LIGHT-INDUCED LINEAR DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS–II. CHROMOPHORE ROTATION IN IMMOBILIZED PHYTOCHROME

-Large phytochrome immobilized via anti-phytochrome immunoglobulin bound to Sepharose beads was irradiated to saturation with unpolarized far-red light. The apparent absorbance level was recorded in a dual wavelength spectrophotometer with both measuring beams set to either 660 or 730 nm and polarized perpendicular to each other. The sample was then irradiated with red polarized light. The apparent change in absorbance obtained after this irradiation indicated that purified phytochrome could show linear dichroism. From the absorbance values obtained it was computed that the direction of the long-wavelength transition moment changes by either 32 or 148^o, when phytochrome is transformed from P_r to P_fr. Considering the model of Hahn and Song (1981) the latter value appears more likely. In light of these results, the conclusions drawn from in vivo experiments on action dichroism in Dryopteris (Etzold, 1965), Adiantum (Kadota et al., 1982) and Mougeoutia (Haupt. 1970), which point to a 90^o rotation. should be reconsidered.     

DYNAMIC LINEAR DICHROISM IN CHROMOPROTEINS

The time-dependent polarized fluorescence and optical density transients arising from electronic excitation transport are derived for solutions of randomly oriented chromoproteins in which the chromophore transition moments have well-defined orientations with respect to the protein. The treatment is extended to oriented monolayers of chromoproteins, in which the molecules are aligned with a particular protein-fixed axis perpendicular to the surface plane and with random azimuthal angles about the surface normal. The initial ratio of parallel to perpendicular transients is invariably 3:1, irrespective of system dimensionality and chromophore organization. The residual anisotropy observed at long times is directly related to the relative orientations of the donor and acceptor chromophores. These calculations form a basis for detailed interpretation of ultrafast fluoresence and pump-probe linear dichroism studies in photosynthetic systems, for which 3-dimensional structures are becoming increasingly available.     

CHROMOPHORE ROTATION IN 124-kD ALTON A vena sativa PHYTOCHROME AS MEASURED BY LIGHT-INDUCED CHANGES IN LINEAR DICHROISM

Abstract— From light-induced changes in linear dichroism, we have calculated the rotation of the long-wavelength-absorbing transition moment that occurs during phototransformation of 124-kilodalton Avena sativa phytochrome. Phytochrome was purified to homogeneity and immobilized onto Sepharose beads covalently coated with antibodies against A. sativa phytochrome. Changes in linear dichroism were induced by plane-polarized red or far-red light and measured by the absorbance differences at 660 and 730 nm using a dual-wavelength spectrophotometer equipped with polarizing filters in the measuring beams. From such measurements, we calculate a rotation angle of 31^o (or 149^o) during photoconversion of Pr to Pfr and 30^o (or 150^o) during photoconversion of Pfr to Pr. These values are similar to the value of 32^o (or 148^o) reported earlier for the rotation of the transition moment of "large" A. sativa phytochrome (∽ 120 kilodalton) isolated under conditions that did not preclude post homogenization proteolysis of the 124 kilodalton molecule.     

LIGHT-INDUCED SYNTHESIS OF ANTHOCYANIN IN CARROT CELLS IN SUSPENSION—IV. THE ACTION SPECTRUM

Abstract— Using carrot cell suspension in 2,4-dichlorophenoxyacetic acid (2,4-D)-depleted culture medium, fluence-response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence-response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence-dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV-B photoreceptor.
Although red (R)-light alone had a minor effect on anthocyanin accumulation, it modulated the action of UV-B light. That is, when carrot cells were irradiated with R-light either before or after UV-B irradiation, anthocyanin formation was greatly enhanced above the level enhanced by UV-B light alone. The most effective wavelength for this enhancement was 660 nm. The effect of R-light on the anthocyanin formation of the UV-B irradiated cells was reversed by immediately following it with far-red light, suggesting the involvement of phytochrome in the R-effect.     

ELECTRONIC ABSORPTION PROPERTIES OF SYMMETRICAL DIALKOXYANTHRACENES. LINEAR DICHROISM AND MAGNETIC CIRCULAR DICHROISM*

Abstract— Linear dichroism (LD) and magnetic circular dichroism (MCD) of symmetrical dialkoxy-anthracenes and some related compounds were recorded in an attempt to elucidate electronic absorption properties and assign the nature of the lowest singlet excited state, recognized to control both the fluorescence and the photodimerization of anthracenes. The spectroscopic measurements proved that the lowest electronic transition is of the ¹L_a type in all the compounds. The peculiar photoreactivity pattern is therefore governed by factors other than an inversion of the order of the ¹L_a and ¹L_b states and may be related to electronic density repartition on the different positions of the anthracene nucleus dictated by the position of the electron-donating substituents.     

