Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl2, have been measured in the wavelength region 460-540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCL concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of p-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

Salt-induced absorbance changes of P-515 in broken chloroplasts

Absorbance changes, caused by adding KCl to a suspension of broken chloroplasts in the presence of a low concentration of MgCl₂, have been measured in the wavelength region 460–540 nm. The magnitude of the KCl-induced absorbance changes is shown to be proportional to the logarithm of the KCl concentration gradient initially induced across the thylakoid membrane. The difference spectrum of these absorbance changes is shown to be identical with the spectrum of the light-induced absorbance changes, which has been attributed to an electrochromic shift of P-515. This is interpreted as evidence that under these conditions salt-induced absorbance changes of P-515 occur in response to a membrane diffusion potential. The results indicate that the electrogenic potential across the thylakoid membrane, generated by a single turnover light flash, is in the range between 15 and 35 mV.     

Shifts to C675-670 and to C696-684 in etiolated leaves illuminated with series of brief flashes

Absorbance changes seen when etiolated primary bean leaves aregiven series of short, low-intensity flashes of light were recorded.Whatever its order in a series, each flash induced an initial,fast increase of the absorbance, producing an absorption bandbetween 660 and 700 nm with a peak at 680 nm. This occurredin less than 30 msec and was followed within seconds by eithera slow dark increase or a slow dark decrease of the leaf absorbance,depending on the order of the flash and the analytical wavelength.The dark absorbance changes were due to shifts of the initialabsorption band produced by the flash, either towards 685 or670 nm. The shift was always towards 670 nm after the firstflash. It appeared to move progressively towards 685 nm fromone flash to the next, this direction becoming predominant aftera certain number (n_inv) of flashes. The (n_inv) depended on thelight dose, but not on the flash frequency. Three proposalsare made to account for the results. (Received May 14, 1977; )     

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

Time-resolved infrared studies of light-induced processes in plant thylakoids and bacterial chromatophore membranes. Evidence for the function of water molecules and the polypeptides in energy dissipation

Spinach thylakoids and chromatophores from the photosynthetic bacterium Rhodopseudomonas capsulata were investigated by means of time-resolved infrared spectroscopy, using thin water-containing membrane films which fully maintained their photochemical activity. Upon flash excitation, reversible infrared absorbance changes were obtained and their difference spectra were recorded. In spinach thylakoids, these transient signals could be described by a sum of two exponential decay functions with half-times of about 2 and 30 ms, respectively. They were insensitive to the addition of benzyl viologen, ferricyanide or ferricyanide + DCMU. They are ascribed by their dependence on intensity and wavelength range of the actinic flash to processes in the antenna pigment-protein complexes. In chromatophores from photosynthetic bacteria, similar infrared signals in the millisecond time range were obtained. Their spectral distribution was investigated for three mutants of the photosynthetic bacterium and is different for membranes lacking carotenoids. Both signals, in thylakoids and chromatophores, reflect the proportion of absorbed flash energy which is neither channelled to the reaction center nor emitted as light, but is dissipated through radiationless decay. A common feature of the difference spectra from spinach thylakoids and bacterial chromatophores are bands identified by deuteration as being due to H₂O. Some bands are interpreted in terms of water going transiently from the hydrogen-bonded to the free state. Other bands are assigned to the polypeptides of the light-harvesting complexes, and thus indicate their participation in energy dissipation. Membranes from photosynthetic bacteria containing a photochemical reaction center show a distinct slow signal component decaying in about 1 s. It saturates at low flash intensity and is abolished upon chemical oxidation of the primary electron donor. Two bands in the difference spectrum of this component are tentatively assigned to the ester C = O and keto C = O vibrations of photooxidized bacteriochlorophylls in the reaction center. The data suggest that chromophoric and non-chromophoric infrared absorbance changes contribute to the difference spectra, and thus may represent a clue to the processes at the active sites of polypeptides in photosynthesis.     

