Interactions of Chlorophyll and Polypeptide Mixture with Bacterial Reaction Centres

In aqueous solutions of chlorophyll (Chl) a with synthesized polypeptides, at high ratios of Chl to polypeptides (about 75–150 µM to 500 µM) clusters of polypeptides and pigment molecules were formed. The main absorption maxima of more than one formed cluster were located at about 500 nm (Soret band) and in the region of 720–806 nm (red band). The formation of these clusters was fairly slow (some hours) at room temperature and even slower at 4 °C. The rate of cluster formation increased with the increase in Chl concentration. The addition of the even low amount of reaction centres (RCs), separated from the purple bacteria Rhodobacter sphaeroides, to the sample of Chl with polypeptides caused a very strong decrease in the efficiency of cluster formation, and a change in concentration ratios of various pigment-polypeptide aggregates. It was probably a competition between the interaction of Chl with polypeptides and with the RCs. The yield of thermal deactivation of the clusters was high, much higher than that for the RCs alone and it was different for various types of cluster. The clusters absorbing at 725–750 nm were fluorescent with maximum of emission at about 770 nm, whereas clusters absorbing at about 800 nm were nonfluorescent.     

Polarized Photoacoustic Spectra of the Cyanobacterium Synechococcus Oriented in Polymer Film

Photoacoustic spectra (PAS) were obtained for the cyanobacterium Synechococcus (Anacystis nidulans) cells embedded in isotropic and stretched polyvinyl alcohol films. The polarized radiation with the electric vector changing in 30° intervals with respect to given direction in a sample plane was used. Two cyanobacterium strains, one with very low biliprotein content, second with normal amount of biliproteins were investigated. The polarized absorption and fluorescence spectra were also measured. Conclusions were drawn about the thermal deactivation occurring in differently oriented pools of chromophores and about mutual orientation of their transition moments. Thermal deactivation in carotenoids (Cars) of both strains was different. The ratio of Car thermal deactivation to the thermal deactivation of chlorophyll (Chl) was higher in cyanobacteria with lower content of biliproteins than in the strain with normal amount of these complexes. Hence biliproteins can play the role in excitation energy transfer from Cars to Chls. For complex biological samples, polarized PAS can be a more sensitive method to investigate the directions of the absorption transition moments than the widely used polarized absorption spectra.     

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ～1.9, ～1.5, and 1.1–1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments — carotenoids, Chl b, and phycobilins — were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.     

Relevance of the Diastereotopic Ligation of Magnesium Atoms of Chlorophylls in the Major Light-harvesting Complex II (LHC II) of Green Plants

The recent high-resolution crystal structure of LHC II [Liu et al. (2004) Nature 428: 287–292] makes possible an unprecedented insight into the stereochemical features of how chlorophylls (Chl)s are bound. The diastereotopic ligation generates four structurally different pigment types, two Chl a and two Chl b, which are distinguished not only by the groups in the 7-position (methyl in Chl a and formyl in Chl b) but also by the face of the tetrapyrrole to which the fifth magnesium ligand is bound. Within a LHC II monomer, out of the eight Chl a six have a ‚normal’ α-coordination and two are β-coordinated while out of the six Chl b only one has the ‚special’ β-coordination. In Photosystem I where a more meaningful statistical analysis could be made, out of 96 Chl a only 14 are β-coordinated, again indicating a preference for the ‚normal’ α-coordination [Balaban et al. (2002) Biochim Biophys Acta Bioenerget 1556: 197–207; Oba and Tamiaki (2002a) Photosynth Res 74: 1–10]. Astonishingly, all the special β-Chls are part of the stromal ring of Chls within the LHC II trimers and occupy key positions for the excitation energy transfer. Sequential energy traps are engineered with one hetero- and three homo-dimers. A careful pairing of carotenoids with the special β-Chls, which could quench their triplet states efficiently, implies a functional relevance of this diastereotopic ligation.     

Phosphorescence study of chlorophyll d photophysics. Determination of the energy and lifetime of the photo-excited triplet state. Evidence of singlet oxygen photosensitization

Chlorophyll d (Chl d) is the major pigment in both photosystems (PSI and II) of the cyanobacterium Acaryochloris marina, whose pigment composition represents an interesting alternative in oxygenic photosynthesis. While abundant information is available relative to photophysical properties of Chl a , the understanding of Chl d photophysics is still incomplete. In this paper, we present for the first time a characterization of Chl d phosphorescence, which accompanies radiative deactivation of the photoexcited triplet state of this pigment. Reliable information was obtained on the energy and lifetime of the Chl d triplet state in frozen solutions at 77?K using diethyl ether and aqueous dispersions of Triton X100 as solvents. It is shown that triplet Chl d is effectively populated upon photoexcitation of pigment molecules and efficiently sensitizes singlet oxygen phosphorescence in aerobic solutions under ambient conditions. The data obtained are compared with the previous results of the phosphorescence studies of Chl a and Pheo a, and their possible biological implications are discussed.     

