STUDIES ON CHROMOPHORE COUPLING IN ISOLATED PHYCOBILIPROTEINS. IV. FEMTOSECOND TRANSIENT ABSORPTION STUDY OF ULTRAFAST EXCITED STATE DYNAMICS IN TRIMERIC PHYCOERYTHROCYANIN COMPLEXES*

The excited state dynamics of trimeric phycoerythrocyanin has been studied by two-color femtosecond transient absorption spectroscopy with a time-resolution better than 200 fs. Upon selective excitation of the short-wavelength phycobiliviolin chromophore at 575 nm absorption bleachings are observed. An isotropic ultrafast decay of the initial bleaching of 585 ± 40 fs has been resolved at short detection wavelength (578 nm). Upon stepwise increase of the detection wavelength up to 617 nm, the bleaching showed a delayed rise above 593 nm with r_rice=380–580 fs. All other isotropic kinetic components in this wavelength range were longer than 100 ps. The ultrafast component is discussed in terms of an energy transfer process from the α-84 phycobiliviolin chromophore to the β-84 chromophore in adjacent monomer subunits of the trimer. It is concluded that the β-155 chromophore is the longest-wavelength chromophore. Exciton relaxation between closely spaced chromophore pairs is discussed as an alternative interpretation for the ultrafast component.     

ISOMERIZATION OF THE RETINYLIDENE CHROMOPHORE OF BACTERIORHODOPSIN IN LIGHT ADAPTATION: INTRINSIC ISOMERIZATION OF THE CHROMOPHORE AND ITS CONTROL BY THE APO-PROTEIN

Abstract— The dependence of the isomeric configuration of the retinylidene chromophore of bacteriorhodopsin on the pH value and on the wavelength of irradiation (in a photostationary state) were examined by high performance liquid chromatographic analyses of extracted retinal. The process of isomerization of the chromophore during light adaptation was also traced. More than 93% of all- trans and less than 5% of 13- cis retinal were extracted in the photostationary state for irradiation at 560 nm in the pH region of5–9 as well as for irradiation in the wavelength region of 400–650 nm at pH 7. Comparison of the above photostationary state composition with that of protonated n -butylamine Schiff base of retinal indicates that strong constraint is applied to the chromophore by the apo-protein. The constraint can be changed at low or high pH by a partial denaturation or transition of the apo-protein, which results in the generation of 11- cis retinal in the extract. At higher photon density, the isomerization process of the chromophore during light adaptation at pH 7 was characterized, as extracted isomeric retinal, by (1) the initial decrease in 13- cis and increase in all- trans , (2) a subsequent, transient toward the above photostationary state composition. The results are discussed in terms of both the photoisomerization pattern inherent in the retinylidene chromophore and the control by the apo-protein.     

ORIENTATION OF RETINYLIDENE CHROMOPHORE OF HYPSORHODOPSIN IN FROG RETINA

Abstract— Hypsorhodopsin and bathorhodopsin were formed in the frog retina by irradiating rhodopsin at liquid He temperature (9 K) with orange light (> 520 nm) and blue light (437 nm), respectively. Hypsorhodopsin was converted to bathorhodopsin in the retina by warming above 32 K in the dark. Similar phenomena were observed in the rod outer segment suspension. A difference spectrum between hypsorhodopsin and bathorhodopsin in the retina produced by warming was almost identical with that in the rod outer segment suspension. This suggests that the transition dipole moment of hypsorhodopsin is parallel to the disk membrane plane which is also parallel to that of bathorhodopsin.     

ENERGETICS OF PROTONATION-DEPROTONATION OF THE CHROMOPHORE IN RETINAL PROTEINS

Abstract— We investigate the energetics of protonation and deprotonation of retinylidene Schiff-base (SB) which is realized in the functioning of retinal proteins. We first calculate the energy difference ΔE between the protonated and unprotonated states of the SB by the ab initio molecular orbital method, using two kinds of molecular model; a counter-ion model where a carboxyl group of Glu or Asp is directly hydrogen-bonded to the SB, and a water-bridge model where a water molecule bridges the carboxyl group and the SB. The calculated results indicate that the protonated SB state is unstable compared with the unprotonated SB state in either model. In addition, we find that coordination of some water molecules to the carboxyl group reduces ΔE significantly. The value of AE for the counterion model with two coordinated water molecules is 0.003 eV. Next, we calculate the electrostatic interaction energy between a tryptophan residue and the SB. We find that the protonated state is more stabilized than the unprotonated state by about 0.1 eV with one tryptophan residue. This fact indicates that if some aromatic amino acid residues work cooperatively, they can contribute to significantly reducing ΔE. We also discuss the possible role of amino acid residues which make hydrogen-bond with the carboxyl group of interest.     