Influence of absorption on polarization effects in light scattering from human red blood cells

Polarization effects in light scattering are sensitive indicators of cell structure and structural changes in time. In the spectral regions where the optical properties of the scatterers are relatively constant, the scattering pattern scales, it contracts or expands in a predictable manner as a function of the wavelength. In the spectral regions where the optical properties are strongly wavelength dependent (near absorption bands, etc.) the scattering curves do not scale, but change drastically in phase and amplitude as the wavelength is varied. Reported here is an empirical study of the magnitude of the influence of absorption on the polarization effects in light scattering. Scattering curves have been obtained for human red blood cells in the absorption band (blue light) and far from the absorption band (red light). The scattering at these wavelengths shows very strong nonscaling differences. These observations suggest the use of polarization effects in light scattering and their wavelength dependence for the studies of structural changes in cell nuclei. Nucleoproteins have strong absorption, optical rotatory dispersion and circular dichroism bands in the ultraviolet region of the spectrum, whereas there is little ψ-dependence in the visible range. There is also the possibility of binding specific chromophoric dyes to cell components, thus introducing absorption bands in the visible range, where scattering instrumentation and laser light sources are more readily available.     

Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase

The mechanism of interaction of O-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity, absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 microM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X 10(3) M-1 s-1) and CD intensity at 430 nm. Concomitantly, there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X 10(-3) s-1) leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures II. Relationship between 515-nm absorbance change and rapid H+ uptake in chloroplasts after a short flash illumination

The absorbance change at 515 nm induced by a short (7.6 µsec)light flash in spinach chloroplasts was studied at sub-roomtemperatures in relation to rapid H⁺ uptake into chloroplasts. Lowering of temperature caused a marked decrease in the rateof recovery of 515-nm absorbance change after a flash illumination.Initial rate of rapid H⁺ uptake, measured with absorbance changeof bromcresol purple (BCP), was also reduced at lower temperatures,in a parallel fashion. Half-recovery time of the absorbancechange at 515 nm and rise-time of the pH-indicating absorbanceincrease of BCP coincided well at each temperature studied.Values of the calculated activation energy for these two processeswere almost the same. The parallelism between the 515-nm absorbance change and therapid H⁺ uptake after a single flash illumination was also observedwhen the electric field decay and/or H⁺ translocation were acceleratedby ionophorous antibiotics, carbonylcyanide m-chlorophenylhydrazoneor phenazine methosulfate. From these results, it is suggestedthat the rapid H⁺ uptake into chloroplast is chemically coupledto electron transfer and at the same time diffusion- (or transport-)controlled. Membrane potential, reflected in the 515-nm absorbancechange is dissipated with the rapid H⁺ influx. A model for theelectron-transfer-coupled H⁺ translocation involving a plastosemiquinoneloop is presented. Dissipation of the illumination-formed inside-positivemembrane potential by the influx of H⁺ is explained by the model. (Received September 17, 1976; )     

Excitonic interactions in wild-type and mutant PSI reaction centers

Femtosecond excitation of the red edge of the chlorophyll a Q(Y) transition band in photosystem I (PSI), with light of wavelength > or = 700 nm, leads to wide transient (subpicosecond) absorbance changes: positive DeltaA between 635 and 665 nm, and four negative DeltaA bands at 667, 675, 683, and 695 nm. Here we compare the transient absorbance changes after excitation at 700, 705, and 710 nm at 20 K in several PSI preparations of Chlamydomonas reinhardtii where amino acid ligands of the primary donor, primary acceptor, or connecting chlorophylls have been mutated. Most of these mutations influence the spectrum of the absorbance changes. This supports the view that the chlorophylls of the electron transfer chain as well as the connecting chlorophylls are engaged in the observed absorbance changes. The wide absorption spectrum of the electron transfer chain revealed by the transient measurements may contribute to the high efficiency of energy trapping in photosystem 1. Exciton calculations, based on the recent PSI structure, allow an assignment of the DeltaA bands to particular chlorophylls: the bands at 675 and 695 nm to the dimers of primary acceptor and accessory chlorophyll and the band at 683 nm to the connecting chlorophylls. The subpicosecond transient absorption bands decay may reflect rapid charge separation in the PSI reaction center.     

