Carotenoid and retinoid--two pigments in a gecko eye lens

The eye lenses of the Moroccan day gecko Quedenfeldtia trachyblepharus contain two different pigments: a retinoid (minor pigment) and a carotenoid (major pigment). The retinoid, all-trans 3, 4-didehydroretinol, is bound to iota-crystallin, which comprises only 2% of the total amount of crystallins. The carotenoid is associated to gammas-crystallin - comprising about 10% of total amount of crystallins--and causes the dark yellow colour of the lens. The absorption spectrum of the isolated carotenoid shows a major, triple-peaked band at 372, 392, and 416 nm and two minor peaks at 284 and 294 nm. This spectrum reminds of that of galloxanthin, a carotenoid found in oil droplets of some avian retinae. The absorption spectrum of the carotenoid-gammas-crystallin complex is shifted 6-8 nm bathochromically. In the lens, this complex absorbs ultraviolet and shortwave blue radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against photodamage. Both the retinoid and the carotenoid are present in eye cups. The lenticular carotenoid of Quedenfeldtia is the first example of a carotenoid in the lens of a terrestrial vertebrate with a sufficiently high concentration to be physiologically effective as a UV-filter. Additionally, it is unique in being the first example of a carotenoid associated with gammas-crystallin.     

Iodopsin

The iodopsin system found in the cones of the chicken retina is identical with the rhodopsin system in its carotenoids. It differs only in the protein-the opsin -with which carotenoid combines. The cone protein may be called photopsin to distinguish it from the scotopsins of the rods. Iodopsin bleaches in the light to a mixture of photopsin and all-trans retinene. The latter is reduced by alcohol dehydrogenase and cozymase to all-trans vitamin A(1). Iodopsin is resynthesized from photopsin and a cis isomer of vitamin A, neovitamin Ab or the corresponding neoretinene b, the same isomer that forms rhodopsin. The synthesis of iodopsin from photopsin and neoretinene b is a spontaneous reaction. A second cis retinene, isoretinene a, forms iso-iodopsin (lambda(max) 510 mmicro). The bleaching of iodopsin in moderate light is a first-order reaction (Bliss). The synthesis of iodopsin from neoretinene b and opsin is second-order, like that of rhodopsin, but is very much more rapid. At 10 degrees C. the velocity constant for iodopsin synthesis is 527 times that for rhodopsin synthesis. Whereas rhodopsin is reasonably stable in solution from pH 4-9, iodopsin is stable only at pH 5-7, and decays rapidly at more acid or alkaline reactions. The sulfhydryl poison, p-chloromercuribenzoate, blocks the synthesis of iodopsin, as of rhodopsin. It also bleaches iodopsin in concentrations which do not attack rhodopsin. Hydroxylamine also bleaches iodopsin, yet does not poison its synthesis. Hydroxylamine acts by competing with the opsins for retinene. It competes successfully with chicken, cattle, or frog scotopsin, and hence blocks rhodopsin synthesis; but it is less efficient than photopsin in trapping retinene, and hence does not block iodopsin synthesis. Though iodopsin has not yet been prepared in pure form, its absorption spectrum has been computed by two independent procedures. This exhibits an alpha-band with lambda(max) 562 mmicro, a minimum at about 435 mmicro, and a small beta-band in the near ultraviolet at about 370 mmicro. The low concentration of iodopsin in the cones explains to a first approximation their high threshold, and hence their status as organs of daylight vision. The relatively rapid synthesis of iodopsin compared with rhodopsin parallels the relatively rapid dark adaptation of cones compared with rods. A theoretical relation is derived which links the logarithm of the visual sensitivity with the concentration of visual pigment in the rods and cones. Plotted in these terms, the course of rod and cone dark adaptation resembles closely the synthesis of rhodopsin and iodopsin in solution. The spectral sensitivities of rod and cone vision, and hence the Purkinje phenomenon, have their source in the absorption spectra of rhodopsin and iodopsin. In the chicken, for which only rough spectral sensitivity measurements are available, this relation can be demonstrated only approximately. In the pigeon the scotopic sensitivity matches the spectrum of rhodopsin; but the photopic sensitivity is displaced toward the red, largely or wholly through the filtering action of the colored oil globules in the pigeon cones. In cats, guinea pigs, snakes, and frogs, in which no such colored ocular structures intervene, the scotopic and photopic sensitivities match quantitatively the absorption spectra of rhodopsin and iodopsin. In man the scotopic sensitivity matches the absorption spectrum of rhodopsin; but the photopic sensitivity, when not distorted by the yellow pigmentations of the lens and macula lutea, lies at shorter wave lengths than iodopsin. This discrepancy is expected, for the human photopic sensitivity represents a composite of at least three classes of cone concerned with color vision.     