CIRCULAR DICHROISM OF THE PHYTOCHROME CHROMOPHORES IN NATIVE AND DENATURED STATE

Abstract— The circular dichroism spectra of oat phytochrome were recorded. Qualitatively, the same spectra were found for large (360 kilodaltons) and small (60 kilodaltons) phytochrome. Quantitative CD data were reported for small P_r and P_fr (photoequilibrium mixture with 20% P_r) in tris buffer (native state) and in acid urea (denatured state). Further, the CD spectra of a phytochromobilinpeptide in acid solution with and without urea were recorded. Differences between the data in native and denatured state are discussed.     

PHOTOREACTIONS OF THE CHROMOPHORE CATIONS OF DENATURED SPECIES OF C-PHYCOCYANIN AND ALLOPHYCOCYANIN

Abstract— C-phycocyanin dissolved in buffer containing 75% ethylene glycol (vol/vol) shows photorevers-ible reactions which are ascribed to the interconversion by light of two species: PC_r, PC_g. After denaturation with formic acid, the chromophore cation of PC_g, can be irreversibly photoconverted to that of PC_r A conversion of the chromophore of denatured PC_g, to that of denatured PC_r also occurs after adjustment of the pH to about 8. Under the same conditions the chromophore of allophycocyanin shows similar reactions. Moreover, the reactions of the chromophores of the denatured species of these phycobiliproteins are similar to the reactions of the chromophore of the denatured species P_fr of photochrome.     

THEORETICAL STUDY OF OPTICAL SPECTRA AND CONFORMATION OF THE CHROMOPHORE OF HYPSORHODOPSIN

Optical spectra of hypsorhodopsin were theoretically analyzed by assuming the unprotonated all-trans form of the Schiff base of the chromophore. The large bathochromic shift of the optical absorption of hypsorhodopsin from that of the retinylidene Schiff base in solution could be easily explained by twisting the double bond of the chromophore; it could not be explained by simple counter anion models. Using the same twisted chromophore conformation for hypsorhodopsin as that of bathorhodopsin obtained by the torsion model, we showed that the calculated absorption wavelength was in fairly good agreement with the experimental value. Our calculated oscillator strengths and rotational strengths were quite similar between hypsorhodopsin and bathorhodopsin. Those theoretical results will be useful when one examines the relation of the chromophore's conformation between hypsorhodopsin and bathorhodopsin experimentally.     

PHYCOBILISOMES AND ISOLATED PHYCOBILIPROTEINS. EFFECT OF GLUTARDIALDEHYDE AND BENZOQUINONE ON FLUORESCENCE

Abstract— The fluorescence of the biliproteins C-phycocyanin from Spirulina platensis , B-phycoerythrin from Porphyridium cruentum and of isolated whole P. cruentum phycobilisomes is quenched in the presence of glutardialdehyde (GA) or benzoquinone (BQ). The kinetics of fluorescence decrease thus induced is biphasic. If GA is used as a quencher, the fluorescence can be recovered at 77 K. Contrary to the GA-effect, only a minor recovery takes place with BQ at 77 K, thus demonstrating a different mechanism of action of GA and BQ on biliproteins.     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

中国煤中铍的分布赋存特征研究

根据对中国不同矿区1018个煤层煤样和生产煤样铍含量数据的统计结果,分析了中国不同成煤时代、不同聚煤区煤中铍的分布特征。同时,根据对10个不同煤田煤样的实验室浮沉试验、煤岩分析等试验研究,运用Solari方法,计算了煤中各组分中铍的理论含量,讨论了中国煤中铍的赋存特征,尤其是铍在各种煤岩组分中的分布。研究表明,铍在煤中富集程度较高,但相对于世界煤中平均水平来说,中国煤中铍含量总体水平较低。煤中铍主要赋存于惰质组及伊利石粘土矿物中,在镜质组及其它矿物中含量较低。同时,中国大多数煤中铍的有机亲和性指数较高,不利于选煤过程中铍的脱除。     