INTERACTION BETWEEN DNA AND 8–METHOXYPSORALEN STUDIED BY LINEAR DICHROISM

Abstract— High sensitivity linear dichroism on DNA oriented by flow is shown to provide conclusive information about the interaction with 8–methoxypsoralen (8–MOP) both in the dark and by irradiation. 8–MOP forms directly in the dark a weak complex with DNA with a structure consistent with the intercalation model of Lerman. When aqueous DNA-psoralen solutions are UV irradiated (30800 nm). a photoadduct is formed. By comparison with experiments in ice matrix, it is suggested that it is the primarily bound 8–MOP that is responsible for the final product. It is inferred that this product is the 4 ¹ ,5 ^I -photoadduct of 8–MOP. The orientation of the photo adduct on DNA is not consistent with intercalation.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

LINEAR DICHROISM STUDY OF METALLOPORPHYRIN TRANSITION MOMENTS IN VIEW OF RADIATIONLESS INTERACTIONS WITH TRYPTOPHAN IN HEMOPROTEINS

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe³⁺-PPIX (ferric-heme) and Fe²⁺CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300–380 nm) and Soret (380450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300–380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300–380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J . 63, 648–653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 from the α-γ meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. I. AN ANALYSIS OF LIGHT-INDUCED ELECTRICAL AND MOTILE RESPONSES IN Stentor coeruleus

Abstract— Light-induced membrane potantial changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellulae microelectrode showed that step-up increase in light induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potetial is dependent on the intensity of light stimulus and the action potetials appears with a lag period (latency) after the onset of light stimuklus. The extent of the membrane established between the latency for te action poitential and the onset of ciliary reversal (stop responses). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. the action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, iondicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to ehich the photoreceptors and the ciliary apparatus is mediated by the membrane potential canges.     

STUDIES ON TETRAPYRROLS PIGMENTS—I. ABSORPTION AND FLUORESCENCE OF BILIVERDIN DIMETHYL ESTERS OF THE IX SERIES

Abstract— Each of the four isomeric biliverdin dimethyl esters of the IX series give a fluorescent spectrum at ambient temperature, the intensity of which increases considerably when these biliverdins are complexed with zinc. A systematic study of the influence of protic and aprotic solvents failed to yield significant results. In methanol. variations in the pH greatly affect the emission absorption spectra of zinc complexes of biliverdins.     

STUDIES ON PLANT BILE PIGMENTS—9. PHOTOOXIDATION OF A-DIHYDROBILINDIONE AS PHYTOCHROME MODEL IN ACID MEDIUM

Abstract— The photooxidation of octaethyl-2, 3-dihydrobilindione ( 2 ) as a model for the P_r-chromophore has been studied in acidic methanol. When 2 is titrated with HCl to the cation and irradiated under N₂ with an excess of I₂, violin and rhodin type pigments are formed bearing methoxy-substituents at either C-4,5 or C-15,16, respectively. The reaction is slowed down as compared to neutral conditions, it is no longer regioselective at the C-5 methine bridge, and the formation of dimers is suppressed. From the data of this and earlier chemical model studies on the photooxidation of 2 , and the known properties of phytochrome, a reaction for the P_r→ P_fr phototransformation is suggested, in which the chromophore is attacked by the protein moiety in a sequence of oxidation and nucleophilic substitution, which may lead to a primary redox signal for the physiological response.     

MECHANISM OF ORIENTATION AND LINEAR DICHROISM OF PHOTOSYNTHETIC PARTICLES IN ELECTRIC FIELDS: CHLOROPLASTS AND CHLOROPHYLL-PROTEIN COMPLEXES

Abstract— The mechanisms of orientation in pulsed and alternating electric fields of thylakoids (derived from the sonication of spinach chloroplasts) and of light-harvesting chlorophyll a/b-protein complexes (CPII) were investigated by utilizing linear dichroism techniques. Comparisons of the linear dichroism spectra of thylakoids and CPII particles suggest that the latter are oriented with their directions of largest electronic polarizabilities (and thus probably their largest dimensions) within the thylakoid membrane planes. At low electric field strengths (< 12 V cm^?1), and at low frequencies of alternating electric fields (< 0.25 Hz), thylakoid membranes tend to align with their normals parallel to the direction of the applied electric field; the mechanism of orientation involves a permanent dipole moment of the thylakoids which is oriented perpendicular to the planes of the membranes. However, at high field strengths and high frequencies of the applied alternating electric fields, the thylakoids tend to orient with their planes parallel to the applied field, thus exhibiting an inversion of the sign of the linear dichroism as the electric field strength is increased. At the higher frequencies and at higher field strengths, the orientation mechanisms of the thylakoids involve induced dipole moments related to anisotropies in the electronic polarizabilities. The polarizability is higher within the plane than along a normal to the plane, thus accounting for the inversion of the dichroism as the electric field strength is increased. The CPII particles align with their largest dimension parallel to the applied field at all field strength, indicating that the induced dipole moment dominates the orientation mechanisms in pulsed electric fields. The magnitude of the absolute linear dichroism of CPII suspensions increases with increasing dilution, indicating that aggregates of lower symmetry are formed at higher concentrations of the CPII complexes.     