A light-sensitive yellow pigment from the housefly

Extraction of house-fly heads with neutral phosphate buffer yielded a dark brown solution from which a number of pigments were separated, either wholly or partially, by chromatography on a column of calcium phosphate mixed with celite. One of the pigments was light-sensitive, and had a yellow color, with a spectral absorption maximum at 437 mmicro in phosphate buffer at pH 6.5. Several consecutively eluted fractions from each chromatogram of the house-fly head extract showed the characteristic absorption curve of this pigment with no trace, spectroscopically, of the other pigments of the extract. The products of bleaching the pigment at pH 6.5 had an absorption curve showing plateaus at 440 to 460 mmicro and 350 to 360 mmicro and an inflection at about 250 mmicro. Above pH 8.0 the pigment bleached in the dark giving an absorption maximum at about 380 mmicro, and inflections at 290 mmicro and at about 250 mmicro. With 2.5 to 5 N HCl or H(2)SO(4) an absorption maximum at 470 to 475 mmicro was obtained with either the unbleached or the bleached pigment. With sulfosalicylic acid, ethanol, or heating at 100 degrees C., a part of the pigment was precipitated, leaving a light-stable yellow supernatant. This light-sensitive house-fly pigment cannot as yet be identified with any of the previously known insect pigments or with the photosensitive pigments of other animals, though these latter compounds exhibit some similarity in their spectroscopic properties.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides: an analysis and interpretation of the band shifts.

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20% of the total carotenoid content, is responsible for the light-induced absorbance changes. The 'remaining' pool absorbs at wavelengths which were about 5 nm lower than those at which the 'changing' pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields. The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 +/- 6 mV per nm shift.     

The light-induced carotenoid absorbance changes in Rhodopseudomonas sphaeroides. An analysis and interpretation of the band shifts

An analysis has been made of the spectrum of the carotenoid absorption band shift generated by continuous illumination of chromatophores of the GlC-mutant of Rhodopseudomonas sphaeroides at room temperature by means of three computer programs. There appears to be at least two pools of the same carotenoid, only one of which, comprising about 20 % of the total carotenoid content, is responsible for the light-induced absorbance changes. The ‘remaining’ pool absorbs at wavelengths which were about 5 nm lower than those at which the ‘changing’ pool absorbs. This difference in absorption wavelength could indicate that the two pools are influenced differently by permanent local electric fields.

The electrochromic origin of the absorbance changes has been demonstrated directly; the isosbestic points of the absorption difference spectrum move to shorter wavelengths upon lowering of the light-induced electric field. Band shifts up to 1.7 nm were observed. A comparison of the light-induced absorbance changes with a KCl-valinomycin-induced diffusion potential has been used to calibrate the electrochromic shifts. The calibration value appeared to be 137 ± 6 mV per nm shift.     

The relationship between chlorophyll and the carotenoids in the algal flagellate,Euglena

1. We have obtained an action spectrum for chlorophyll formation in Euglena gracilis. This action spectrum is similar to the absorption spectrum of protochlorophyll. However, efforts to isolate and identify this pigment have been unsuccessful. 2. Porphyrins have been extracted from both the normal and dark-adapted Euglena and a chlorophyll-free mutant. 3. The "action" spectra for chlorophyll and carotenoid synthesis have been found to almost coincide, indicating that the same porphyrin-like molecule may influence the synthesis of both pigments. 4. It is indicated that two porphyrin-like systems are in operation simultaneously, one concerned with carotenoid "removal" and another involved in carotenoid and chlorophyll synthesis.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides II. Analysis of the band shifts of carotenoid and bacteriochlorophyll