在Raney Fe催化剂上F-T合成产物的分布和选择性

在浆态相Ｆ－Ｔ合成的连续搅拌釜反应器中,研究了ＲａｎｅｙＦｅ系列催化剂和其它工业铁基催化剂的性能。结果发现,ＲａｎｅｙＦｅ的前驱体对其性能影响很大。由Ａ１／Ｆｅ比为３的合金得到的ＲａｎｅｙＦｅ表现出了良好的活性稳定性,常规条件下ＣＯ的转化接近完全,其较大的中孔分布可能对初级产物的扩散限制较小,因而生成的２－烯烃和异构烃就少。也比较分析了催化剂固载量和粒度对ＲａｎｅｙＦｅ上的烷烃选择性的影响。此外,从产物分布的规律来看,在ＲａｎｃｙＦｅ催化剂上以生成高碳数烷烃和低碳数烯烃为主。     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

PHOTOCHEMICAL AND PHOTOBIOLOGICAL PROPERTIES OF KETOPROFEN ASSOCIATED WITH THE BENZOPHENONE CHROMOPHORE

Abstract Irradiation of ketoprofen in neutral aqueous medium gave rise to 3-ethylbenzophenone as the major photoproduct. Its formation is justified via protonation of a benzylic carbanion or hydrogen abstraction by a benzylic radical. Minor amounts of eight additional compounds were isolated. Four of them are derived from the benzylic radical: 3-(1-hydroperoxyethyl)benzophenone, 3-(1-hydroxyethyl)benzophenone, 3-acetylbenzophenone and 2,3-bis- (3-benzoylphenyl)butane. The other four products involve initial hydrogen abstraction by the excited benzophenone chromophore of ketoprofen: 1,2-bis-(3-ethylphenyl)-1,2-diphenyl-1,2-ethanediol, 2-(3-benzoylphenyl)-1-(3-ethylphenyl)-1 -phenylpropan-1 -01,α -(3-ethylphenyl)phenylmethanol, 1,2-bis-[3-(2-hydroxycarbonylethyl)phenyl]-1,2-di-phenyl-1,2-ethanediol. The latter process was found to mediate the photoperoxidation of linoleic acid through a type I mechanism, as evidenced by the inhibition produced by the radical scavengers butylated hydroxyanisole and reduced glutathione. The major photoproduct, which contains the benzophenone moiety but lacks the propionic acid side chain, also photosensitized linoleic acid peroxidation. Because lipid peroxidation is indicative of cell membrane lysis, the above findings are highly relevant to explain the photobiological properties of ketoprofen.     

PHYCOERYTHROCYANINS FROM Westiellopsis prolifica AND Nostoc rivulare: CHARACTERIZATION OF THE PHYCOVIOLOBILIN CHROMOPHORE IN BOTH STATES*

Abstract— Phycoerythrocyanin or fractions enriched in it have been isolated from the filamentous cyanobacteria, Westiellopsis prolifica ARM 365 and Nostoc rivulare ARM 212. Both show the photoreversible photochromism (difference maxima at 503 and 570 nm) characteristic of this pigment, which is related to the phycoviolobilin chromophore on the α-subunit. Native phycoerythrocyanin and its β-subunit show little if any reversible photochemistry in the 600–620 nm region, where the phycocyanobilin chromophores absorb maximally. Instead the phycocyanobilin chromophores are bleached irreversibly. At the same time, the data show that reversible photochemistry is a useful analytical tool to detect phycoerythrocyanin in cyanobacterial extracts. Fluorescence measurements indicate that: (i) the 510 nm absorbing isomer of the violobilin chromophore has only little fluorescence; and (ii) the energy transfer from the violobilin chromophores to the cyanin chromophores is efficient only in the 570 nm form.     