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution

Under strong light conditions, long-lived chlorophyll triplets (³Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench ³Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of ³Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching ³Chl.     

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: Comparison with the native PCP complex from Amphidinium carterae

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at ∼ 620 and ∼ 640 nm in the Triplet-minus-Singlet (T − S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.     

Altering the exciton landscape by removal of specific chlorophylls in monomeric LHCII provides information on the sites of triplet formation and quenching by means of ODMR and EPR spectroscopies

The triplet states populated under illumination in the monomeric light-harvesting complex II (LHCII) were analyzed by EPR and Optically Detected Magnetic Resonance (ODMR) in order to fully characterize the perturbations introduced by site-directed mutations leading to the removal of key chlorophylls. We considered the A2 and A5 mutants, lacking Chls a612(a611) and Chl a603 respectively, since these Chls have been proposed as the sites of formation of triplet states which are subsequently quenched by the luteins. Chls a612 and Chl a603 belong to the two clusters determining the low energy exciton states in the complex. Their removal is expected to significantly alter the excitation energy transfer pathways. On the basis of the TR- and pulse EPR triplet spectra, the two symmetrically related pairs constituted by Chl a612/Lut620 and Chl a603/Lut621 were both possible candidate for triplet-triplet energy transfer (TTET). However, the ODMR results clearly show that only Lut620 is involved in triplet quenching. In the A5 mutant, the Chl a612/Lut620 pair retains this pivotal photoprotective role, while the A2 mutant was found to activate an alternative pathway involving the Chl a603/Lut621pair. These results shows that LHCII is characterized by a robust photoprotective mechanism, able to adapt to the removal of individual chromophores while maintaining a remarkable degree of Chl triplet quenching. Small amounts of unquenched Chl triplet states were also detected. The analysis of the results allowed us to assign the sites of “unquenched” chlorophyll triplets to Chl a610 and Chl a602.     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for an O2-barrier in the light-harvesting chlorophyll-a/b-protein complex LHC II

An O₂-barrier in the intact light-harvesting complex LHC II protects chlorophylls (Chl) and xanthophylls (Car) from photooxidation. Direct evidence for the limited access of O₂ to pigment sites comes from the decay kinetics of the first excited triplet state of Car (³Car-). The LHC-bound ³Car- in air-saturated solution decays mono-exponentially with a lifetime of 6.7-7.1 µs as compared to the approx. 1.2 µs of the -carotene triplet in hexane and the 8.8-9 µs observed for both systems under anaerobiosis. Further properties of the photostable complex are the limited access of protons to pigment sites and the efficient energy transfer from ¹Car- to Chl-a and from ³Chl- to Car. Fatty acids with increasing chain length increasingly lower both, the efficiency of the O₂ barrier and the photo- and acid stability of the LHC-bound pigments while singlet and triplet energy transfer between the pigments is maintained. Therefore, the close proximity of Chl and Car is not sufficient to protect the pigments from photooxidation; in addition, an O₂-barrier limiting the access of O₂ to pigment sites is required for efficient photoprotection. Structural properties of the photostable LHC II possibly underlying its O₂-barrier function are discussed.     

Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes

This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes.Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets.Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments.The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.     

Photoinhibition of Photosynthesis: Role of Carotenoids in Photoprotection of Chloroplast Constituents

Exposure of plants to irradiation, in excess to saturate photosynthesis, leads to reduction in photosynthetic capacity without any change in bulk pigment content. This effect is known as photoinhibition. Photoinhibition is followed by destruction of carotenoids (Cars), bleaching of chlorophylls (Chls), and increased lipid peroxidation due to formation of reactive oxygen species if the excess irradiance exposure continues. Photoinhibition of photosystem 2 (PS2) in vivo is often a photoprotective strategy rather than a damaging process. For sustainable maintenance of chloroplast function under high irradiance, the plants develop various photoprotective strategies. Cars perform essential photoprotective roles in chloroplasts by quenching the triplet Chl and scavenging singlet oxygen and other reactive oxygen species. Recently photoprotective role of xanthophylls (zeaxanthin) for dissipation of excess excitation energy under irradiance stress has been emphasised. The inter-conversion of violaxanthin (Vx) into zeaxanthin (Zx) in the light-harvesting complexes (LHC) serves to regulate photon harvesting and subsequent energy dissipation. De-epoxidation of Vx to Zx leads to changes in structure and properties of these xanthophylls which brings about significant structural changes in the LHC complex. This ultimately results in (1) direct quenching of Chl fluorescence by singlet-singlet energy transfer from Chl to Zx, (2) trans-thylakoid membrane mediated, pH-dependent indirect quenching of Chl fluorescence. Apart from these, other processes such as early light-inducible proteins, D1 turnover, and several enzymatic defence mechanisms, operate in the chloroplasts, either for tolerance or to neutralise the harmful effect of high irradiance.     