An analysis was made of the changes of pigment absorption upon illumination of chromatophores of Rhodopseudomonas sphaeroides at ?35 °C, described in the preceding paper (de Grooth, B. G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 237–246). Comparison of the light-induced difference spectra in the carotenoid region obtained without additions, and in the presence of N-methylphenazonium methosulphate and ascorbate as donor-acceptor system showed that the latter spectrum was not only about 10 times larger in amplitude, but also red-shifted with respect to the first one. Together with the shape of the difference spectrum, this indicated that the spectrum obtained in the presence of a donor-acceptor system is due to an electrochromic shift of the absorption spectrum of a carotenoid by a few nm towards longer wavelength, caused by a delocalized potential across the chromatophore membrane. The results of an analysis of the kinetics of the absorbance changes near the zero points of the spectrum were in quantitative agreement with the extent of the red shift and indicated a shift of 0.25 nm for a single electron transfer per reaction center, and shifts of up to 4 nm when the electron transport is stimulated by a donor-acceptor system. For bacteriochlorophyll B-850 the shift is three times smaller.Analysis of the overall absorption spectrum showed that there are at least two pools of carotenoid. The carotenoid that shows electrochromism has absorption bands at 452, 481 and 515 nm, and comprises about one-third of the total carotenoid present; the remaining pool absorbs at about 7 nm shorter wavelength and does not show an electrochromic response to illumination. Both pools presumably consist of spheroidene; the differences in band location may be explained by the assumption that only the first pool is subjected to a local electric field which induces an electric dipole even at zero membrane potential. Similar results were obtained at room temperature and with a mutant of Rps. sphaeroides (G1C)-containing neurosporene.     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment-protein complexes: peridinin-chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin-chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment-protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

Near edge X-ray absorption fine structure spectroscopy (NEXAFS) of pigment–protein complexes: Peridinin–chlorophyll a protein (PCP) of Amphidinium carterae

Peridinin–chlorophyll a protein (PCP) is a unique water soluble antenna complex that employs the carotenoid peridinin as the main light-harvesting pigment. In the present study the near edge X-ray absorption fine structure (NEXAFS) spectrum of PCP was recorded at the carbon K-edge. Additionally, the NEXAFS spectra of the constituent pigments, chlorophyll a and peridinin, were measured. The energies of the lowest unoccupied molecular levels of these pigments appearing in the carbon NEXAFS spectrum were resolved. Individual contributions of the pigments and the protein to the measured NEXAFS spectrum of PCP were determined using a “building block” approach combining NEXAFS spectra of the pigments and the amino acids constituting the PCP apoprotein. The results suggest that absorption changes of the pigments in the carbon near K-edge region can be resolved following excitation using a suitable visible pump laser pulse. Consequently, it may be possible to study excitation energy transfer processes involving “optically dark” states of carotenoids in pigment–protein complexes by soft X-ray probe optical pump double resonance spectroscopy (XODR).     

The effects of light on a circadian rhythm of conidiation in neurospora

The expression of a circadian rhythm of conidiation by timex, a strain of Neurospora crassa, is inhibited by growth in continuous white light. The action spectrum for this effect has a strong peak (with minor subpeaks) in the blue region of the visible spectrum, and a broad shoulder in the near ultraviolet. This action spectrum suggests that a carotenoid or flavin compound may be the photoreceptor, but does not allow one to determine conclusively whether the receptor is indeed a carotenoid, flavin, or some other unrelated pigment. Two lines of evidence suggest that a carotenoid is not the photoreceptor. First, the in vivo absorption spectrum of timex (representing the sum of the spectra of the individual pigments present, predominantly carotenoids) has peaks at wavelengths 10 to 20 mμ longer than those of the action spectrum peaks. Second, an albino-timex has normal photosensitivity, a situation requiring that the photoreceptor, if carotenoid, be a quantitatively minor constituent of the total carotenoid complement.

The magnitude and direction of phase-shift resulting from a standard dose of white light given at different times in the daily cycle of timex varies in the manner reported for other organisms. Additional phase-shift experiments have shown that there are no major transients in the attainment of a new equilibrium after a phase-shifting perturbation, and that 2 light reactions (rapidly and slowly saturating) may be involved in the phase-shift response.

     

The synthesis from vitamin A1 of retinene 1 and of a new 545 m-mu chromogen yielding light-sensitive products

