Correlation of the 680 to 672 nm Spectral Shift and the Halving of the Apparent Molecular Weight for Chlorophyll(ide) Holochrome from Barley

Protochlorophyll(ide) holochrome was isolated from dark-grown barley (Hordeum vulgare L.) leaves and photoconverted. When the chlorophyll(ide) absorption maximum had decreased from 680 to 676 nm the preparation was chromatographed on a Sephadex-gel column under conditions which strongly inhibited a further decrease in the absorption maximum. The absorption properties of the column fractions and the shape of the chlorophyll(ide) elution-profile indicated the presence of two distinct chlorophyll(ide)-bearing molecular species with apparent molecular weights of c. 74,000 and 29,000 and absorption maxima at 680 and 672 nm, respectively. It is concluded that: (1) no long-lived species with intermediate absorption maximum is formed during the 680 to 672 nm shift of the absorption maximum of newly photoconverted holochrome; (2) no long-lived pigment-protein complexes with intermediate molecular weights are formed during the approximate halving of the molecular weight; (3) the shift in the absorption maximum and the decrease in molecular weight are closely correlated.     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m^?2) was investigated for the chlorophyll(ide) pigments formed after the primary photoreduction of the protochlorophyll(ide) in dark grown leaves of wheat. After photoreduction, most of the chlorophyll(ide) exists in a form with an absorption maximum at 684 nm. This form is gradually transformed into a form with an absorption maximum at 673 nm (the Shibata shift). It was possible to ascribe a specific photostability to each of the pigment forms. This photostability was higher for the 673-form than for the 684-form. A red-shift in the absorption maximum following upon the Shibata shift, reflects the successive transformation of the 673-form into other pigment forms, which were quite photostable at the intensity used.     

Photoconvertible protochlorophyll(ide) in vivo: A single species or two species in dynamic equilibrium?

The decreasing absorbances in vivo of protochlorophyll(ide) at 635 and 650 nm bear the same relationships to one another during photoconversion to chlorophyll(ide) a in the leaves of dark-grown barley seedlings, regardless of whether the actinic light is absorbed primarily at 630, 640 or 671 nm. Accordingly, the absorption bands at 635–637 and 650 nm of photoconvertible protochlorophyll(ide) are attributed to a single species of membrane-bound protochlorophyll(ide) molecule or, alternatively, to two species which are in dynamic equilibrium.     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coefficient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ 1 mole^–1 cm^–1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assuming that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.Abbreviations PChl(ide) protochlorophyll(ide) - Chl(ide) chlorophyll(ide)     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coeffecient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ l mole^-1 cm^-1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assurning that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells I. Chlorophyll Accumulation and Phototransformation of Protochlorophyll(ide) in Callus Cells under Blue and Red light

A marked accumulation of chlorophyll was observed in calluscells of Nicotiana glutinosa when they were grown under bluelight, while under strong red light no chlorophyll accumulated.This blue light effect saturated at an intensity of about 500mW.m^–2. The effects of white, blue and red light on the transformationof protochlorophyll (ide) (Pchl) accumulated in dark-grown calluscells were studied by following the changes in the intensityof fluorescence emitted by Pchl and different forms of chlorophyll(ide) (Chi). Pchl with a fluorescence maximum at 633 nm (absorptionmaximum: 630 nm) decreased slowly, concomitant with an increasein Chl having a fluorescence maximum at 677 nm (absorption maximum:675 nm), which was subsequently transformed, independently oflight, to Chi with a fluorescence maximum at 683 nm (absorptionmaximum: 680 nm). Both blue and red light of low intensitieswere effective for the phototransformation, while red light,but not blue light, of high intensities caused significant destructionof Pchl. An action spectrum for this photodestruction showedthat the maximum destruction took place at 630 nm. White lightof high intensities was effective for the photoreduction withonly slight destruction of Pchl, suggesting that blue lightcounteracts the destructive effect of red light. At low temperatures,however, blue light as well as red light of low intensitiescaused photodestruction of Pchl. It was inferred that blue lightenhances a certain step or steps involved in the productionof a reductant required for the photoreduction of Pchl to Chl. (Received July 3, 1981; Accepted November 11, 1981)     

Effect of ligands on cytochromed fromAzotobacter vinelandii

Spectra of oxidized and reduced cytochromed in particles ofA. vinelandii were studied in the presence of the ligands CO, azide, and NH₂OH under oxidizing, reducing, and turnover conditions. Under oxidizing conditions, spectral changes were observed on oxidized cytochromed (absorption maximum at 648 nm) in the presence of CO and NH₂OH showing a shift of the maximum to shorter wavelengths (639 and 645 nm, respectively) and a broadening of the half-band width. Under reducing conditions, spectral changes were observed on reduced cytochromed (absorption maximum at 631 nm) in the presence of CO (absorption maximum at 636 nm), NO, NO^– ₂, and NH₂OH (absorption maximum at 642 nm in the presence of dithionite). The spectral changes of cytochromed in the presence of NH₂OH or with dithionite and NO^– ₂ were ascribed to the formation of the NO-cytochromed compound. Under turnover conditions CO, NH₂OH, and azide cause a spectral shift of the absorption maximum of cytochromed from 648 nm to 636, 645, and 655 nm, respectively. With NH₂OH and azide a broadening of the half-band width of 7 and 6 nm, respectively, was also observed. The spectral changes caused by CO and NH₂OH were interpreted as a binding of the ligands to cytochromed changing its conformation from the oxidized state absorbing at 648 nm into a more stable liganded form. Since azide does not affect the spectral bands of oxidized and reduced cytochromed, the spectral change during turnover in the presence of azide were ascribed to a preferential binding of azide to enzymically active conformation of cytochromed (cytochromed _x).     

Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ)₆ hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Microspectrophotometric characterization and localization of three visual pigments in the compound eye ofNotonecta glauca L. (Heteroptera)

Summary The absorption maxima ( _max) of the visual pigments in the ommatidia ofNotonecta glauca were found by measuring the difference spectra of single rhabdomeres after alternating illumination with two different adaptation wavelengths. All the peripheral rhabdomeres contain a pigment with an extinction maximum at 560 nm. This pigment is sensitive to red light up to wavelengths > 700 nm. In a given ommatidium in the dorsal region of the eye, the two central rhabdomeres both contain one of two pigments, either a pigment with an absorption maximum in the UV, at 345 nm, or — in neighboring rhabdoms — a pigment with an absorption maximum at 445 nm. In the ventral part of the eye only the pigment absorbing maximally in the UV was found in the central rhabdomeres. The spectral absorption properties of various types of screening-pigment granules were measured.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

CO-reacting haemoproteins of neutrophils: Evidence for cytochrome b-245 and myeloperoxidase as potential oxidases during the respiratory burst

Room temperature, CO-difference spectra of intact rat polymorphonuclear leucocytes (neutrophils) revealed the presence of a number of CO-binding haemoproteins. Absorption maxima at 413, 540 and 570 nm were attributed to the CO-complex of cytochromeb-245 whereas an absorption maximum at 595 nm was assigned to the contribution from a myeloperoxidase complex, since an identical absorption maximum was observed in CO-difference spectra of purified myeloperoxidase in the presence of H₂O₂. Photochemical action spectra for the relief of CO-inhibited O₂ uptake revealed contributions from both cytochromeb-245 and myeloperoxidase. The potential of these two O₂- and CO-binding haemoproteins to function as oxidases during the respiratory burst is discussed.     

Spectroscopic and Biochemical Analyses of UV Effects on Phycobiliproteins of Anabaena sp. and Nostoc carmium

The effects of UV (280–400 nm) irradiation on phycobiliprotein composition have been studied in two N₂-fixing cyanobacteria, Anabaena sp. and Nostoc carmium, isolated from rice paddy fields in India. Phycobiliproteins were isolated and separated by sucrose density gradient centrifugation. After UV exposure the top fraction mainly contained carotenoids (absorption maximum at 485 nm), which first showed an increase in intensity and absorption and then a gradual decrease with increasing UV exposure in Anabaena sp., whereas, in Nostoc carmium this fraction showed a steady increase over the whole exposure time. The bottom fraction of both organisms mainly contained phycocyanin (absorption peak at 620 nm) which showed a steady decline in intensity, as well as absorption. Fluorescence excitation at 620 nm resulted in an emission at 650 nm which underwent a shift towards shorter wave-lengths with increasing UV-exposure time, indicating a disassembly of the phycobilisomal complex and of impaired energy transfer from accessory pigments to the reaction centers. SDS PAGE analysis of the fractions revealed a loss of high molecular mass linker proteins and low molecular mass (αβ monomers indicating that the phycobiliproteins, which function as accessory pigments for the operation of photosystem II, disassemble during UV irradiation.     

Change of speciation of Cu(II) in growth medium due to uptake of ammonia byPseudomonas testosteroni during growth

Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH₃)₃(H₂O)₃]²⁺. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH₃)₂(H₂O)₄]²⁺, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.     

Chlorophyll Formation in the YG-6 Mutant of Chlorella regularis: Accumulation of Protochlorophyllide and Protochlorophyll Esterified with Geranylgeraniol

A chlorophyll-less mutant, YG-6, was produced by UV treatmentof the wild strain of Chlorella regularis (S-50). Cells grownfor 3 days in darkness showed a red absorption maximum at 634nm and a shoulder near 650 nm, indicative of the accumulationof at least two spectral forms of protochlorophyll(ide). Protochlorophyllide(Pchlide), and one species only of Pchlide ester, protochlorophyllesterified with geranylgeraniol (Pchl GG) were separated, thelatter with absorption maxima in diethyl ether at 438, 574 and624 nm. Spectroscopically, Pchl GG was identical with divinyl-protochlorophyll.The content of Pchlide was 10 to 13 times that of Pchl GG. Bothpaper and high-performance liquid chromatography showed thephototransformation of Pchlide, but no Pchl GG was present.This suggests that Pchl GG is not a direct precursor of chlorophylla esterified with geranylgeraniol. (Received June 4, 1983; Accepted November 11, 1983)     

Retinal receptor types inAglais urticae andPieris brassicae (Lepidoptera), revealed by analysis of the electroretinogram obtained with Fourier interferometric stimulation (FIS)

Summary Electroretinograms obtained in the butterfliesAglais urticae andPieris brassicae by the procedure of Fourier interferometric stimulation (FIS) were used to construct spectral sensitivity curves. These curves, representing the combined responses of several receptor types, were approximated by summation of spectral sensitivity curves for individual pigments, and the presence of these pigments was corroborated by chromatic adaptation experiments. The results show that the retina in the compound eye ofAglais urticae contains 3 photopigments, with maximal absorption at ca. 360 nm, 460 nm and 530 nm, respectively (Fig. 5). The retina in the compound eye ofPieris brassicae has two subdivisions. In the dorsal region of the eye 3 photopigments were found, with maxima at ca. 360 nm, 450 nm and 560 nm (Fig. 8). In the medioventral region pigments with essentially the same maxima are present together with an additional, fourth long-wavelength component with effective maximal absorption at ca. 620 nm (Fig. 11). Its absorption curve is considerably narrower than would be expected for a rhodopsin with the same absorption maximum, and presumably results from the spectral combination of a photopigment and a photostable screening pigment.Abbreviations FIS Fourier interferometric stimulation - WLP White-light position - ERG Electroretinogram