An improved spectrophotometric assay for prostaglandin synthetase based on the oxidation of epinephrine

The spectrophotometric assay method for prostaglandin synthetase from Takeguchi and Sih (1) was improved by monitoring the absorption change at 320 nm instead of at 480 nm during the enzymatic synthesis. The measurement at 320 nm is more sensitive and more consistent than the A₄₈₀ measurement. The improvement resulting from the measurement at 320 nm is attributed to a combination of factors, including a higher extinction coefficient, a more inclusive measurement of other epinephrine oxidative product(s) and lower interference due to the product of the further oxidation of adrenochrome. The validity of this spectrophotometric method was also verified in this report.     

Flash spectrophotometric identification of a fourth rhodopsin-like pigment in Halobacterium halobium

A fourth retinal-containing pigment in Halobacterium halobium cell membrane was examined by flash spectrophotometry. The absorption maximum of this pigment was at about 480 nm. Flash light caused a photoreaction cycle with a half recovery time of about 300 ms at room temperature. The photoreaction cycle involved at least two photo-intermediates. The absorption maximum of the first one was at about 350 nm and that of the second was at around 530 nm. The spectral properties of this pigment and the content of the cells correlate with the sensitivity of photo-repellent response to the light around 480 nm. We suggest a name 'phoborhodopsin' for this new pigment.     

Spectrophotometric study on the oxidation of rutin by horseradish peroxidase and characteristics of the oxidized products

Rutin (3',4',5,7-tetrahydroxyflavone-3-rutinoside) was oxidized by a horseradish peroxidase-H2O2 system to an ascorbate-reducible product which had an absorption maximum at about 290 nm and a shoulder at about 440 nm at pH 4. At pH 7.8, ascorbate-reducible compounds and sodium hydrosulfite-reducible and -nonreducible compounds were formed by the oxidation. The ascorbate-reducible compounds consisted of at least two components, the absorption bands of which were at 460-480 nm and about 620 nm. The sodium hydrosulfite-reducible compounds also consisted of two components, and one of the components which had an absorption maximum at about 480 nm seems to be formed from the ascorbate-reducible component of an absorption maximum at the blue region by a nonenzymatic reaction. A mixture of oxidized products of rutin formed by tert-butyl hydroperoxide-dependent oxidation was similar to that formed by the enzymatic reaction. It is discussed that the 3'- and 4'-OH groups of rutin were oxidized by the horseradish peroxidase-H2O2 system and that the oxidized product which could be reduced by ascorbate is an o-quinone derivative.     

Photoreaction cycle of phoborhodopsin studied by low-temperature spectrophotometry.

The photochemical and subsequent thermal reactions of phoborhodopsin (pR490), which mediates the negative phototaxis (phobic reaction) of Halobacterium halobium, were investigated by low-temperature spectrophotometry. At room temperature, the absorption spectrum of pR490 displayed vibrational structure with a maximum at 490 nm and a shoulder at 460 nm, which were remarkably sharpened by cooling, resulting in the appearance of two well-separated peaks. On irradiation of pR490 at -170 degrees C, a photo-steady-state mixture composed of pR490 and two photoproducts, P520 and P480, was formed. P480 had an absorption maximum at 480 nm and thermally converted to pR490 above -160 degrees C, while P520 had an absorption maximum at 515 nm and thermally converted to P350, the next intermediate, above -60 degrees C. Above -30 degrees C, P350 was converted to P530, and then reverted to pR490. P520, P350, and P530 may correspond to K, M, and O intermediates of bacteriorhodopsin, respectively, on the basis of their absorption spectra, but the intermediates corresponding to L and N intermediates were not observed. On the basis of these results, a new scheme of the photoreaction cycle of pR490 was presented.     

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.     

Effect of lipid-protein interaction on the color of bacteriorhodopsin

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.     

Escherichia coli DNA photolyase is a flavoprotein

Escherichia coli DNA photolyase (photoreactivating enzyme) was purified to homogeneity from a strain that greatly overproduces the protein. The purified enzyme has absorption peaks at 280 and 380 nm, a fluorescence emission peak at 480 nm and, upon denaturation, releases a chromophore that has the spectroscopic properties of flavin adenine dinucleotide (FAD), indicating that FAD is an intrinsic chromophore of the enzyme.     

Spectroscopic observation of intermediates formed during the oxidative half-reaction of copper/topa quinone-containing phenylethylamine oxidase.

