Fluorescence anisotropy controlled by light quenching

A retrospective review of 94 humeral shaft fractures stabilized with retrograde Ender nails was performed to determine the safety and efficacy of this technique. Follow-up averaged 71 weeks. Eighty-six (91.5%) healed with no additional intervention (average: 14 weeks). There were eight (8.5%) nonunions. The functional results were good in 74 (81%), fair in 12 (13%), and poor in 5 (6%). There was one radial nerve palsy secondary to the operative procedure. The reoperation rate, including the repair of nonunions, was 19%. We conclude that intramedullary Ender nail fixation is a relatively simple and quick procedure, especially for the multiply injured patient, that adequately stabilizes most humeral fractures, yielding overall good results.     

Nonphotochemical quenching of excitation energy in photosystem II. A picosecond time-resolved study of the low yield of chlorophyll a fluorescence induced by single-turnover flash in isolated spinach thylakoids

Chlorophyll a fluorescence emission is widely used as a noninvasive measure of a number of parameters related to photosynthetic efficiency in oxygenic photosynthetic organisms. The most important component for the estimation of photochemistry is the relative increase in fluorescence yield between dark-adapted samples which have a maximal capacity for photochemistry and a minimal fluorescence yield (F0) and light-saturated samples where photochemistry is saturated and fluorescence yield is maximal (Fm). However, when photosynthesis is saturated with a short (less than 50 micro(s)) flash of light, which induces only one photochemical turnover of photosystem II, the maximal fluorescence yield is significantly lower (Fsat) than when saturation is achieved with a millisecond duration multiturnover flash (Fm). To investigate the origins of the difference in fluorescence yield between these two conditions, our time-resolved fluorescence apparatus was modified to allow collection of picosecond time-resolved decay kinetics over a short time window immediately following a saturating single-turnover flash (Fsat) as well as after a multiturnover saturating pulse (Fm). Our data were analyzed with a global kinetic model based on an exciton radical pair equilibrium model for photosystem II. The difference between Fm and Fsat was modeled well by changing only the rate constant for quenching of excitation energy in the antenna of photosystem II. An antenna-based origin for the quenching was verified experimentally by the observation that addition of the antenna quencher 5-hydroxy-1,4-naphthoquinone to thylakoids under Fm conditions resulted in decay kinetics and modeled kinetic parameters very similar to those observed under Fsat conditions in the absence of added quinone. Our data strongly support the origin of low fluorescence yield at Fsat to be an antenna-based nonphotochemical quenching of excitation energy in photosystem II which has not usually been considered explicitly in calculations of photochemical and nonphotochemical quenching parameters. The implications of our data with respect to kinetic models for the excited-state dynamics of photosystem II and the practical applications of the fluorescence yield parameters Fm and Fsat to calculations of photochemical yield are discussed.     

Fluorescence quenching by metal ions in lipid bilayers

Fluorescence quenching of perylene by Co2+ and Ni2+ ions has been investigated both below and above the phase transition temperature in small unilamellar DPPC vesicles. Classic F?rster type energy transfer was observed for perylene quenched by Co2+ ions below the phase transition when the effects of donor-donor energy transfer are taken into account. In the liquid crystalline phase a simple diffusion theory incorporating energy transfer was found to model the system well. For quenching by Ni2+ ions both above and below the phase transition temperature in lipid bilayers and in glycerol the data did not follow classic F?rster type energy transfer but indicated that an additional quenching mechanism was present. A mechanism other than F?rster behaviour was also observed for the quenching by Cr3+ ions in glycerol.     

Femtosecond transient absorption study of carotenoid to chlorophyll energy transfer in the light-harvesting complex II of photosystem II

Singlet energy transfer between the carotenoids (Cars) and chlorophylls (Chls) in the light-harvesting complex II (LHC II) from higher plants has been studied using ultrafast transient absorption spectroscopy by exciting the Cars directly in the 475-515 nm wavelength range. LHC II trimers from Arabidopsis thaliana with well-defined Car compositions have been used. From HPLC, the wild type (WT) monomer contains two luteins (Ls), one neoxanthin (N), and a trace of violaxanthin (V) per 12 Chls. The ABA-3 mutant contains 1.4 Ls and 0.6 zeaxanthin (Z) per monomer. Though exploitation of the difference in Car constitution and exciting the WT at 475 and 490 nm, and the ABA-3 mutant at 490 and 515 nm, the different Car contributions to energy transfer have been probed. Evidence for energy transfer mainly from the Car to Chl b is observed in the WT. In the mutant, additional transfer from Car to Chl a correlates with the presence of Z. The results imply predominant energy transfer from the central Ls to Chl b which requires a modification of the currently accepted arrangement of Chl pigments in LHC II.     