Ball, Goodwin, and Morton (1946) have reported that vitamin A(1) in contact with solid manganese dioxide is transformed slowly into a substance which displays spectroscopic properties of retinene(1). The latter is known to be the precursor of vitamin A(1) in the rhodopsin cycle of the retinal rods. The synthetic product is here referred to as "retinene(1)." In the present experiments this observation is confirmed. The procedure is recast in the form of a chromatographic oxidation. Manganese dioxide is packed in a column, vitamin A(1) solution poured in at the top, and the product drawn off in the filtrate. Depending upon the proportions of manganese dioxide and vitamin A(1), the product is either "retinene(1)," or a new substance which yields with antimony chloride a wine-red product with maximal absorption at 545 mmicro (545 mmicro chromogen). This procedure is an example of a potentially important class of chromatographic reactions. The synthetic "retinene(1)" is virtually identical with the natural substance in absorption spectrum and antimony chloride reaction. It lacks the pH indicator properties of crude natural retinene(1). The 545 mmicro chromogen possesses absorption maxima at 380 and 290 mmicro in chloroform; at 376 and 290 mmicro in ethanol; and at 361 and 277 mmicro in hexane. It is non-fluorescent. It has no acidic character, but on the contrary is mildly basic, being extracted from hexane by sulfuric or hydrochloric acids to form orange-red products. In partition between petroleum ether and aqueous methanol it is highly hypophasic. It is adsorbed strongly on calcium carbonate. Certain peculiarities in spectral behavior indicate the presence of a carbonyl group in the 545 mmicro chromogen, and support Morton's proposal that such a group occurs in retinene(1). Other properties of the 545 mmicro chromogen indicate hydroxyl groups. This substance therefore appears to be a hydroxy-carbonyl derivative of vitamin A(1). The red products which the 545 mmicro chromogen forms with antimony chloride or with sulfuric or hydrochloric acids are all markedly light-sensitive. They appear to be formed by the condensation of two molecules with loss of water; and to bear a close generic relation to the prosthetic groups of the visual photopigments.     

Light‐induced pigment degradation in leaves and ripening fruits studied in situ with reflectance spectroscopy

Pigment breakdown mediated by activated oxygen species is a consequence and a general symptom of oxidative stress and injury to plants. We have attempted to estimate the patterns of pigment bleaching and follow pigment susceptibility to irradiation as related to the process of senescence/ripening. Light‐induced pigment breakdown was studied in situ in the leaves of a shade‐requiring plant, wax flower ( Hoya carnosa R. Br.), as well as in apple ( Malus domestica Borlh. cv. Zhigulevskoe) and lemon ( Citrus limon Burm. cv. Pavlovsky) fruits, using reflectance spectroscopy. It was found that the sensitivity of plant pigments to photobleaching increases as ripening progresses in lemon fruit. Kinetic analysis showed that in all systems a rapid breakdown of the pigment occurs after a lag‐phase. The signature analysis revealed a common pattern of chlorophyll and carotenoid changes, but degradation of the individual pigments was found to be inhomogeneous. Both in lemon and apple fruits a decrease in reflectance in the band of carotenoid absorption preceded pigment photodestruction. In the fruits, the bulk of chlorophyll b and the long‐wavelength chlorophyll a forms were degraded at early stages of the process whereas the breakdown of both chlorophylls in H. carnosa leaves was more synchronous. Prolonged irradiation induced bleaching of the main chlorophyll a band with maximum at 678 nm in the difference spectra, as well as carotenoids. Some features of reflectance spectra in the bands of chlorophyll and carotenoid absorption were found to be suitable for the differentiation of photo‐induced pigment breakdown from the transformation of the pigments taking place during senescence.     

Antheraxanthin, a light harvesting carotenoid found in a chromophyte alga

The pigments of the chromophyte freshwater alga, Chrysophaera magna Belcher were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) to reveal the presence of chlorophylls a and c, β-carotene, fucoxanthin, and antheraxanthin. The presence of antheraxanthin was verified by comparison of TLC R_F values, HPLC retention times, and absorption features to those of authentic, synthetic antheraxanthin. Antheraxanthin accounted for about 15% of the total carotenoid content of C. magna. The molar ratio of the major carotenoids was antheraxanthin:fucoxanthin:β-carotene, 1:2.3:3.3. The whole-cell absorption spectrum revealed a broad band between 470 and 520 nanometers which was attributed to fucoxanthin and antheraxanthin in vivo. Upon extraction in hydrocarbon, this broad absorption region was lost. The in vivo fluorescence excitation spectrum for 680 nm emission revealed the energy transfer activities and light harvesting roles of chlorophylls a and c, and fucoxanthin. In addition, an excitation band was resolved at 487 nanometers which could be attributed only to antheraxanthin. Comparison of whole-cell fluorescence excitation spectra of C. magna with the diatom Phaeodactylum tricornutum, which possesses fucoxanthin but not antheraxanthin, supports the assignment of the 487 nm band to antheraxanthin. This is the first report of a photosynthetic light harvesting function of the xanthophyll, antheraxanthin. This carotenoid broadens the absorption cross-section for photosynthesis in C. magna and extends light harvesting into the green portion of the spectrum.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