On the long-wavelength spectral forms of chlorophyll a in Photosystem I: Spectroscopic and immunological investigations on a greening mutant of the green alga Scenedesmus obliquus

The origin of the long-wavelength chlorophyll (Chl) absorption (_peak > 680 nm) and fluorescence emission (_peak > 685 nm) has been investigated on Scenedesmus mutants (C-2A-series, lacking the ability to synthesize chlorophyll in the dark) grown at 0.3 (LL), 10 (ML) and 240 µE s^–1 m^–2(HL). LL cells are arrested in an early greening state; consequently, Chl availability determines the phenotype. LL thylakoids are totally lacking long-wavelength Chl; nonetheless, PS I and PS II are fully functional. Gel electrophoresis and Western blots indicate that four out of seven resolved LHC polypeptides seem to require a high Chl availability for assembly of functional chlorophyll-protein complexes. The PS I core-complex of ML and HL thylakoids contains long-wavelength chlorophylls, but in the PS I core-complex of LL thylakoids these pigments are lacking. We conclude that long-wavelength pigments are only present in the PS I core in the case of high Chl availability. The following hypothesis is discussed: Chl availability determines not only the LHC polypeptide pattern, but also the number of bound Chl molecules per individual pigment-protein complex. Chl-binding at non-obligatory, peripheral sites of the pigment-protein complex results in long-wavelength Chl. In the case of low Chl availability, these sites are not occupied and, therefore, the long-wavelength Chl is absent.     

Differences in pigment composition, photosynthetic rates and chlorophyll fluorescence images of sun and shade leaves of four tree species.

The differential pigment composition and photosynthetic activity of sun and shade leaves of deciduous (Acer pseudoplatanus, Fagus sylvatica, Tilia cordata) and coniferous (Abies alba) trees was comparatively determined by studying the photosynthetic rates via CO(2) measurements and also by imaging the Chl fluorescence decrease ratio (R(Fd)), which is an in vivo indicator of the net CO(2) assimilation rates. The thicker sun leaves and needles in all tree species were characterized by a lower specific leaf area, lower water content, higher total chlorophyll (Chl) a+b and total carotenoid (Cars) content per leaf area unit, as well as higher values for the ratio Chl a/b compared to the much thinner shade leaves and needles that possess a higher Chl a+b and Cars content on a dry matter basis and higher values for the weight ratio Chls/Cars. Sun leaves and needles exhibited higher rates of maximum net photosynthetic CO(2) assimilation (P(Nmax)) measured at saturating irradiance associated with higher maximum stomatal conductance for water vapor efflux. The differences in photosynthetic activity between sun and shade leaves and needles could also be sensed via imaging the Chl fluorescence decrease ratio R(Fd), since it linearly correlated to the P(Nmax) rates at saturating irradiance. Chl fluorescence imaging not only provided the possibility to screen the differences in P(N) rates between sun and shade leaves, but in addition permitted detection and quantification of the large gradients in photosynthetic rates across the leaf area existing in sun and shade leaves.     

Effects of Cold-Hardening on Chilling-Induced Photoinhibition of Photosynthesis and on Xanthophyll Cycle Pigments in Sweet Pepper