The catalytic reaction of copper/topa quinone (TPQ) containing amine oxidase consists of the initial, well-characterized, reductive half-reaction and the following, less studied, oxidative half-reaction. We have analyzed the oxidative half-reaction catalyzed by phenylethylamine oxidase from Arthrobacter globiformis (AGAO) by rapid-scan stopped-flow measurements. Upon addition of dioxygen to the substrate-reduced AGAO at pH 8.2, the absorption bands derived from the semiquinone (TPQ(sq)) and aminoresorcinol forms of the TPQ cofactor disappeared within the dead time (<1 ms) of the measurements, indicating that the reaction of the substrate-reduced enzyme with dioxygen is very rapid. Concomitantly, an early intermediate exhibiting an absorption band at about 410 nm was formed, which then decayed with a rate constant of 390 +/- 50 s(-1). This intermediate was detected more prominently in the reaction in D2O buffer (pD 8.1) and was assigned to a Cu(II)-peroxy species. The assignment was based on the observation that addition of H2O2 to the substrate-reduced AGAO under anaerobic conditions led to the formation of a new band at about 415 nm, accompanied by partial quenching of absorption bands derived from TPQ(sq). Other intermediates exhibiting absorption bands at about 310 and 340 nm were also observed in the oxidative half-reaction. Kinetics of the disappearance of these latter bands did not correspond with that of the Cu(II)-peroxy band at 410 nm but did well with that of the increase of the 480 nm absorption band due to the reoxidized TPQ. Rapid increase of the absorption in the 320-370 nm region was also observed for the reaction of the substrate-reduced, Ni-substituted enzyme with dioxygen. On the basis of these results, a possible mechanism is proposed for the oxidative half-reaction of the bacterial copper amine oxidase.     

Photosynthetic enhancement spectra in higher plants

Enhancement spectra for photosynthesis of intact leaves of higherplants were investigated by means of the rate of CO₂ absorptionunder atmospheric conditions. Enhancement spectra for photosystem(system)II measured with a reference beam of 700 nm had twopronounced peaks at about 480 and 650 nm and lower humps at540–600 nm in all of the nine species tested. By the useof a rice mutant which lacks chlorophyll b, it was revealedthat the 650-nm peak and the middle humps in the spectrum canbe attributed mostly to chlorophyll b absorption, whereas the480-nm peak must be due to the absorption of carotenoids andchlorophyll b. Enhancement for system I in wheat had a peakat about 715 nm, and the maximum was much higher than that ofthe enhancement for system II. Enhancement between a red anda farred light for wheat was much greater for the farred lightthan for the red light in the presence of an excess amount ofthe other light. These results demonstrate that the enhancementphenomenon in higher plants is essentially the same as thatin green algae. (Received November 30, 1977; )     

赤潮异弯藻在铁限制条件下的光谱特性

由活体吸收光谱可见,赤潮异弯藻在叶绿素c靠近红光区的吸收峰处,由铁丰富条件下的632nm向蓝漂移2nm．由于类胡萝卜素相对于叶绿素a的比值在铁限制的细胞内增大,因而受铁限制的细胞活体吸收光谱在480nm左右类胡萝卜素的吸收峰处增加了一个吸收峰．赤潮异弯藻细胞低温荧光发射光谱在685nm处有一明显的发射峰。与铁丰富条件(10μmol.L-1)相比,缺铁(5nmol·L-1)和低铁(100nmol·L-1)细胞在685nm处的荧光得率分别升高了2倍和1．4倍．补铁48h后荧光得率则明显降低。表明细胞在铁限制条件下存在大量能量耗散,降低了细胞光合作用效率．     

假根羽藻外周天线色素分子间能量传递

假根羽藻外周天线捕光色素蛋白复合物(L ight-harvesting Comp lex II,LHC II)在不同聚集态的情况下,它所包含色素分子间的能量传递是不同的。采用荧光发射光谱和激发光谱技术对不同聚集态(单体、三聚体和寡聚体)的LHC II进行研究,发现三聚体中色素分子间的能量传递效率比较高,单体要小一些。520 nm激发下,类胡萝卜素分子向叶绿素a分子的能量传递效率:三聚体约为64%、单体约为56%;650 nm激发下,叶绿素b分子向叶绿素a分子的能量传递效率:三聚体约为89%、单体约为78%。寡聚体的能量传递要复杂些,从光谱分析出它包含两种不同吸收光谱特性的叶绿素b分子,吸收峰分别为480 nm和468 nm,其中蓝区吸收峰为480 nm的叶绿素b分子向发射685 nm荧光的叶绿素a分子的能量传递效率要小于75%。     

Effects of Nano-anatase TiO<Subscript>2</Subscript> on Absorption,Distribution of Light,and Photoreduction Activities of Chloroplast Membrane of Spinach

