Picosecond primary photoprocesses of bilirubin bound to human serum albumin.

We measure the fluorescence quantum yield of bilirubin bound to its highest-affinity site on human serum albumin to increase from about 0.001 near room temperature to 0.5 at 77 K. The quantum yield for configurational (Z leads to E) photoisomerization about the meso double bonds concomitantly decreases from about 0.22 to less than 0.01 over the same temperature range in reciprocal relationship to the fluorescence yield. Transient absorption spectra recorded after excitation with a 0.5-ps pulse of 305-nm light decay with a lifetime of 19 +/- 3 ps at 22 degrees C and 35 +/- 7 ps at 2 degrees C. Bilirubin undergoes the same photoisomerization reaction in chloroform solution, in which a similar short-lived (17 +/- 3 ps at 22 degrees C) transient is observed. From these and other data we conclude that configurational isomerization of bilirubin is the predominant nonradiative pathway that competes with pigment fluorescence, that photoisomerization proceeds via a short-lived (much less than 18 ps) partially twisted excited-singlet-state intermediate, and that bilirubin remains relatively unihibited with respect to photoisomerization when bound to human serum albumin.     

Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.     

Nanometers-Thick Ferromagnetic Surface Produced by Laser Cutting of Diamond

In this work, we demonstrate that cutting diamond crystals with a laser (532 nm wavelength, 0.5 mJ energy, 200 ns pulse duration at 15 kHz) produced a ≲20 nm thick surface layer with magnetic order at room temperature. We measured the magnetic moment of five natural and six CVD diamond crystals of different sizes, nitrogen contents and surface orientations with a SQUID magnetometer. A robust ferromagnetic response at 300 K was observed only for crystals that were cut with the laser along the (100) surface orientation. The magnetic signals were much weaker for the (110) and negligible for the (111) orientations. We attribute the magnetic order to the disordered graphite layer produced by the laser at the diamond surface. The ferromagnetic signal vanished after chemical etching or after moderate temperature annealing. The obtained results indicate that laser treatment of diamond may pave the way to create ferromagnetic spots at its surface.     

Cone visual pigments are present in gecko rod cells.

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.     

Evidence that a distribution of bacterial reaction centers underlies the temperature and detection-wavelength dependence of the rates of the primary electron-transfer reactions

The rates of the primary electron-transfer processes in Rhodobacter sphaeroides reaction centers have been examined in detail by using 150-fs excitation flashes at 870 nm. At room temperature the apparent time constants for both initial charge separation (P* --> P+BPhL-) and subsequent electron transfer (P+BPhL- --> P+QA-) are found to encompass a range of values (approximately 1.3-4 ps and approximately 100-320 ps, respectively), depending on the wavelength at which the kinetics are followed. We suggest this reflects a distribution of reaction centers (or a few conformers), having differences in factors such as distances or orientations between the cofactors, hydrogen bonding, or other pigment-protein interactions. We also suggest that the time constants observed at cryogenic temperatures (approximately 1.3 and approximately 100 ps, respectively, with much smaller or negligible variation with detection wavelength) do not reflect an actual increase in the rates with decreasing temperature but rather derive from a shift in the distribution of reaction centers toward those in which electron transfer inherently occurs with the faster rates.     

Ultrafast spectroscopy of the visual pigment rhodopsin.

We report on time-resolved absorption studies of the bovine visual pigment rhodopsin with subpicosecond resolution at room temperature. Our data show that bathorhodopsin, rhodopsin's early photoproduct, is photochemically formed in 3.0 +/- 0.7 ps. The data suggest that bathorhodopsin formation is kinetically preceded by two species along the rhodopsin-to-bathorhodopsin reaction coordinate. The first is identified with the vertically excited Franck-Condon state. This decays with an approximately 200-fs lifetime to an intermediate, which then decays to bathorhodopsin in 3.0 ps. We assign this intermediate to be an excited state transient near 90 degrees along the 11-12 torsional coordinate of rhodopsin's chromophore. Exchange of rhodopsin's exchangeable protons for deuterons does not affect the observed dynamics. These observations are both qualitatively and quantitatively consistent with molecular dynamics calculations, which model the rhodopsin to bathorhodopsin phototransition as a cis-trans isomerization along the 11-12 torsional coordinate of rhodopsin's chromophore.     