ACTION SPECTRA FOR LIGHT-INDUCED SPORULATION OF THE FUNGI PLEOSPORA HERBARUM AND ALTERNARIA DAUCI*,†

Abstract— Action spectra for light induced sporulation were determined for conidia of Alternaria dauci , and conidia and perithecia of Pleospora herbarum (Imperfect stage is Stemphylium botryosum ). Only radiation less than 370 mμ induced formation of conidia, and less than 390 mμ formation of perithecia. The action spectra showed increased effectiveness in the 230 and 290 mμ regions, and possibly in the 260–270 mμ regions. Below 280 mμ these action spectra may not be representative of the "true" absorption spectra of the photoreceptors involved because of possible superimposed effects caused by nucleic acid absorption. Though the action spectra were not identical, the number of characters in common were suggestive of a similar photoreceptor. Similarities between action spectra for sexual and asexual reproduction of P. herbarum indicate the possibility of a single photoreceptor for both processes.     

A LINEAR DICHROISM STUDY OF THE ORIENTATION OF AROMATIC PROTEIN RESIDUES IN MAGNETICALLY ORIENTED BOVINE ROD OUTER SEGMENTS

Abstract— Linear dichroism measurements using magnetic field oriented bovine visual rod outer segments have been made in the UV and visible spectral regions. The results indicate that the planes of the aromatic amino acid residues of rhodopsin tend to be oriented normal to the membrane plane both before and after bleaching. In contrast, the retinal chromophore which tends to be oriented with its absorption oscillator parallel to the membrane plane before bleaching is randomly oriented about 10min after bleaching whereas the membranes remain oriented. Estimates of the anisotropy in the diamagnetic susceptibility of rhodopsin aromatic residues indicate that the anisotropic magnetic properties of these protein residues are sufficient to account for the observed orientation of visual rod outer segments in a homogenous magnetic field.     

硫增感AgBr I T颗粒乳剂光电子行为研究

本文利用微波吸收相敏检测技术,同时获得了硫增感AgBrIT颗粒乳剂,在不同增感条件下自由光电子和浅俘获光电子的时间衰减曲线,分析了不同的硫增感产物的陷阱效应.结果表明:开始时,增感产物起电子陷阱作用,至45 min时,浅电子陷阱作用最佳.如增感时间进一步增加,硫增感产物将变为深电子陷阱.本文还讨论了浅电子陷阱中浅俘获光电子衰减时间与阱深的依存关系.     

VISUAL PIGMENTS—10. SPECTROSCOPY AND PHOTOPHYSICAL DYNAMICS OF RETINOL AND RETINYL ETHER

Abstract— The absorption and emission spectra, lifetimes and quantum yields of all-trans retinol have been examined as a function of solvent and temperature. In addition, the spectroscopy of retinyl ether and 2 other polyene alcohols have been determined. Based on the results obtained, we conclude that the singlet excited state of retinol and retinyl cther is of a forbidden character and of the type loosely called ¹A^-_g. Retinol forms a dimer in an alkane solvent when cooling from 298 to 77 K. A general structure for the dimer is proposed.     

SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N-FORMYLKYNURENINE ON BOVINE α-CRYSTALLIN

Abstract— Fluorescence and circular dichroic properties of bovine a-crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N-formylkynurenine. Methylene blue-sensitized photooxidation causes a change in the tertiary structure as manifested in the near-UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non-polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near-UV CD (tertiary structure) suffers a drastic alteration whereas the far-UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron-ments, orientation, and intermolecular interactions of different amino acids. N-formylkynurenine also shows some changes in the near-UV CD, presumably, caused by H₂O₂ generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near-UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N-formylkynurenine and air.     

LIGHT-INDUCED ABSORBANCE CHANGES IN CHLOROPLASTS AT -196°C*

Abstract— Absorbance changes induced by irradiating chloroplasts at — 196°C were measured in the region of 525–575 nm with a single-beam spectrophotometer. Irradiation at low temperature caused a bleaching at 556 nm due to oxidation of cytochrome b₅₅₉ but little or no change of cytochrome f. There occurred in addition a loss of absorbance at 547 nm and an increase at 543 nm. The bleaching at 547 nrn (and possibly the increase at 543 nm) could be induced chemically with dithionite or borohydride but not ascorbate. Subchloroplast particles with only Photosystem I activity showed no light-induced absorbance changes, while particles containing combined Photosystem I and Photosystem II activities showed the same changes as whole chloroplasts. Scenedesmus mutant No. 11 cells showed no absorbance changes while mutant No. 8 and wild-type cells showed the normal changes. It is concluded that the photooxidation of cytochrome b₅₅₉ and the photoreduction causing the bleaching at 547 nm are both mediated by Photosystem II.