The effects of nano-anatase TiO₂ on light absorption, distribution, and conversion, and photoreduction activities of spinach chloroplast were studied by spectroscopy. Several effects of nano-anatase TiO₂ were observed: (1) the absorption peak intensity of the chloroplast was obviously increased in red and blue region, the ratio of the Soret band and Q band was higher than that of the control; (2) the great enhancement of fluorescence quantum yield near 680 nm of the chloroplast was observed, the quantum yield under excitation wavelength of 480 nm was higher than the excitation wavelength of 440 nm; (3) the excitation peak intensity near 440 and 480 nm of the chloroplast significantly rose under emission wavelength of 680 nm, and F ₄₈₀ / F ₄₄₀ ratio was reduced; (4) when emission wavelength was at 720 nm, the excitation peaks near 650 and 680 nm were obviously raised, and F ₆₅₀ / F ₆₈₀ ratio rose; (5) the rate of whole chain electron transport, photochemical activities of PSII DCPIP photoreduction and oxygen evolution were greatly improved, but the photoreduction activities of PSI were a little changed. Together, the studies of the experiments showed that nano-anatase TiO₂ could increase absorption of light on spinach chloroplast and promote excitation energy to be absorbed by LHCII and transferred to PSII and improve excitation energy from PSI to be transferred to PSII, thus, promote the conversion from light energy to electron energy and accelerate electron transport, water photolysis, and oxygen evolution.     

Optical characteristics of the phototroph Thiocapsa roseopersicina and implications for real-time monitoring of the bacteriochlorophyll concentration.

Optical characteristics of a Thiocapsa roseopersicina culture and environmental samples containing T. roseopersicina were investigated in the spectral range of 400 to 1,100 nm (absorption coefficient, diffuse attenuation coefficient, and reflectance). Specific absorption coefficients of T. roseopersicina at wavelengths of 480, 520, 550, 580, 805, 860, and 880 nm were determined. It is suggested that the optical properties of T. roseopersicina in the near-infrared range of 800 to 930 nm, confirmed in this study, may be used for development of remote sensing techniques for real-time monitoring of T. roseopersicina and other bacteriochlorophyll a-containing microbes.     

Bi‐modal sensitivity to monochromatic light by the mud snail Ilyanassa obsoleta †

Under laboratory conditions the mud snail Ilyanassa obsoleta exhibits a pronounced positive behavioral response to monochromatic light. The response spectrum for‐this positive phototaxis has maxima at 480 nm and 560–580 nm. The threshold intensities for this response are 2.06 x 10^‐7 /μW/cm² for 480 nm and 2.18 × 10^‐7 μW/cm² for 580 nm. These results are suggestive of Ilyanassa's possessing two visual pigments with different absorption maxima.     

Optical assessment of phytoplankton nutrient depletion

The ratio of light absorption at 480 and 665 nm by 90% acetoneextracts of marine phytoplankton pigments has been examinedas a potential indicator of phytoplankton nutritional statusin both laboratory and field studies. The laboratory studiesdemonstrated a clear relationship between nutritional status(carbon/nitrogen ratio) and the absorption ratio that was independentof light and temperature climate. The absorption ratio for nutrient-repletecells was shown to vary between taxonomic groups. However, theinter-specific variation was less than the differences observedbetween nutrient-replete and nutrient-depleted cells. The fielddata suggest that the absorption ratio may be a useful indicatorof nutritional status of natural phytoplankton populations,and can be used to augment the interpretation of other data.     

The inhibition of mitosis in tissue culture cells by blue light

Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.     

The active sites of cytochromes P450 IA1, IIB1, IIB2, and IIE1. Topological analysis by in situ rearrangement of phenyl-iron complexes

The reactions of cytochromes P450 IA1, IIB1, IIB2, and IIE1 with phenyldiazene yield complexes with absorption maxima at either 474 or 480 nm. Anaerobic extraction of the prosthetic group from the phenyldiazene-treated proteins followed by its exposure to oxygen and strong acid produces an equal mixture of the four possible N-phenylprotoporphyrin IX regioisomers. Exposure of the anaerobically extracted heme complexes to oxygen in the absence of strong acid results in their decomposition to heme and products other than N-phenylprotoporphyrin IX. These results show that the 474/480 nm absorption maxima are due to sigma phenyl-iron complexes. Treatment of the intact hepatic cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 474/480 nm band. Extraction of the prosthetic group then yields only the two N-phenylprotoporphyrin IX regioisomers with the N-phenyl group on pyrrole rings A and D. The same regioisomer pattern is obtained if the cytochrome P450IA1 phenyl-iron complex is simply warmed to 37 degrees C, but this thermal rearrangement occurs much less readily, if at all, with the complexes of the other isozymes. The regioisomers with the N-phenyl on pyrrole rings A and D are obtained in a 2:1 ratio with isozyme IA1, 1:1 with IIB2, 1:1.7 with IIB1, and 1:2 with IIE1. These results establish that the active sites of these cytochrome P450 isozymes have a common architecture despite their gross differences in substrate specificity. In this architecture, the region directly above pyrrole rings A and D is relatively open whereas that above pyrrole rings B and C is occluded by protein residues.