The Spectral Sensitivity of Crayfish and Lobster Vision

(1) The spectral sensitivity function for the compound eye of the crayfish has been determined by recording the retinal action potentials elicited by monochromatic stimuli. Its peak lies at approximately 570 mµ. (2) Similar measurements made on lobster eyes yield functions with maxima in the region of 520 to 525 mµ, which agree well with the absorption spectrum of lobster rhodopsin if minor allowances are made for distortion by known screening pigments. (3) The crayfish sensitivity function, since it is unaffected by selective monochromatic light adaptation, must be determined by a single photosensitive pigment. The absorption maximum of this pigment may be inferred with reasonable accuracy from the sensitivity data. (4) The visual pigment of the crayfish thus has its maximum absorption displaced by 50 to 60 mµ towards the red end of the spectrum from that of the lobster and other marine crustacea. This shift parallels that found in both rod and cone pigments between fresh water and marine vertebrates. In the crayfish, however, an altered protein is responsible for the shift and not a new carotenoid chromophore as in the vertebrates. (5) The existence of this situation in a new group of animals (with photoreceptors which have been evolved independently from those of vertebrates) strengthens the view that there may be strong selection for long wavelength visual sensitivity in fresh water.     

Light-induced blue shift of the carotenoid spectrum in chromatophores of Chromatium vinosum strain D

In Chromatium chromatophores, the response of part of the carotenoid complement to a light-induced membrane potential is a shift to the blue of its absorption spectrum, as indicated by the characteristics of the light-minus-dark difference spectrum. The spectrum in the dark of the population of carotenoid which responds to a light-induced membrane potential is located at least 1–2 nm to the red in comparison to the total carotenoid absorption. The results indicate that the proposed permanent electric field affecting the responding population has a polarity with respect to the chromatophore membrane opposite to that in Rhodopseudomonas sphaeroides chromatophores. The carotenoid absorption change interferes seriously with measurements of cytochrome c-555 redox changes at its α band.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Optical activity of octopus metarhodopsins.

The optical activity of octopus rhodopsin, acid metarhodopsin and alkaline metarhodopsin was studied by a sensitive and rapid CD apparatus. For sometime it has been thought that cephalopod metarhodopsins do not have any optical activity associated with their main absorption band. However, the present work shows that acid metarhodopsin in digitonin has a positive CD band at 498 nm and a negative CD band at 436 nm and alkaline metarhodopsin has a negative CD band at 381 nm. Detergent affected the wavelength of the CD peak of the visual pigments though the pattern of the spectrum was similar. From these results it is concluded that the conformation of all-trans retinal in octopus metarhodopsin is influenced by the asymmetric conformation of the protein near the retinal and therefore inducing optical activity.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

Veränderung der Lichtabsorption eines Carotinoids im Enzym (De-epoxidase)-Substrat(Violaxanthin)-Komplex

Summary The enzyme violaxanthin de-epoxidase catalysing the transformation of the xanthophyll violaxanthin to zeaxanthin has been isolated from spinach chloroplasts.Special properties of the enzyme make it possible for the carotenoid to be bound without initiation of any catalytic reaction; the isolation of an enzyme-substratecomplex is thereby greatly facilitated. After addition of cofactors to this complex the transformation of violaxanthin to zeaxanthin takes place.In this complex the light-absorption of violaxanthin is changed drastically: the normal three-peak absorption curve in the blue region of the spectrum is strongly decreased but in the uv-region around 380 nm a new absorption maximum appears.Recently a similar spectrum has been determined in vivo in the phototropic sensitive region of the sporangiophores of Phycomyces (Wolken, 1969) with the aid of microspectrophotometry.From these results it is concluded that part of the carotenoids occurring in plants is present in a protein-bound form and that these pigments show a considerably changed light absorption in comparison with the isolated pigment. The simultaneous occurrence of differently bound carotenoids may lead to the formation of 4-peak absorption curves (similar to those of flavines) with 3 maxima in the blue region and 1 maximum in the UV around 370–380 nm. These 4-peak curves are characteristic for many action spectra.It is emphasized that the strong absorption changes of carotenoids occurring during the binding of these pigments to proteins should be considered in analyzing difference spectra.