Light absorption by carotenoid peridinin in zooxanthellae cell and setting down of hermatypic coral to depth

Carotenoid peridinin absorbs ocean light which could penetrate deep into the water. Absolute and relative contents of symbiotic dinoflagellatae zooxanthellae are increased with depth of habitat of germatypic corals. To estimate whether the presence of peridinin in corals is chromatic adaptation or not, the absorbance of solar radiation by different amounts of peridinin and chlorophyll in natice zooxanthellae cells was evaluated. Calculations have shown that at the great depths the peredinin absorbance corresponds to 42% of total cell absorbance and that the increase of light absorbance correlating with changes of its spectral characteristics is entirely determined by presence of this carotenoid. The increase of amount of peridinin in cell is as much important as important the increase of all other pigments taken together. However, at the same time selective and preferential accumulation of peridinin and the change of its native state in the limits naturally occurred in zooxanthellae cells have only low impact on the light absorbance. The presence of peridinin could be considered as manifestation of chromatic adaptation of organism. The comparison of light absorption by zooxanthellae with different content of peridinin (or without peridinin) reveals that this pigment expands the habitat of hermatypic corals in ocean waters at 8-17 meters into the deep.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Computer-assisted indirect coulometric titrations of biological redox components

The optically coupled, indirect coulometric titration (ICT) method has been applied to assist in the characterization of several redox biocomponents such as heme proteins. This method has provided rapid and repetitive evaluation of redox stoichiometry and energeties with a high degree of precision. The usual manual ICT procedure requires operator control of the repetitive incremental additions of electrochemical charge, the on/off of the magnetic stirrer between such additions, and the recording of optical spectra after each addition. The sequence and timing of events in the above procedure can be fixed which then lends itself ideally to computer control. In this paper, a computer-controlled ICT intsrumentation is described. The hardware and software developed for the computerization permit versatility of displaying ICT data in various formats, i.e., conventional absorbance or derivative spectra and dual wavelength or differences between dual-wavelength spectra. Also, absorbance at a given wavelength could be obtained from the spectra as a function of the incremental addition of charge. The three-dimensional plots of spectral absorbance as a function of wavelength and incremental charge could also easily be obtained. To demonstrate the capability and versatility of the computer-controlled instrumentation, the ICT of the redox components, cytochrome c and cytochrome c oxidase, and of intact mitochondria is presented.     

Cytochrome f function in photosynthetic electron transport.

The questions of whether the stoichiometry of the turnover of cytochrome f, and the time-course of its reduction subsequent to a light flash, are consistent with efficient function in noncyclic electron transport have been investigated. Measurements were made of the absorbance change at the 553-nm alpha-band maximum relative to a reference wavelength. In the dark cytochrome f is initially fully reduced, oxidized by a 0.3-s flash, and reduced again in the dark period after the flash. In the presence of gramicidin at 18 degrees C, the dark reduction was characterized by a half-time of 25-30 ms, stoichiometries of cytochrome f:chlorophyll and P700:chlorophyll of 1:670 and 1:640, respectively, and a short time delay. The time delay in the dark reduction of cytochrome f, which is expected for a component in an intermediate position in the chain, becomes more apparent in the presence of valinomycin and K+. Under these conditions the half-time for cytochrome f dark reduction is 130-150 ms, and the delay is approximately equal to 20 ms. The measured value for the activation energy of the dark reduction of cytochrome f (11 +/- 1 kcal/mol) is the same as that for noncyclic electron transport in steady-state light. A sigmoidal time-course for the reduction of cytochrome f has been calculated for a simple linear electron transport chain. The kinetics for reduction of cytochrome f predicted by the calculation, in the presence of valinomycin and K+, are in reasonably good agreement with the experimental data. There is an appreciable amount of data in the literature to document complex properties of cytochrome f after illumination with short flashes, and evidence for complexity in a light-minus-dark transition. The data presented here, obtained after a long flash that should establish steady-state conditions, either fulfill or are consistent with the basic criteria for efficient function of cytochrome f in noncyclic electron transport.     