PHYCOBILIPROTEINS: COMPARISON OF SOLUTION AND SINGLE CRYSTAL FLUORESCENCE FOR C-PHYCOCYANIN AND B-PHYCOERYTHRIN*

Abstract— Trimeric and hexameric solution forms of C-phycocyanin (CPC) from the cyanophyte Agme-nellum quadruplicatum have been isolated and their spectral properties compared to those obtained from single crystals. Although the absorbance peak of a suspension of small C-phycocyanin crystals is red-shifted only 7 nm relative to the solution forms, the single crystal fluorescence is red-shifted 60 nm relative to the solution forms. The crystal fluorescence spectrum exhibits a single peak at LD_max= 708 nm when excited at 514.5 or 530.9 nm and two peaks (LD_max= 661 and 708 nm) when excitation occurs at 568.2 nm. Fluorescence depolarization measurements indicate that extensive energy transfer could occur for both solution and crystal forms with the latter being dependent upon the relative orientation of the crystal with respect to the excitation dipole. Similar results were obtained with B-phycoerythrin (BPE) from the red alga Porphyridium cruentum where the single crystal fluorescence is red-shifted =50nm relative to the solution spectra with two peaks (LD_max= 583 and 617 nm) observed whose relative intensities are dependent on the excitation wavelength (LD_max– 514.5 and 530.9 nm). Single crystal fluorescent lifetimes exhibited considerable shortening relative to that observed for the solution forms. The implications of these results are discussed with respect to the possible relationships of the crystalline structures to the assembly forms present within phycobilisomes.     

润滑剂抗氧剂、总酸值和总碱值的快速测定

对润滑剂工作状况的新监测技术－伏安测定技术作了详细的介绍，应用这种技术可以快速对润滑剂中抗氧剂、总酸值和总碱值进行测定，测定结果与常规技术测定结果具有一致性，利用这种技术可以有效地评价润滑剂的使用寿命和设备工作状况。     

SPECTRAL PROPERTIES OF PHYCOBILISOMES AND PHYCOBILIPROTEINS FROM THE BLUE-GREEN ALGA-NOSTOC SP.*

Abstract— An improved method for phycobilisome isolation from a blue-green alga Nostoc sp. was developed using 1% Triton X-100. The phycobilisome preparations showed little fragmentation and had structures similar in size to those observed in thin sections of the organism. Phycobiliproteins isolated from phycobilisomes and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, had subunits with the following molecular weights: phycoerythrin (PE), 20,000 and 16,900; phycocyanin (PC), 14,700 and 16,300; and allophycocyanin (APC), 14,000. Isoelectric focusing of each phycobiliprotein resulted in major bands isoelectric at the following pH values: PE, 4.43, 4.45; PC 4.32; APC, 4.38. Absorption spectra at -196°c showed maxima at 551 and 566 nm for PE; 598 and 631 nm for PC; and 590, 600, 629 and 650 nm for APC. Concentrated vs dilute difference spectra of phycobiliproteins showed increased absorption at 574 nm (PE), 630 nm (PC) and 651 nm (APC) suggesting that spectral changes resulted from aggregation. Fluorescence analysis of each phycobiliprotein and of intact phycobilisome preparations showed that energy absorbed by phycoerythrin is transferred to allophycocyanin, possibly by a resonance transfer mechanism. These observations support a model where allophycocyanin forms the base of the phycobilisome which is attached to the photosynthetic membrane. The next layer is assumed to be phycocyanin, which in turn is followed by a phycoerythrin layer that is the outermost layer (on the stroma side) of the phycobilisome.     

REVERSIBLE PHOTOCHEMISTRY IN THE α-SUBUNIT OF PHYCOERYTHROCYANIN: CHARACTERIZATION OF CHROMOPHORE AND PROTEIN BY MOLECULAR DYNAMICS AND QUANTUM CHEMICAL CALCULATIONS

The ground and excited state properties of two regions in the Δ15-configurational space of the phycoviolobilin chromophore in the α-subunit of phycoerythrocyanin are analyzed. Molecular dynamics calculations reveal that the chromophore geometry determines the active-site dynamics. The excited state torsional potential surface shows a negative barrier for isomerization and trapping of an activated complex. Strong coupling of excited states localized in the chromophore and charge transfer states from the surrounding polar residues provides favorable prerequisites for fast excited state surface crossing in competition with other deactivation processes. The formation of a photoreduced intermediate following the photoinduced charge transfer may trigger subsequent chemical reactions.