Thor Bernt Melø

Fluorescence from pilosebaceous follicles

SummaryFluorescence studies were performed on the extrusions from pilosebaceous follicles. Pressure extractions produced follicle samples which showed fluorescence under Woods 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.

Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection.

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.

Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants.

Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be quenched by proline is singlet oxygen ((1)O(2))-molecular oxygen in its lowest energy electronically excited state-constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline cannot quench (1)O(2) in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against (1)O(2).

Uptake of protoporphyrin and violet light photodestruction of Propionibacterium acnes.

Abstract 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 protoporphyrinreceptor complexes formed dimers. The final step in the overall uptake process of protoporphyrin by the cells from the surroundings consisted in a jum p of such dimers from waterlike to lipidlike com partm ents in the cell mem brane where protoporphyrin became fluorescent. The lipidlike com partm ents 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 from the lipidlike compartm ents. The relevance of these results to the therapy of Acne vulgaris is also discussed.

HYDROGEN ABSTRACTION BY TRIPLET FLAVINS. I : TIME-RESOLVED MULTI-CHANNEL ABSORPTION SPECTRA OF FLASH-IRRADIATED RIBOFLAVIN SOLUTIONS IN WATER

Abstract Multichannel detection with μs time resolution has been used for recording flash-induced changes in the absorbance of deaerated solutions of riboflavin in phosphate buffer at pH 6.8. By comparing the spectra of solutions containing, in addition to riboflavin (Fv), different substrates (ascorbic acid, EDTA, indole acetic acid, tryptophan), and by recording the absorption spectrum of the neutral tryptophyl radical (produced by flash photolysis of aqueous solutions of tryptophan), the absorption spectrum and the extinction coefficient of FvH are ascertained. Under the experimental conditions pertaining to the present investigation (solute concentration (7.5±2.5)×10−5 M, ca. 10% conversion into triplets), quenching of Fv† (a riboflavin triplet) by Fv0 (an unexcited riboflavin molecule) was found to be insignificant; most triplets appear to decay through triplet–triplet annihilation, the loss of two triplets being accompanied by the gain of one neutral semiquinone radical (FvH ). It is proposed that (i) triplets deactivate mainly through triplet–triplet annihilation, (ii) the annihilation event leads to the formation of an ion pair, Fv†+Fv†→Fv++Fv−, (iii) the anion rapidly converts to FvH , and (iv) the cation ejects a proton and splits into two neutral products, a molecule whose ground-state absorption spectrum resembles that of Fv0, and an odd-electronic species containing the remainder of the aliphatic side chain.

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.

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, ΔA(λ; 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 ΔA(λ; t) shows, besides a signal attributable to triplet Car (with a half‐life of about 4 μs), 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 μs (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.

Triplet excited states of some thiophene and triazole substituted platinum(II) acetylide chromophores

The photophysical properties of a series of platinum(II) acetylide compounds (trans-Pt(PBu(3))(2)(C[triple bond]C-R)(2)) with the R group consisting of two or three aryl rings (phenyl, phenyl/thiophenyl, phenyl/triazolyl) linked together with ethynyl groups were systematically investigated. Four new structurally similar compounds are reported with: (i) a bithiophene unit in the ligands, (ii) methyl or (iii) methoxy substituents on the aryl ring ligands that promote a more twisted conformation along the long axis of the molecule, and (iv) with two different alkynylaryl ligands giving rise to an asymmetric substitution with respect to the photoactive metal ion center. The spectroscopic studies include optical absorption, spectrally and time-resolved luminescence, as well as transient absorption spectra. The ground-state UV absorption between 300 and 420 nm gave rise to fluorescence with quantum efficiencies in the range of 0.1-1% and efficient intersystem crossing to triplet states. Phosphorescence decay times were in the order of 10-500 micros in oxygen-evacuated samples. The triplet states also lead to strong broadband triplet-triplet absorption between 400 and 800 nm. The complex with asymmetric substitution was found to populate two triplet states of different structure and energy.

Multichannel Flash Spectroscopy of the Reaction Centers of Wild-type and Mutant Rhodobacter sphaeroides: BacteriochlorophyllB-mediated Interaction Between the Carotenoid Triplet and the Special Pair†,¶

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)‐containing and Car‐less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light‐induced spectral changes in the 350–1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870+QA−, and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870†) are formed. The time‐resolved triplet‐minus‐singlet (TmS) spectrum of Car‐less RC shows a deep bleaching at 870 nm, which belongs to P ‡870, and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 μs). The bleaching at 800 nm arises from the pigment interaction between P870† and the accessory bacteriochlorophylls on A and B branches (BA, B). In Car containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals—a sharp peak at 809 ± 2 nm and a broad band at 870 nm—which decay at the same rate as the TmS spectrum of Car (ca 10 μs). The former is ascribed to the perturbation, by Car†, of the absorption spectrum of BB; the latter, to the TmS spectrum of P870†, a species that appears to be in approximate thermal equilibrium with Car†. These assignments are consistent with the absorption‐detected magnetic resonance spectra obtained by other workers at low temperatures.

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II(slow) and Signal II(fast) of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y(Z) and Y(D) of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043+/-0.0003 and a line width of 10+/-1G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q(A) in the absence of magnetic interaction with the non-heme Fe2+. We conclude that peroxynitrite impairs PSII electron transport in the Q(A)Fe2+ niche.