Direct observation of vibrational coherence in bacterial reaction centers using femtosecond absorption spectroscopy.

It is shown that vibrational coherence modulates the femtosecond kinetics of stimulated emission and absorption of reaction centers of purple bacteria. In the DLL mutant of Rhodobacter capsulatus, which lacks the bacteriopheophytin electron acceptor, oscillations with periods of approximately 500 fs and possibly also of approximately 2 ps were observed, which are associated with formation of the excited state. The kinetics, which reflect primary processes in Rhodobacter sphaeroides R-26, were modulated by oscillations with a period of approximately 700 fs at 796 nm and approximately 2 ps at 930 nm. In the latter case, at 930 nm, where the stimulated emission of the excited state, P*, is probed, oscillations could only be resolved when a sufficiently narrow (10 nm) and concomitantly long pump pulse was used. This may indicate that the potential energy surface of the excited state is anharmonic or that low-frequency oscillations are masked when higher frequency modes are also coherently excited, or both. The possibility is discussed that the primary charge separation may be a coherent and adiabatic process coupled to low-frequency vibrational modes.     

Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium.

Mutant Halobacterium halobium strains deficient in all previously reported rhodopsin-like pigments show phototaxis responses comparable to those of wild-type strains. Spectroscopic analysis reveals the presence of a third retinal-containing pigment in the cells and their membrane fractions. It undergoes a photoreaction cycle with a half-time of approximately equal to 1 sec at room temperature and at physiological light intensities the photostationary state of the pigment consists of two species, one absorbing in the 580- to 590-nm region and the other at 373 nm, both of which are photoactive. Illumination of the long-wavelength species generates the 373-nm intermediate, which upon photoexcitation reconverts to the long-wavelength form. Therefore, changes in the relative light intensities in the long- and short-wavelength regions of the visible spectrum cause opposing shifts in the photostationary state. The spectral sensitivity of this pigment correlates with the color-discriminating phototaxis sensitivities of this organism and strongly suggests that it is the sensory photoreceptor.     

Fluorescent products from aging Drosophila melanogaster: an indicator of free radical lipid peroxidation damage

The fluorescent pigments in chloroform-methanol extracts of adult Drosophila melanogaster maintained at three temperatures increased linearly with age. The rate of pigment accumulation was highly significant, was greater for males than for females, and was greater for flies kept at 26·7°C and 30·0°C than at 22·2°C. Maximum levels of pigment were attained earliest in flies maintained at 30·0°C, although a clear relationship between temperature and fluorescence was not shown.The fluorescence spectra had excitation maxima at 368–370 nm and emission maxima at 445–448 nm. Chemical characterization of the pigments suggested a Schiff-base chromophore, a product of lipid peroxidation. This conclusion was based upon the selective extraction of the pigments with chloroform-methanol; the concurrence of spectral peaks with those of known Schiff-base chromophores; and the characteristic decrease in fluorescence in the presence of base or the metal chelate, europium (III)-2,2,6,6-tetramethyl heptane-1,3-dione.     