The modification of induced genetic change in yeast by an amino acid analogue

Summary Treatment of diploid yeast cultures with the amino acid analogue, para-fluorophenylalanine (PFPA), at concentrations which caused inhibition of growth, resulted in up to 5 fold increases in the frequency of mitotic gene conversion at two different heteroallelic loci. With haploid yeast cultures, growth in PFPA increased the rate of forward mutation to canavanine resistance by at least 2 fold.Growth of diploids in PFPA prior to exposure to the deaminating agent nitrous acid, the cross-linking agent mitomycin C, the alkylating chemical ethylmethanesulphonate (EMS) and UV light resulted in significant changes in the potency of these diverse mutagens to induce intragenic recombination. For all four mutagens, increased frequencies of gene convertants/viable cell were observed in those cultures which had been exposed to the amino acid analogue prior to mutagen treatment. In haploid WT yeast cells, amino acid analogue incorporation resulted in an enhanced frequency of UV induced forward mutation to canavanine resistance whilst in a DNA repair deficient rad 6 mutant this interaction between UV and PFPA was abolished.The results have been interpreted on the basis of incorporation of the analogue into enzymes involved with DNA replication with a consequent loss of fidelity of such enzymes and increased errors in base incorporation.     

Comparison of chlorophyll fluorescence emission characteristics of wheat leaf tissue and isolated thylakoids as a function of excitation wavelength

Abstract. Chlorophyll fluorescence emission spectra and the kinetics of 685 mm fluorescence emission from wheat leaf tissue and thylakoids isolated from such tissue were examined as a function of excitation wavelength. A considerable enhancement of fluorescence emission above 700 nm relative to that at 685 nm was observed from leaf tissue when it was excited with 550 nm rather than 450 nm radiation. Such excitation wavelength dependent changes in the emission spectrum occurred over an excitation spectral range of 440–660 nm and appeared to be directly related to the total quantity of radiation absorbed at a given excitation wavelength. Experiments with isolated thylakoid preparations demonstrated that changes in the fluorescence emission spectrum of the leaf were attributable to the optical properties of the leaf and were not due to the intrinsic characteristies of the thylakoid photochemical apparatus. This was not the case for the observed excitation wavelength dependent changes in the 685 nm fluorescence induction curve obtained from leaf tissue infiltrated with DCMU. Excitation wavelength dependent changes in the ratio of the variable to maximal fluorescence emission and the shape of the variable fluorescence induction were observed for leaf tissue. Isolated thylakoid studies showed that such changes in the leaf fluorescence kinetics were representative of the way in which the photochemical apparatus in vivo was processing the absorbed radiation at the different excitation wavelengths. The results are considered in the context of the use of fluorescence emission characteristics of leaves as non-destructive probes of the photochemical apparatus in vivo.     

pH-induced hysteretic transitions of ovoperoxidase

Ovoperoxidase, the enzyme that catalyzes the dityrosine cross-linking of fertilization membranes of eggs from the sea urchin Stronglyocentrotus purpuratus, exhibits slow changes in catalytic activity upon alterations of pH, with attendant changes in spectral properties. For ovoperoxidase pre-equilibratated at pH 8, abrupt decreases in pH are accompanied by a slow loss in activity that is temporally associated with a change in absorbance at the Soret band. With enzyme pre-equilibrated at pH 4.5 and then shifted to higher pH, there was a slow increase in catalytic activity following a rapid change in the Soret band absorbance. These changes were reversible and led to the same equilibrium state, regardless of the direction of pH shift. The rate of approach to the equilibrium state of ovoperoxidase was independent of enzyme concentration, the presence of substrates, or temperature (from 6.5 to 39.7 degrees C). The pH-induced interconversions of catalytic and spectral properties indicate that ovoperoxidase undergoes hysteretic transitions, in which alterations in the heme environment accompany, but are not sufficient for, the expression of catalytic activity. We present a kinetic mechanism for the hysteretic relaxations and suggest how these transitions may have relevance to the assembly of the fertilization membrane in vivo.     

