Single shot laser flash photolysis with a fibre-coupled reference beam monitor.

In the standard nanosecond laser photolysis method for kinetic studies, a Q-switched laser generates transient species, and absorption spectrophotometry provides a measure of their concentrations. The sample is placed between the monitoring source (a pulsed xenon arc or a flash lamp) and a monochromator, and a photomultiplier tube (PMT) is used for measuring the intensity of the light leaving the exit slit of the monochromator. With this (single-beam) arrangement, the laser-induced change in the absorbance of the sample, ΔA, can be calculated only if the intensity of the monitoring beam remains constant during the time interval of interest. When this condition is not fulfilled, a second measurement of the PMT output is made after blocking the path of the laser beam, but shot-to-shot variations in the output of the monitoring source vitiate the analysis when ΔA is small. To overcome this problem, double-beam versions were developed in the last century, but the single-beam version still enjoys greater popularity. With a view to making the double-beam method easily implementable, some simple modifications are introduced, which permit the conversion of an existing laser kinetic spectrometer into a double-beam variant (with one or two monochromators).

Photomorphogenesis and pigment induction in lentil seedling roots exposed to low light conditions.

Although roots are normally hidden in soil, they may inadvertently be exposed to low light levels in experiments or in natural conditions through cracks or light transmittance through the soil. Light has been implicated in root morphogenesis. Thus, effects of low light conditions on lentil (Lens culinaris L. cv. Verte du Puy) root morphology and root pigmentation were studied. Lentil seedlings were grown in peat or transparent, nutrient-fortified agar at a 12-h light (PAR 240 µmol · m(-2) · s(-1)), 12-h dark cycle. Roots were exposed to low levels (≈ 1-10 µmol · m(-2) · s(-1)) of broadband white light, either directly or indirectly by aboveground light penetrating the growth medium. Control roots were grown in darkness. In situ spectroscopy was used to measure transmittance and reflectance spectra of intact root tissue by mounting the upper part of the primary root directly in a spectrophotometer equipped with an integrating sphere attachment. The transmittance and reflectance spectra were used to calculate the in situ root absorbance spectrum. Absorbance bands were found in the regions 480-500 nm and 650-680 nm, possibly due to low levels of root-localised carotenoids and chlorophylls, respectively. Low light levels (≈ 1-10 µmol · m(-2) · s(-1) ) transmitted through the growth medium significantly increased root pigment concentration and root biomass, and altered root morphology by enhancing lateral root formation and inhibiting root elongation relative to roots grown in complete darkness. The light-induced changes in root morphogenesis and pigmentation appear to be primarily due to upper root light perception.

Triplets, radical cations and neutral semiquinone radicals of lumiflavin and riboflavin: an overhaul of previous pump-probe data and new multichannel absolute absorption spectra.

Though long-lived transient photoproducts of flavins have received much attention over the last five decades, their spectroscopic characterization remains rudimentary and altogether inconclusive. Lumiflavin and riboflavin have therefore been reexamined, using nanosecond laser photolysis and multichannel detection over a wide spectral range (230-900 nm); differential absorption spectra of flavin solutions in aqueous buffer at neutral pH have been recorded; radical cations and semiquinone radicals were generated by quenching the triplets with C (NO(2))(4) (electron acceptor) and NO(2)(-) (electron donor), respectively. The problem of estimating the extent of ground state depletion after a single flash has been solved by developing a new strategy that provides reliable lower and upper bounds for the depletion (and for the molar absorption coefficients of the transient species of interest). The spectra of the neutral radicals and triplets of the two flavins are almost identical, but those of the two cations are not.

Comparison of the photochemical behaviors of α-tocopherol and its acetate in organic and aqueous micellar solutions.