Reduction of laser-induced pathologic tissue injury using pulsed energy delivery

Continuous-wave (CW) laser irradiation of cardiovascular tissues is characterized by 2 distinctive histologic findings: a superficial zone of coagulation necrosis and a subjacent zone of polymorphous lacunae. The present investigation was designed to determine whether such injury could be eliminated by altering the temporal profile of laser energy delivery. One hundred forty-five myocardial slices were irradiated with an air-tissue interface using CW laser irradiation at wavelengths of 488 to 515 nm (argon), 1,064 nm (Nd-YAG) and 10,600 nm (CO2). Pulsed laser irradiation included 248 nm (excimer); 355, 532 and 1,064 nm (Nd-YAG); and 515 nm (mode-locked argon). Energy profiles in the pulsed mode included a range of repetition rates (1 Hz to 256 MHz), pulse duration (0.2 to 358 ns) and pulse energies (2 nJ to 370 mJ). Resultant average powers were 0.1 to 38 W. Grossly visible charring of myocardial tissue was observed at all laser wavelengths when the laser energy profile was CW or pulsed at high repetition rates (more than 2 KHz) and low pulse energies (less than 3 mJ) independent of the wavelengths used. In contrast, when laser energy was pulsed at low repetition rates (less than 200 Hz) and large pulse energies (more than 10 mJ), neither gross nor histologic signs of thermal injury were observed. Pathologic injury associated with laser-induced tissue ablation may thus be substantially reduced by use of pulsed energy delivery at low repetition rates. Potential advantages of pulsed laser energy include a more benign healing process, a less thrombogenic surface, and improved preservation of structural tissue integrity.     

The interaction between excimer laser energy and vascular tissue

The effects of XeF1 excimer laser on isolated normal and atherosclerotic aorta were studied. Experiments were performed in flowing water at constant temperature, flow rate, water depth, pulse width (10 nsec), wavelength (351 nm), beam size (1 mm2) and focal length (50 cm). The number of pulses, the pulse energy, and the pulse frequency were varied, and the vascular tissue was studied histologically. The following observations were made: tissue ablation required a minimum threshold pulse energy and was nonlinearly proportional to the number of pulses and the pulse energy delivered; precise tissue ablation occurred at low pulse frequencies, but changes resembling a thermal process were seen as pulse frequency increased; calcified plaque was more photoresistant than atheroma or normal vessel; excimer laser energy was markedly attenuated by blood; and the time interval between pulses and high peak power are related to the precision of ablation by pulsed excimer laser. It is concluded that excimer laser can rapidly and precisely ablate vascular tissue by a photothermal process.     

Energy transfer and trapping in photosystem I reaction centers from cyanobacteria.

A mutant strain of the cyanobacterium Synechocystis 6803, TolE4B, was constructed by genetic deletion of the protein that links phycobilisomes to thylakoid membranes and of the CP43 and CP47 proteins of photosystem II (PSII), leaving the photosystem I (PSI) center as the sole chromophore in the photosynthetic membranes. Both intact membrane and detergent-isolated samples of PSI were characterized by time-resolved and steady-state fluorescence methods. A decay component of approximately 25 ps dominates (99% of the amplitude) the fluorescence of the membrane sample. This result indicates that an intermediate lifetime is not associated with the intact membrane preparation and the charge separation in PSI is irreversible. The decay time of the detergent-isolated sample is similar. The 600-nm excited steady-state fluorescence spectrum displays a red fluorescence peak at approximately 703 nm at room temperature. The 450-nm excited steady-state fluorescence spectrum is dominated by a single peak around 700 nm without 680-nm "bulk" fluorescence. The experimental results were compared with several computer simulations. Assuming an antenna size of 130 chlorophyll molecules, an apparent charge separation time of approximately 1 ps is estimated. Alternatively, the kinetics could be modeled on the basis of a two-domain antenna for PSI, consistent with the available structural data, each containing approximately 65 chlorophyll a molecules. If excitation can migrate freely within each domain and communication between domains occurs only close to the reaction center, a charge separation time of 3-4 ps is obtained instead.     

The first step in vision occurs in femtoseconds: complete blue and red spectral studies.

Femtosecond transient absorption measurements of the cis-trans isomerization of the visual pigment rhodopsin clarify the interpretation of the dynamics of the first step in vision. We present femtosecond time-resolved spectra as well as kinetic measurements at specific wavelengths between 490 and 670 nm using 10-fs probe pulses centered at 500 and 620 nm following a 35-fs pump pulse at 500 nm. The expanded spectral window beyond that available (500-570 nm) in our previous study [Schoenlein, R. W., Peteanu, L. A., Mathies, R. A. & Shank, C. V. (1991) Science 254, 412-415] provides the full differential absorption spectrum of the photoproduct as a function of delay time after photolysis. The high time-resolution data presented here contradict an alternative interpretation of the rhodopsin photochemistry offered by Callender and co-workers [Yan, M., Manor, D., Weng, G., Chao, H., Rothberg, L., Jedju, T. M., Alfano, R. R. & Callender, R. H. (1991) Proc. Natl. Acad. Sci. USA 88, 9809-9812]. Our results confirm that the red-shifted (lambda max approximately 570 nm) photo-product of the isomerization reaction is fully formed within 200 fs. Subsequent changes in the differential spectra between 200 fs and 6 ps are attributed to a combination of dynamic ground-state processes such as intramolecular vibrational energy redistribution, vibrational cooling, and conformational relaxation.     