Insect pupil mechanisms

Summary The spectral characteristics of the pupil mechanism in blowfly photoreceptors and their dependence on light intensity have been investigated together with the intensity dependence of the receptor potential. The threshold for the pupil response as measured by reflectance is found at an intensity at which the peak of the receptor potential is about half maximal and the plateau potential starts to saturate. The reflectance saturates at about 3 log-units above threshold. The reflectance spectrum peaks near 620 nm, and its shape is independent of adaptation intensity. The absorbance change, measured by transmission, is extreme in the blue, at about 470 nm. The shape of the absorbance spectrum is slightly intensity dependent, presumably due to optical waveguide effects. The dynamic ranges of the light-induced reflectance and absorbance changes do not coincide. The reflectance change shows saturation at least 1 to 1.5 log units before the absorbance change saturates.     

Determination of the labeling density of fluorophore-biomolecule conjugates with absorption spectroscopy

Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.     

Effects of a triterpenoid saponin on spectral properties of undegraded pea phytochrome

Soyasaponin I, a triterpenoid saponin isolated from etiolated pea (Pisum sativum cv. Alaska) shoots and identified as Pfr killer, was examined for its effects on spectral properties of undegraded pea phytochrome. When soyasaponin I in concentrations of 100 micromolar or lower was added to Pr in the dark, the spectrum of Pr was not significantly affected, whereas in the presence of 120 micromolar or higher concentrations the absorption maximum of Pr shifted from 666 to 658 nanometer with slight decrease of absorbance. After a brief exposure of the mixture to red light, the increase in absorbance at 666 nanometers that occurs in the dark was inhibited at 26 micromolar and higher soyasaponin I concentrations; the maximum effect being reached at about 180 micromolar. The decrease in absorbance at 724 nanometers in the dark after red light irradiation was somewhat inhibited by 60 micromolar and totally prevented by 410 micromolar soyasaponin I. When P₆₅₈ was irradiated with red light in the presence of 220 micromolar or higher soyasaponin I concentrations, a bleached form (P_bl) was produced instead of Pfr. P_bl showed no dark spectral changes, and the phototransformation of P_bl to P₆₅₈ required a significantly high irradiance of far-red light. When the saponin was added to Pfr in the dark, none of the above-described spectral changes occurred, although the same effects were observed after the mixture was exposed briefly to far-red light followed by red light.     

Euphausiid visual pigments. The rhodopsins of Euphausia superba and Meganyctiphanes norvegica (Crustacea, Euphausiacea)

The rhabdoms of Euphausia superba contain one digitonin-extractable rhodopsin, lambda max 485 nm. The rhodopsin undergoes unusual pH- dependent spectral changes: above neutrality, the absorbance decreases progressively at 485 nm and rises near 370 nm. This change is reversible and appears to reflect an equilibrium between a protonated and an unprotonated form of the rhodopsin Schiff-base linkage. Near neutral pH and at 10 degrees C, the rhodopsin is partiaLly converted by 420-nm light to a stable 493-nm metarhodopsin. The metarhodopsin is partially photoconverted to rhodopsin by long-wavelength light in the absence of NH2OH; in the presence of NH2OH, it is slowly converted to retinal oxime and opsin. The rhodopsin of Meganyctiphanes norvegica measured in fresh rhabdoms by microspectrophotometry has properties very similar to those of the extracted rhodopsin of E. superba. Its lambda max is 488 nm and it is partially photoconverted by short wavelength irradiation to a stable photoconvertible metarhodopsin similar to that of E. superba. In the presence of light and NH2OH, the M. norvegica metarhodopsin is converted to retinal oxime and opsin. Our results indicate that previous determinations of euphausiid rhodopsin absorbance spectra were incorrect because of accessory pigment contamination.