using quantum theory in harmonic approximation. MD simulation is used for studying the excited state dynamics. Stilbene photoinduced isomerization is modeled. Model potential energy surfaces (PES) for the ground and first excited singlet states are obtained using experimental absorption spectra in supersonic jet. Using a symmetrical along the torsional coordinate PES, it is shown that cis-stilbene undergoes the first stage of the isomerization reaction, i.e. transition to the twisted configuration, much faster than trans- stilbene, only due to the specific conformational properties.

To provide an insight how this only one more (or less) hydration site influences the pattern of hydration of this nucleic acid building blocks, computer simulation studies have been undertaken. The objectives of the molecular dynamics (MD) simulation studies of Cyd, dCyd and their phosphate salts in aqueous solutions were: 1. to compare the hydration pattern of the studied compounds in the ribo and deoxyribo series at the level of microscopic interactions, 2. to study intermolecular interactions in these molecular systems, 3. to explain the unexpectedly big difference in solubiity between the studied compounds in the ribo and deoxyribo series.

We have studied the kinetics of irreversible diffusion-limited aggregation of particles, randomly walking on a square lattice. Diffusion coefficients of particle aggregates varied with size according to Saffmann-Delbruck equation. For three initial concentrations of particles, we present the time dependences of cluster size and mobility distributions, and of the zeroth and first moments of particle distributions. We show that time variation of the number of clusters can be, to a good approximation, described by a Smoluchowski-type kinetics for the bimolecular reaction A + A yields A in two dimensions.

Fast conformational changes in B-DNA and Z-DNA have been studied using MD simulation techniques. The double helix right-handed B-DNA is simulated in a high-salt aqueous NaCl (2.5 M) solution, while the double helix left-handed Z-DNA is put in a non-salt solution with only potassium ions, enough to electro-neutralize the phosphates. The fast conformational changes are followed by studying the changes in the backbone and sugar torsional angles, as well as, the glycosidic bond angle. The B-DNA goes over to A form during the simulation of 30 pico seconds and the Z-DNA exhibits a Z(I) yields Z(II) transition within 70 pico seconds.

In order to calculate the characteristics of vibrational quantum beats we use the Heisenberg (time-domain) approach to the nonlinear properties of vibronic spectra. While using this approach, the intensity of emission may be expressed as the convolution-type integral that contains the nonlinear-response function. To evaluate this integral the Laplace method is applied. In so doing the value of the Stokes shift is used as a natural great parameter. This allowed the simple expressions describing the manifestations of vibrational quantum beats to be obtained.

The mathematical model is presented of low-intensity laser radiation influence on the dynamics of the X-irradiated bone marrow tissue erythropoiesis. The process of recovering in postradiation stage of the cells of erythroid series has been investigated for various parameters of laser emission.

This report addresses the energy migration problem in the light-harvesting antenna systems of photosynthetic objects. Both linear and nonlinear relaxation cases are discussed. In the linear case, the spectral inhomogeneity is taken into account by averaging the Master equation for the excitation over the spectrally inhomogeneous distribution of pigment molecules. That reformulates the Master equation as the excitation diffusion in the energy space. The stationary solution of this equation enables the determination of the Stokes shift dependence on homogeneous and inhomogeneous spectral bandwidths. The nonlinear relaxation is considered at the singlet-singlet annihilation conditions. The analysis of the fluorescence quantum yield, the excitation delay kinetics as well as the changes of spectral properties of the object under consideration as a function of the excitation intensity, demonstrates the possibility to determine the energy migration parameters. An important conclusion of this analysis is that the real pulse-shape of the excitation as well as the population of higher excited states during the nonlinear annihilation have to be taken into account.

The diffusion of a single molecule through a sharply focused laser beam has been observed. The diffusion was observed by recording photoelectron bursts of single molecules in transit through the laser beam with Gaussian intensity profile. Up to 20 photoelectrons were detected for Rhodamin 6G in a transit time of 40 .ts which corresponds to the diffusion time through a volume element of 2x10'61. From the intensity correlation the probability for the occurence of one molecule in the volume element of observation is determined. The existence of one molecule events is also verified by the measured and calculated photoelectron number distribution.

With infrared transient absorption spectroscopy we have studied how energy migrates through the light-harvesting antenna of photosynthetic purple bacteria, and how the energy is trapped by the reaction center. In Bchl a-containing purple bacteria the light-harvesting (LH) antenna is highly heterogeneous, consisting of several spectroscopically distinct pigments. Energy transfer among these pigments occurs on several different time-scales. The overall trapping time at room temperature is about 60 ps, while energy transfer from the high-energy to the low-energy pigments of the lightharvesting antenna only takes 10 ps (measured at 77 K). This implies that trapping is relatively slow, 35 ps at 77 K and probably not much faster at room temperature. Energy transfer among neighboring Bchl molecules within a pigment-protein complex occurs on the 1 ps to subpicosecond time scale. For the Bchl b-containing purple bacterium Rps.viridis the results show that the antenna is homogeneous, and trapping can approximately be described by a randomwalk model, provided that the light-harvesting antenna and reaction center are more tightly coupled than in the Bchl a containingpurple bacteria.