Formation and Decay of Prelumirhodopsin at Room Temperatures

We have excited detergent-solubilized bovine rhodopsin at room temperature with 530-nm light pulses from a mode locked laser, and have observed the appearance and decay of a transient species that absorbs more strongly at 560 nm than does ground-state rhodopsin. Our data show that the absorbing intermediate appears in a time that is at least as short as the experimental resolution (about 6 psec) and decays with a life time of about 30 nsec. The extremely fast risetime supports the hypothesis that prelumirhodopsin is the product of the primary photoprocess.     

Photochemistry and spectroscopy of a five-chlorophyll reaction center of photosystem II isolated by using a Cu affinity column

A reaction center of photosystem II was isolated from Pisum sativum by using immobilized metal affinity chromatography. This reaction center is photochemically active and has a room temperature Qgamma chlorophyll (Chl) absorption band peaking at 677.5 nm. From HPLC analysis, the pigment stoichiometry was suggested to be 5 Chls per 1 beta-carotene per 2 pheophytins. Low-temperature absorption measurements at 77 K were consistent with the removal of one of the Chls associated with the usual form of the reaction center isolated by using ion-exchange chromatography. Transient absorption spectroscopy on the picosecond time scale indicated that the Chl removed belongs to a pool of Chl absorbing at approximately 670 nm (C670II) that transfers energy relatively slowly to the primary donor P680 in support of our recently proposed model. The results also support the previous conclusion that radical pair formation is largely associated with a 21-ps time constant when P680 is directly excited and that the identity of C670II is likely to be peripherally bound Chls possibly ligated to conserved His residues at positions 118 on the D1 and D2 proteins.     

Primary electron transfer reactions in modified reaction centers from Rhodopseudomonas sphaeroides

Absorption spectra were measured by means of an optical multichannel analyzer in Rhodopseudomonas sphaeroides R-26 reaction centers (RCs) modified by treatment with NaBH₄ at various times (≥1 ps) after the onset of a short excitation flash at 880 nm. Most of these RCs (75-95%) have only one “monomeric” bacteriochlorophyll-800 (B₁) molecule and are as active as the original RCs. The duration of the excitation and measuring pulses was ≈33 ps. If the center of the excitation pulse preceded the center of the measuring pulse by 36-40 ps, the formation of a state P^E (early state), which is converted to the state P^F (P⁺ bacteriopheophytin^-) in 4 ± 1 ps (1/e time), was observed. Also the kinetics and the spectrum of the stimulated emission (reflecting the kinetics and the emission spectrum of the excited state P^*) were determined. The difference spectrum of the state P^E approximately equals the sum of the spectra of the states P^* (≈65%) and ¹[P⁺B₁^-] (≈35%). This indicates that B₁^- is an intermediate in the electron transfer from P^* to bacteriopheophytin, H₁, transferring this electron with a rate constant of (4 × 0.35 ps)^-1 = 7 × 10¹¹ s^-1.     