Photoionization is known to take place when α-tocopherol (TOH) is excited to the S(1) state in a polar medium. It has been previously suggested that TO(•) is formed only as a result of proton release by TOH(•+), a process that is expected to occur, in a protic solvent, on the subnanosecond time scale. Recent redeterminations of the molar absorption coefficients of e(aq)(­) (Hare J. Phys. Chem. A 2010, 114, 1766) and of TOH(•+) and TO(•) (Naqvi J. Phys. Chem. A 2010, 114, 10795) have paved the way for testing the above suggestion, even if subnanosecond time resolution is not available, since it implies the equality of [e(aq)(­)](0) and [TO(•)](0), where [···](0) denotes the concentration of the enclosed species immediately after a nanosecond laser pulse. Nanosecond pump-probe spectroscopy of TOH in aqueous micellar solution (AMS) and two organic solvents with similar polarities (acetonitrile and methanol) has revealed that prompt formation of TO(•) through dissociation (TOH + hν → TO(•) + H(•)) is not negligible even in AMS. In acetonitrile, TOH(•+) and TO(•) are formed with comparable yields, and the former converts quantitatively into TO(•) within 15 µs. In methanol, TO(•) was observed, but no evidence was found for electron ejection from TOH. Only one photoproduct, namely TO(•), could be detected when α-tocopherol acetate (TOAc) was excited to the S(1) state in several polar and nonpolar solvents; TOAc has been found to be a more efficient energy degrader than TOH.

Spectroscopic characterization of neutral and cation radicals of α-tocopherol and related molecules: a satisfactory denouement.

Notwithstanding the facile occurrence of one-electron oxidation in α-tocopherol and its acetate (TOH and TOAc, respectively), and despite the remarkable stability, under appropriate conditions, of the oxidation products (TOH(•+), TO(•), and TOAc(•+)), their spectroscopic characterization is in an unsatisfactory state, calling for a fresh attempt to acquire reliable data. A new, model-free method is developed for analyzing time-resolved spectra showing the progress of the reaction TOH + R(•) → TO(•) + RH, where R(•) is a stable free radical. The resulting absorption coefficients of TO(•) in dichloromethane and hexane are in severe disagreement with some recent values derived from stopped-flow spectrophotometry. The discrepancy is traced to the imposition of boundary conditions that do not take proper account of the dead time of the apparatus; when multiplied by a factor of two, the stopped-flow data fall mostly in the range ε = (7.5 ± 0.5) × 10(3) M(-1) cm(-1), conforming with the results of this study and the values found by Boguth and Niemann in 1969. Absorption spectra of the radical cations produced (electro)chemically are found to be reliable only in the visible region. Incomplete conversion of the parent compound to the radical cation, an obstacle to the determination of absorption coefficients from electrochemical studies, is circumvented by combining EPR and optical spectroscopy. The absorption coefficients of TOH(•+) and TOAc(•+), determined in this manner, are found to be, respectively, 1.6 × 10(4) and 1.3 × 10(4) M(-1) cm(-1), in accord with the values found first through similar means.

Estimation of leaf transmittance in the near infrared region through reflectance measurements.

A method, which allows one to compensate for the incomplete collection of transmitted light (T) by an integrating sphere, has recently been developed, and shown to be reliable provided that the absorptance (A) of the leaf in the NIR region (750-800 nm) can be neglected, allowing one to set R+T=1, where R denotes the reflectance; this implies that proper compensation can only be applied to healthy leaves, which do not absorb in the NIR region. To overcome this limitation, the feasibility of an alternative, requiring neither measurements of T nor an elaborate analysis of radiative transport through a leaf, is explored. Not surprisingly, this simplistic alternative provides results which (in general) do not agree with those found by using the compensation method, but the two approaches converge in the spectral regions where absorptance is low (that is, where R+T> or =0.9). The "T-through-R" method, as described here, thus provides an additional check on the correction factor used in conjunction with the integrating sphere, and extends the applicability of the compensation method to situations where NIR absorptance is not negligible, e.g., in the presence of 'browning' pigments produced upon the oxidation of polyphenols during leaf senescence.

Absorption and scattering of light by suspensions of cells and subcellular particles: an analysis in terms of Kramers-Kronig relations.

An analysis of light scattering from suspensions of pigmented cells and particles is undertaken, and a practicable method, requiring only the experimentally measured extinction spectra, is documented. The analysis is based on two premises: Absorption and selective scattering from a single pool of pigments satisfy the Kramers-Kronig relations, which imply that one can be derived from the other; pigment-free domains contribute only nonselective scattering. This approach succeeds in simulating the spectra of many systems (human erythrocytes, chloroplasts and subchloroplast particles, algal cells) over a wide spectral range. Other, less favourable, cases are also examined, but even here the apparent discrepancy between theory adn experiment provides some clues that cannot be gleaned from absorption data alone.