Two cultivars of Capsicum annuum L. were acclimated for 5 d at sub-optimal temperature (14 °C) and irradiance of 250 µmol m^–2 s^–1. This cold-hardening resulted in some reduction in the extent of photoinhibition during an 8 h exposure to high irradiance at 4 °C. Obvious differences were observed between non-hardened leaves (NHL) and cold-hardened leaves (CHL) in the recovery under low irradiance at room temperature. The CHL of both cultivars recovered faster than NHL, especially during the initial fast phase of recovery. Compared with NHL, the total content of carotenoids (Cars), based on chlorophyll, Chl (a+b), and the proportions of xanthophyll cycle pigments referred to total Cars increased in CHL, mainly due to an increase of violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) content per mol Chl (a+b). Faster development and a higher non-photochemical quenching (NPQ) of Chl fluorescence, related to a stronger deepoxidation of the larger xanthophyll cycle pool in NHL, could act as a major defence mechanism to reduce the formation of reactive oxygen species during severe chilling. This is suggested by higher content of Z or Z+A in photoinhibition as well as by its rapid decline during the initial fast phase of recovery. In contrast to the chilling-sensitive cv. 0004, the chilling-tolerant cv. 1141 did more easily acclimate its photosynthetic apparatus to low temperatures.     

Comparative Characterization of the Pigment Complex in Emergent, Floating, and Submerged Leaves of Hydrophytes

The content of chlorophylls (Chls) and carotenoids was studied in the leaves of 42 species of boreal aquatic plants with different degree of submergence (emergent, floating, and submerged) and isopalisade, dorsoventral, and homogenous types of mesophyll structure. Hydrophytes were shown to have a low Chl content (1–2 mg/g fr wt) and low Chls/carotenoids ratio (2.3–3.5) as compared to terrestrial plants. The pigment content per dry wt unit and unit leaf area was dependent on the type of mesophyll structure. It was a consequence of the changes in the parameters of leaf mesophyll structure characterizing the density of photosynthetic elements. In a sequence emergent floating submerged forms, the content of Chls and carotenoids decreased, and the photosynthetic capacity decreased due to a reduction in the chloroplast number per unit leaf area. Adaptation of submerged leaves to low illumination and slow CO₂ diffusion changed the functional properties of chloroplasts. An increase in the pigment content in the chloroplasts of submerged leaves (7 × 10^–9 mg Chl, 2 × 10^–9 mg carotenoids) as compared to emergent and floating leaves was accompanied by a decline in the photosynthetic capacity per Chl comprising 1.6 mg CO₂/(mg Chl h) versus 3.9 and 3.8 mg CO₂/(mg Chl h) in emergent and floating leaves, respectively.     

The water bloom of Cyanobacterial picoplankton in Lake Biwa,Japan

Unicellular autofluorescent picoplankton ranging from 0.4 to 1.5 µm in diameter were found to be a significant component of phytoplankton in the North Basin of Lake Biwa during early summer in 1989 and 1990. The abundance of these picoplankton varied seasonally by about three orders of magnitude with one maximum of up to 10⁶ cells ml^–1. Bloom-forming picoplankton were isolated by dilution and further cultivated in liquid medium. Three clones were found to be representative species of the bloom. Using epifluorescence and electron microscopy as well as absorption and fluorescence emission spectroscopy, we examined these clones according to shape and pigment composition. They have ringlike thylakoids, are photosynthetically active and have no nuclear envelope. The cyanobacterial clones isolated represent three types containing phycobilisomes with either phycocyanin or phycoerthrin as the dominant accessory pigment. They are described here as three new species, two phycoerythrin-rich types and one phycocyanin-rich type, all of them belonging to the Synechococcus group. The differences found by fluorescence emission of isolated clones are discussed with respect to in situ strain identification.     

The Origin of the Low-Energy Form of Photosystem I Light-Harvesting Complex Lhca4: Mixing of the Lowest Exciton with a Charge-Transfer State

The peripheral light-harvesting complex of photosystem I contains red chlorophylls (Chls) that, unlike the typical antenna Chls, absorb at lower energy than the primary electron donor P700. It has been shown that the red-most absorption band arises from two excitonically coupled Chls, although this interaction alone cannot explain the extreme red-shifted emission (25 nm, ∼480 cm⁻¹ for Lhca4 at 4 K) that the red Chls present. Here, we report the electric field-induced absorption changes (Stark effect) on the Q_y region of the Lhca4 complex. Two spectral forms, centered around 690 nm and 710 nm, were necessary to describe the absorption and Stark spectra. The analysis of the lowest energy transition yields a high value for the change in dipole moment, Δμ_710nm ≈ 8 Df⁻¹, between the ground and excited states as compared with monomeric, Δμ = 1 D, or dimeric, Δμ = 5 D, Chl a in solution. The high value of the Δμ demonstrates that the origin of the red-shifted emission is the mixing of the lowest exciton state with a charge-transfer state of the dimer. This energetic configuration, an excited state with charge-transfer character, is very favorable for the trapping and dissipation of excitations and could be involved in the photoprotective mechanism(s) of the photosystem I complex.     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.