Bistable UV pigment in the lamprey pineal

Lower vertebrates can detect UV light with the pineal complex independently of eyes. Electrophysiological studies, together with chromophore extraction analysis, have suggested that the underlying pigment in the lamprey pineal exhibits a bistable nature, that is, reversible photoreaction by UV and visible light, which is never achieved by known UV pigments. Here we addressed the molecular identification of the pineal UV receptor. Our results showed that the long-hypothesized pigment is a lamprey homologue of parapinopsin, which exhibits an absorption maximum at 370 nm, in the UV region. UV light causes cis-trans isomerization of its retinal(2) chromophore, forming a stable photoproduct having an absorption maximum at 515 nm, in the green region. The photoproduct reverts to the original pigment upon visible light absorption, showing photoregeneration of the pigment. In situ hybridization showed that parapinopsin is selectively expressed in the cells located in the dorsal region of the pineal organ. We successfully obtained the hyperpolarizing responses with a maximum sensitivity of approximately 380 nm from the photoreceptor cells at the dorsal region, in which the outer segment was clearly stained with anti-parapinopsin antibody. These results demonstrated that parapinopsin is the pineal UV pigment having photointerconvertible two stable states. The bistable nature of the parapinopsin can account for the photorecovery of the pineal UV sensitivity by background green light in the lamprey. Furthermore, we isolated the parapinopsin homologues from fish and frog pineal complexes that exhibit UV sensitivity, suggesting that parapinopsin is a common molecular basis for pineal UV reception in the vertebrate.     

Differential effects of light wavelength in phase advancing the melatonin rhythm

Shorter wavelength light has been shown to be more effective than longer wavelengths in suppressing nocturnal melatonin and phase delaying the melatonin rhythm. In the present study, different wavelengths of light were evaluated for their capacity to phase advance the saliva melatonin rhythm. Two long wavelengths, 595 nm (amber) and 660 nm (red) and three shorter wavelengths, 470 nm (blue), 497 nm (blue/green), and 525 nm (green) were compared with a no-light control condition. Light was administered via a portable light source comprising two light-emitting diodes per eye, with the irradiance of each diode set at 65 microW/cm(2). Forty-two volunteers participated in up to six conditions resulting in 15 per condition. For the active light conditions, a 2-hr light pulse was administered from 06:00 hr on two consecutive mornings. Half-hourly saliva samples were collected on the evening prior to the first light pulse and the evening following the second light pulse. The time of melatonin onset was calculated for each night and the difference was calculated as a measure of phase advance. The shorter wavelengths of 470, 495 and 525 nm showed the greatest melatonin onset advances ranging from approximately 40-65 min while the longer wavelengths produced no significant phase advance. These results strengthen earlier findings that the human circadian system is more sensitive to the short wavelengths of light than the longer wavelengths.     

Interaction of glyoxal and methylglyoxal with biogenic amines.

Glyoxal and methylglyoxal interact with biogenic amines and form biologically active free radicals. Electron spin resonance absorption of the radical at room temperature is characterized by a signal at g equals 2.004 with peak-to-peak width of 29 G. An optical absorption at 400 nm with molar absorptivity of 23,000 accompanies the formation of the radical. The dry powdered preparation of the same reaction, which is considered to be the seconardy product, gives an electron spin resonance signal much narrower and 1/200 in intensity compared with the one in solution. Similarly the 400 nm absorption intensity is 1/8 that of the primary product. Possible biological significance of the primary and the secondary product, in relation to muscular dystrophy and photophosphorylation, is discussed.     

Tunable Laser Resonance Raman Spectroscopy of Bacteriorhodopsin

Bacteriorhodopsin is a rhodopsin-like protein found in the cell membrane of Halobacterium halobium. It shows an absorption maximum at 570 nm and, in the light, undergoes cyclic spectral changes which include a relatively long-lived complex absorbing maximally at 412 nm. Excitation profiles have been obtained with several laser frequencies for two vibrations in the resonance Raman spectrum of bacteriorhodopsin. The results show that the Schiff base retinylidene lysine linkage is protonated in the 570 nm complex and that in the 412 nm complex it is unprotonated. The 412 nm complex must be present at appreciable concentrations when bacteriorhodopsin is exposed to high-energy argon ion laser light of the Raman spectrophotometer at room temperature. We conclude that the observed C=N stretch at 1622 cm(-1) in the room temperature spectra, which in an earlier study by Mendelsohn was interpreted as evidence for an unprotonated linkage in bacteriorhodopsin, results from the presence of the 412 nm complex.