Does a leaf absorb radiation in the near infrared (780-900 nm) region? A new approach to quantifying optical reflection, absorption and transmission of leaves.

The following question is addressed here: do healthy leaves absorb, as the spectra published over the last 50 years indicate, some 5-20% of incident radiation in the 780-900 nm region? The answer is found to be negative, and previous findings result from incomplete collection of the transmitted light by the detection system (even when the leaf is placed next to, but outside, the entrance port of an integrating sphere). A simple remedy for this inherent flaw in the experimental arrangement is applied successfully to leaves (of 10 unrelated species) differing in thickness, age and pigment content. The study has shown that, from an optical standpoint, a leaf tissue is a highly scattering material, and the infinite reflectance of a leaf is exceedingly sensitive to trace amounts of absorbing components. It is shown that water contributes, in a thick leaf (Kalanchoe blossfeldiana), an easily detectable signal even in the 780-900 nm region. The practical benefits resulting from improved measurements of leaf spectra are pointed out.

Nanosecond laser photolysis studies of chlorosomes and artificial aggregates containing bacteriochlorophyll e: evidence for the proximity of carotenoids and bacteriochlorophyll a in chlorosomes from Chlorobium phaeobacteroides strain CL1401.

Time-resolved, laser-induced changes in absorbance, delta A(lambda; t), have been recorded with a view to probing pigment-pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes delta A(lambda; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 microseconds), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 microsecond (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.

Electronic energy transfer involving carotenoid pigments in chlorosomes of two green bacteria: Chlorobium tepidum and Cholroflexus aurantiacus.

Electronic energy transfer processes in chlorosomes isolated from the green sulphur bacterium Chlorobium tepidum and from the green filamentous bacterium Chloroflexus aurantiacus have been investigated. Steady-state fluorescence excitation spectra and time-resolved triplet-minus-singlet (TmS) spectra, recorded at ambient temperature and under non-reducing or reducing conditions, are reported. The carotenoid (Car) pigments in both species transfer their singlet excitation to bacteriochlorophyll c (BChlc) with an efficiency which is high (between 0.5 and 0.8) but smaller than unity; BChlc and bacteriochlorophyll a (BChla) transfer their triplet excitation to the Car's with nearly 100% efficiency. The lifetime of the Car triplet states is approximately 3 micros, appreciably shorter than that of the Car triplets in the light-harvesting complex II (LHCII) in green plants and in other antenna systems. In both types of chlorosomes the yield of BChlc triplets (as judged from the yield of the Car triplets) remains insensitive to the redox conditions. In notable contrast the yield of BChlc singlet emission falls, upon a change from reducing to non-reducing conditions, by factors of 4 and 35 in Cfx. aurantiacus and Cb. tepidum, respectively. It is possible to account for these observations if one postulates that the bulk of the BChlc triplets originate either from a large BChlc pool which is essentially non-fluorescent and non-responsive to changes in the redox conditions, or as a result of a process which quenches BChlc singlet excitation and becomes more efficient under non-reducing conditions. In chlorosomes from Cfx. aurantiacus whose Car content is lowered, by hexane extraction, to 10% of the original value, nearly one-third of the photogenerated BChlc triplets still end up on the residual Car pigments, which is taken as evidence of BChlc-to-BChlc migration of triplet excitation; the BChlc triplets which escape rapid static quenching contribute a depletion signal at the long-wavelength edge of the Qy absorption band, indicating the existence of at least two pools of BChlc.

Optical absorption studies of the kinetics of UV- and self-initiated autoxidation of linoleate micelles.

Autoxidation of linoleate micelles in aqueous alkaline solutions was studied by absorption spectroscopy. Autoxidation was initiated in either a UV-induced or a spontaneous decomposition (self-initiation) of hydroperoxides, initially present as trace impurity. In both cases, the hydroperoxides, the primary oxidation products, increased quadratically with time. In the presence of vitamin E, UV irradiation initially caused an exponential photodecomposition of hydroperoxides, with a rate constant of 2.6 center dot 10(-3) s-1, towards a stationary level. The period of inhibition of autoxidation by vitamin E was in proportion to the amount of vitamin E added to the solution. In the course of autoxidation, when the concentration of hydroperoxides exceeded a certain critical level, the micelles collapsed, which was monitored by light scattering. A model for the consumption of vitamin E, in the case of UV initiation, was developed and from which an initiation rate of 4.05 center dot 10(-7) M center dot s-1 was derived. From the initiation rate and the coefficient of the second-order dependence of hydroperoxides with time, an oxidizibility of 1.28 (M center dot s)-1/2 was derived.