Modulation of quantum yield of primary radical pair formation in photosystem II by site-directed mutagenesis affecting radical cations and anions

Pigment-protein interactions play a significant role in determining the properties of photosynthetic complexes. Site-directed mutants of Synechocystis PCC 6803 have been prepared which modify the redox potential of the primary radical pair anion and cation. In one set of mutants, the environment of P680, the primary electron donor of Photosystem II, has been modified by altering the residue at D1-His198. It has been proposed that this residue is an axial ligand to the magnesium cation. In the other set, the D1-Gln130 residue, which is thought to interact with the C9-keto group of the pheophytin electron acceptor, has been changed. The effect of these mutations is to alter the free energy of the primary radical pair state, which causes a change in the equilibrium between excited singlet states and radical pair states. We show that the free energy of the primary radical pair can be increased or decreased by modifications at either the D1-His198 or the D1-Gln130 sites. This is demonstrated by using three independent measures of quantum yield and equilibrium constant, which exhibit a quantitative correlation. These data also indicate the presence of a fast nonradiative decay pathway that competes with primary charge separation. These results emphasize the sensitivity of the primary processes of PS II to small changes in the free energy of the primary radical pair.     

Fluorescence quenching study of melittin-membrane interactions

The bee venom peptide melittin is a popular object for studying lipid-protein interactions. In this paper we show that binding of melittin to the bilayer is a complex process involving several steps. We were able to resolve those steps by utilizing a new approach in the quantitative analysis of depth-dependent fluorescence quenching in membranes. The "distribution analysis" technique (DA) employed here provides not only the most probable depth of the fluorophore but also allows the estimation of its conformational heterogeneity and accessibility to the lipid phase. A model for melittin interaction with the membranes is suggested.     

UV-B-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components

Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.     

Carotenoid-dependent oligomerization of the major chlorophyll a/b light harvesting complex of photosystem II of plants

Under many environmental conditions, plants are exposed to levels of sunlight in excess of those required for photosynthesis. Then, a regulated increase in the rate of nonradiative dissipation of excess excitation energy in the thylakoid membrane correlates with the conversion of the carotenoid violaxanthin into zeaxanthin and provides protection from the damaging effects of excessive irradiation. The hypothesis that these carotenoids specifically control the oligomerization of the light harvesting complexes of photosystem II was tested by investigating the effects of violaxanthin and zeaxanthin on the behavior of the major complex, LHCIIb, on sucrose gradients; it was found that zeaxanthin stimulated the formation of LHCIIb aggregates with reduced chlorophyll fluorescence yield whereas violaxanthin caused the inhibition of such aggregation and an elevation of fluorescence. Measurements of 77 K fluorescence indicated that zeaxanthin was not exerting an additional direct quenching of chlorophyll fluorescence. These effects can explain the physiological control of the light harvesting system by the xanthophyll cycle.     

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.     

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants

Collectively, the xanthophyll class of carotenoids perform a variety of critical roles in light harvesting antenna assembly and function. The xanthophyll composition of higher plant photosystems (lutein, violaxanthin, and neoxanthin) is remarkably conserved, suggesting important functional roles for each. We have taken a molecular genetic approach in Arabidopsis toward defining the respective roles of individual xanthophylls in vivo by using a series of mutant lines that selectively eliminate and substitute a range of xanthophylls. The mutations, lut1 and lut2 (lut = lutein deficient), disrupt lutein biosynthesis. In lut2, lutein is replaced mainly by a stoichiometric increase in violaxanthin and antheraxanthin. A third mutant, aba1, accumulates normal levels of lutein and substitutes zeaxanthin for violaxanthin and neoxanthin. The lut2aba1 double mutant completely lacks lutein, violaxanthin, and neoxanthin and instead accumulates zeaxanthin. All mutants were viable in soil and had chlorophyll a/b ratios ranging from 2.9 to 3.5 and near wild-type rates of photosynthesis. However, mutants accumulating zeaxanthin exhibited a delayed greening virescent phenotype, which was most severe and often lethal when zeaxanthin was the only xanthophyll present. Chlorophyll fluorescence quenching kinetics indicated that both zeaxanthin and lutein contribute to nonphotochemical quenching; specifically, lutein contributes, directly or indirectly, to the rapid rise of nonphotochemical quenching. The results suggest that the normal complement of xanthophylls, while not essential, is required for optimal assembly and function of the light harvesting antenna in higher plants.     

beta-Carotene quenches singlet oxygen formed by isolated photosystem II reaction centers

By measuring time-resolved luminescence emission at 1270 nm, we have detected singlet oxygen formation by illuminated, reaction centers of photosystem II isolated from Pisum sativum, which is in agreement with earlier work (Macpherson, A. N., Telfer, A., Barber, J., & Truscott, T. G. (1993) Biochim. Biophys. Acta 1143, 301-309). In this paper we show that the yield of singlet oxygen is significantly increased if the number of beta-carotene molecules bound per isolated complex is reduced from two to one. We conclude, therefore, that beta-carotene can act as an effective quencher of singlet oxygen in the photosystem II reaction center. This conclusion is supported by the finding that the rate of light-induced irreversible bleaching of chlorins in the reaction center is increased with decreasing beta-carotene levels. The results demonstrate the direct intermediacy of singlet oxygen in causing photooxidative damage within a biological environment and are discussed, specifically, in terms of the role of beta-carotene in protecting photosystem II against photoinhibition.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Fluorescence quenching studies of the rat ovarian LH/hCG receptor

Fluorescence quenching method providing information about the structure and dynamics of proteins, ligand-protein and protein-lipid interactions was used in a study of the rat ovarian LH/hCG receptor. The efficiency of two different quenchers, acrylamide and iodide, was tested. Acrylamide was significantly more effective in quenching of intrinsic fluorescence of ovarian membranes than iodide and therefore it was used in all of the following experiments. Both acrylamide and iodide were not effective in quenching of membranes labelled with fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH). In the process of desensitization of ovarian LH/hCG receptors the administration to rats of hCG modified the quenching rate of protein fluorescence and intrinsic fluorescence spectral properties of membranes. Alteration in the quenching of intrinsic fluorescence of ovarian membranes was observed after chemical modification of LH/hCG receptors by 2-hydroxy-5-nitrobenzyl bromide (HNB-Br). The accessibility of tryptophan fluorophores was increased in HNB-Br-treated membranes. Delipidation of the LH/hCG receptor modified the quenching of protein fluorescence characteristic for control proteoliposomes. These results demonstrate that fluorescence quenching technique can be successfully applied in the study of the LH/hCG receptor.     

Structure and thermal stability of photosystem II reaction centers studied by infrared spectroscopy

The secondary structure of photosystem II reaction centers isolated from pea has been deduced from quantitative analysis of the component bands of the infrared amide I spectral region, determined by FTIR spectroscopy. The analysis shows the isolated complex to consist of 40% alpha-helix, 10% beta-sheet, 14% beta-strands (or extended chains), 17% turns, 15% loops, and 3% nonordered segments. These structural protein elements were determined for samples in H2O, in D2O, and in dried films. The isolated reaction center, composed of proteins D1,D2,cytochrome b559, and PsbI, has been predicted to contain a total of 13 transmembrane alpha-helices, which conveys a percentage of this type of structure congruent with the structural determination deduced from FTIR spectra. The process of thermal destabilization of the reaction centers has also been studied by FTIR spectroscopy, showing a clear main conformational transition at 42 degrees C, which indicates a high thermal sensitivity of the secondary structure of this protein complex. Such thermal instability may correlate with the well-described high sensitivity of photosystem II to damage and may relate to the process of rapid protein degradation that photosystem II suffers during photoinhibition of plants.     

Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer. The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural charasteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H2O to phenyl-p-benzoquinone and ascorbate-Cl2Ind to NADP+ electron transport rates indicate a ratio of around six between Photosystem II and I. The total fractionation procedure could be completed within 2-3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP+. These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Demonstration of thermal dissipation of absorbed quanta during energy-dependent quenching of chlorophyll fluorescence in photosynthetic membranes

Generally biglycan, a small proteoglycan, has been thought to play a role as an extracellular matrix and/or a reservoir for other factors, such as TGF-beta and collagens. Recently, we have found that a soluble 100 kDa biglycan, produced from the rat thymic myoid cells and the brain glial cells, predominantly stimulates growth and differentiation of monocytic lineage cells from various lymphatic organs, including microglias. In the present study, we attempted to identify biological significance of the corresponding molecules in human, using five myasthenic thymuses (three with hyperplasia and two with thymoma) that had been surgically removed for therapeutic purpose. With immunohistochemistry, many biglycan positive cells were detected in the germinal center of the three hyperplastic thymuses, but not in the two thymuses associated with lymphocytic thymoma. Biglycan purified from the hyperplastic thymuses by an immunoaffinity column was found as a monomer with apparent molecular size of 95-100 kDa and self associated oligomers of greater than 201 kDa. The purified biglycan markedly stimulated the growth and differentiation of monocytic cells from haemopoietic stem cells of the rat bone marrow. These results suggest that particular cells, which produce haemopoietic biglycan, play significant roles in generation and maintenance of the hyperplastic changes associated with myasthenia gravis.     

5’-硝基-水杨基荧光酮荧光熄灭法测定煤中微量锗

用荧光熄灭法研究了 5’-硝基-水杨基荧光酮 ( 5’-NSAF) -TritonX 10 0 -Ge 体系的反应条件和测定方法。在0.0 0 2～ 0.0 8mol/L的硫酸介质中 ,TritonX 10 0存在下 ,Ge 与 5’ NSAF反应生成 1∶4的红色络合物 ,使 5’ NSAF溶液的荧光明显熄灭。其激发波长λex=5 0 5nm ,发射波长λem=5 3 3nm。线性范围为 8～ 2 10ng/mL ,检出限为 8ng/mL ,本法灵敏度高 ,选择性好 ,可不经分离直接测定煤中微量锗