UV-initiated autoxidation of methyl linoleate in micelles studied by optical absorption.

The UV-induced dissociation of lipid hydroperoxides, naturally present in methyl linoleate and methyl linoelaidate, was monitored by absorption spectroscopy. The decay of hydroperoxides in homogeneous solution, for a fluence rate of 0.82 mW.cm-2 at 240 nm, followed an exponential course with an average rate constant of k = (2.0 +/- 0.1) . 10(-3) s-1. Addition of alpha-tocopherol led to an increase in the magnitude of k until it reached a limiting value of (3.4 +/- 0.2) . 10(-3)s-1. When methyl linoleate (containing adventitous traces of hydroperoxides) was incorporated in SDS micelles, sustained UV irradiation caused initially an increase in the amounts of lipid hydroperoxides, due to linoleate autoxidation, followed by an exponential decrease due to photodissociation. From the initial rate of hydroperoxide formation (due to linoleate autoxidation) and the rate constant of the subsequent hydroperoxide decay, an oxidizibility of 0.61 . 10(-2) (Ms)-1/2 of methyl linoleate in micelles was deduced. It is suggested that UV irradiation and optical absorption afford an easy and reliable means for studying autoxidation of polyunsaturated fatty acids.

Near-UV-induced radicals in Propionibacterium acnes, studied by electron spin resonance spectrometry at 77 K.

Suspensions of Propionibacterium acnes were UV irradiated and the induced radicals were measured at 77 K by electron spin resonance (ESR) spectrometry. Two types of radical were formed during irradiation and stabilized in the frozen suspensions. The relative yield of each radical was studied as a function of irradiation wavelength. The first radical, which was a singlet with a peak-to-peak width of 20 G, was insensitive to the deoxygenation of the samples and to the exchange of solvent water by heavy water. The action spectrum was similar to the absorption spectrum of NADPH. The second type of radical was not formed in deoxygenated samples and the shape of the ESR spectrum was characteristic of the superoxide radical. This radical was only formed at wavelengths below 340 nm.

The effect of the antioxidant spermine on the photophysical reactions of tryptophan in aqueous solution at 77 K.

Fluorescence, phosphorescence and electron paramagnetic resonance techniques were used to investigate the effect of the antioxidant spermine on the initial photophysical reactions of tryptophan (Trp) in aqueous salt solutions at 77 K. At low concentrations of Trp (3.5 X 10(-5) M) a ground state complex was formed between one Trp and two spermine molecules (a 1:2 complex). Complexed Trp was photodegraded at a rate 65% lower than the free molecule due to a change in the charge-transfer character of the excited 1La state. At high concentrations of Trp (3.5 X 10(-3) M) the phosphorescence was almost completely quenched due to hydrogen-bond formation between two neighbouring Trp molecules. A strong complex was formed between this Trp dimer and one spermine molecule on addition of spermine (a 2:1 complex). Spermine enhanced intersystem crossing in one of the two Trp molecules in the 2:1 complex and phosphorescence was observed. From this triplet state the tryptophyl radical was formed with high efficiency by hydrogen-atom transfer. The yield of radical formation from the triplet state in the 2:1 complex was much larger than from the excited singlet state in the 1:2 complex.

Fluorescence studies on dopamine beta-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+.

The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.

Fluorescence from pilosebaceous follicles.

Fluorescence studies were performed on the extrusions from pilosebaceous follicles. Pressure extractions produced follicle samples which showed fluorescence under Wood's light. The samples were then analysed in a fluorometer giving corrected excitation spectra. The structured emission spectra achieved were interpreted as being due to porphyrins produced by Propionibacterium acnes (P. acnes). Details in the spectra showed close resemblance to spectra from cultured P. acnes cells. The emission spectra showed distinct features in all the four subjects investigated (who were different with respect to age, sex, follicle sampling area, and tendency to acne) and dominant peaks due to at least three porphyrins were found. The concentrations of these porphyrins vary from case to case. Excitation spectra were recorded and supported the assumption that the fluorescent emission was partly due to coproporphyrins and metalloporphyrins in the samples. Free protoporphyrins did not seem to be present in the extrusions. The excitation spectra, in particular, vary from person to person but seem to be constant over time in one and the same subject.

Uptake of protoporphyrin and violet light photodestruction of Propionibacterium acnes.

The uptake of protoporphyrin IX by Propionibacterium acnes in suspension has been studied by fluorescence spectroscopy. Protoporphyrin, after it was injected into a cell suspension, was firstly bound to receptors on the cell surface and in this state protoporphyrin was non-fluorescent. Subsequently, probably as a result of lateral diffusion in the cell wall, these protoporphyrin-receptor complexes formed dimers. The final step in the overall uptake process of protoporphyrin by the cells from the surroundings consisted in a jump of such dimers from waterlike to lipidlike compartments in the cell membrane where protoporphyrin became fluorescent. The lipidlike compartments in the cells had a limited binding capacity of protoporphyrin. The fraction of surviving cells versus light dose has also been studied for varying amounts of protoporphyrin added to the cell suspensions. The survival curves were exponentially decaying with the irradiation time and there was a direct proportionality between the inverse slope of the survival curves and the intensity of protoporphyrin fluorescence form the lipidlike compartments. The relevance of these results to the therapy of Acne vulgaris is also discussed.

The physicochemical state of protoporphyrin IX in aqueous solution investigated by fluorescence and light scattering.

Fluorescence spectros copy and light scattering have been used to investigate the physicochemical behaviour of protoporphyrin IX in aqueous solutions. In the alkaline range large micelles are formed with a hydrodynamic radius of 130 nm and a molecular mass of 5.0 x 10(7) Da. The micelles are fluorescent with an emission maximum at 620 nm. A pH lowering caused quenching of the micelle fluorescence. On a collision encounter these micelles will disintegrate and they are reformed by nucleation of collision fragments. From measurements of the fluorescence intensity of the micelles versus total concentration an equilibrium constant of 4.0 x 10(6) M(-1) was found for this collision-nucleation process. In the pH range between 6 and 3 another micelle type of twice the size of those in the alkaline range was stable with respect to the solute. These micelles have free base porphyrin fluorescence with an emission maximum at 634 nm. A lowering of the pH below unity causes disintegration of these micelles and monomer fluorescence from the protoporphyrin dication was observed.

Photodestruction of endogenous porphyrins in relation to cellular inactivation of Propionibacterium acnes.

During growth of Propionibacterium acnes on Eagles medium protoporphyrin was accumulated inside the cells and coproporphyrin, both as a free base as metalcontaining, outside the cells. The photochemical processes in the endogenous porphyrins were studied by fluorescence spectroscopy during continuous irradiation of Propionibacterium acnes in suspension. The irradiation caused initially an increase in the content of protoporphyrin in the cells in comparison to that which had been accumulated during growth. Maximum light induced protoporphyrin production was achieved in 5 days old cultures. In old cultures where there was practically no initial protoporphyrin release, the fluorescence intensities from all the porphyrins present in the culture vanished exponentially with the irradiation time. The metal containing form of fluorescent coproporphyrin, with a maximum emission at 580 nm, was photobleached about ten times faster than the free base forms of coproporphyrin and protoporphyrin. Among these three fluorescent substances in the cell culture only the free base forms of the porphyrins have longer lifetimes than the cells themselves irradiated at the same conditions.

Photodestruction of Propionibacterium acnes porphyrins.

The fluorescence spectra of colonies of Propionibacterium acnes were studied under various experimental conditions. The spectra contained peaks at 580 nm and 620 nm. These bands were due to two different components; the 580 nm component was likely to be a metalloporphyrin, and there are indications that the 620 nm component could be a coproporphyrin. The 580 nm fluorescence was destroyed by the combined action of light and oxygen (no destruction under strict anaerobic conditions). A dark period interrupting the bleaching light stopped the destruction of this component for the time of the dark period. The initial production of the 620 nm component was due to the oxygen exposure. Upon light irradiation this component was later destroyed by the combined action of oxygen and light.