Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons,caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast.     

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons, caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Réoxydation du quencher de fluorescence “Q” en présence de 3-(3,4-dichlorophényl)-1,1-diméthylurée

Reoxidation of the fluorescence quencher “Q” in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

Reoxidation of the fluorescence quencher Q in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea shows the following properties:

It is sensitive to very low concentrations of hydroxylamine (a few μM).

It corresponds to a back reaction between Q⁻ and the primary oxidant Z⁺ formed in the light. A part of this back reaction gives rise to luminescence emission.

Within the range we studied the kinetic of reoxidation is second order with regards to Q⁻.     

Assignment of the low-temperature fluorescence in oxygen-evolving Photosystem II

Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b₅₅₉ subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a ‘slow-transfer’ pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 ‘slow-transfer’ states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680–700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.     

Fluorescence emission spectra of plant leaves and plant constituents

Summary The UV-B radiation (e.g. 337 nm) induced blue fluorescence (BF) and red chlorophyll fluorescence spectra (RF) of green leaves from plants with different leaf structure were determined and the possible nature and candidates of the blue fluorescence emission investigated. The blue fluorescence BF is characterized by a main maximum in the 450 nm region and in most cases by a second maximum/shoulder in the 530 nm region. The latter has been termed green fluorescence GF. The red chlorophyll fluorescence RF, in turn, exhibits two maxima in the 690 and 730 nm region. In general, the intensity of BF, GF and RF emission is significantly higher in the lower than the upper leaf side. The ratio of BF to RF emission (F450/F690) seems to vary from plant species to plant species. BF and GF emission spectra appear to be a mixed signal composed of the fluorescence emission of several substances of the plant vacuole and cell wall, which may primarily arise in the epidermis. Leaves with removed epidermis and chlorophyll-free leaves, however, still exhibit a BF and GF emission. Candidates for the blue fluorescence emission ( max near 450 nm) are phenolic substances such as chlorogenic acid, caffeic acid, coumarins (aesculetin, scopoletin), stilbenes (t-stilbene, rhaponticin), the spectra of which are shown. GF emission ( max near 530 nm) seems to be caused by substances like the alkaloid berberine and quercetin. Riboflavine, NADPH and phyllohydroquinoneK ₁ seem to contribute little to the BF and GF emission as compared to the other plant compounds. Purified natural-carotene does not exhibit any blue fluorescence.     

Phase-sensitive fluorescence spectroscopy: A new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores

A novel phase fluorometric method is described which permits direct recording of individual emission spectra from a mixture of two flourescent compounds. Additionally, the lifetimes of each component may be determined by examination of the phase-sensitive fluorescence spectra. The method utilizes phase-sensitive detection of the sinusoidally modulated emission from a phase fluorometer. Resolution of the individual emission spectra in the mixture requires different fluorescence lifetimes for each components. Determination of the individual lifetime requires knowledge of the steady-state emission spectra of the components. Use of low-frequency (≈ 10 Hz) cross-correlated signals eliminates the need for high-frequency frequency (≈10⁶ Hz) phase-sensitive detection. A mixture of 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) and 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) was used to demonstrate the possibility of phase resolution of fluorophore mixture and to confirm theoretical predictions. A mixture of dibenzo[a,h]anthracene and dibenzo[c,g]carbazole was used to demonstrate that phase resolution is possible for spectra which overlap strongly and which are highly structured. In addition, the possibility of using phase-sensitive emission spectra for the resolution of excited-state reactions was demonstrated with anthracene and its diethylaniline exciplex. From a sample whose steady-state emission displayed both components we directly recorded the emission spectrum of anthracene monomer and the exciplex. For all these samples the dependence of the individual intensities on the phase angle of the detector agreed precisely with that expected on the basis of the individual fluorescence lifetimes. The detector phase angles chosen for suppression of each component in the mixture also agreed with the measured lifetimes. Thus, phase-sensitive fluorescence spectra can reveal individual spectral distributions or lifetimes. This method will be useful in the analysis fluorescence emissions which frequently occur from proteins, membranes and other biological samples.     

A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures

A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735?nm, and a 2-band spectrum with maxima at 685 and 740?nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.     

Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries

Low-temperature (77 K) fluorescence emission spectra of intact cells of a cyanobacterium, Synechocystis sp. PCC 6714, and a green alga, Chlamydomonas reinhardtii, were quantitatively analyzed to examine differences in PS I/PS II stoichiometries. Cells cultured under different spectral conditions had various PS I/PS II molar ratios when estimated by oxidation-reduction difference absorption spectra of P700 (for PS I) and Cyt b-559 (for PS II) with thylakoid membranes. The fluorescence emission spectra under the Chl a excitation at 435 nm were resolved into several component bands using curve-fitting methods and the relative band area between PS II (F685 and F695) and PS I (F710 or F720) emissions was compared with the PS I/PS II stoichiometries of the various cell types. The results indicated that the PS I/PS II fluorescence ratios correlated closely with photosystem stoichiometries both in Synechocystis sp. PCC 6714 and in C. reinhardtii grown under different light regimes. Furthermore, the correlation between the PS I/PS II fluorescence ratios and the photosystem stoichiometries is also applicable to vascular plants.     

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.     

Fluorescence-quenching-resolved spectroscopy of proteins

A new procedure is described for using fluorescence-quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern-Volmer quenching plot is determined at each particular emission wavelength and iterative non-linear least-squares fitting procedure allowed to resolve the steady-state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two-tryptophan-containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single-photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.     

Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus.

Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.     

Fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor

Microbial products formed in biological wastewater treatment systems are closely related to system performance and status, and many of them have fluorescence spectral characteristics. In this work, the fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor were studied using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. Since the components of the microbial products are complex, the parallel factor analysis (PARAFAC) method was used to extract the real spectra from the overlapped spectra. Two principal components were identified from the EEM spectra. The peaks at excitation–emission maxima of 280/350 and 350/440 nm were, respectively, attributed to the fluorescence of proteins and NADH. Their real concentrations were quantified using the PARAFAC coupled with the second-order calibration method. Results show that the formation rate of proteins was correlated to the production rate of hydrogen and volatile fatty acids, as well as the substrate degradation rate. A close correlation between the hydrogen partial pressure and the two fluorophores was found out. This study provides a reliable and convenient approach, which could be potentially used for monitoring the wastewater treatment reactor performance through measuring the fluorescence spectra of the supernatant.     

Fluorescent Fingerprints of Endolithic Phototrophic Cyanobacteria Living within Halite Rocks in the Atacama Desert

Halite deposits from the hyperarid zone of the Atacama Desert reveal the presence of endolithic microbial colonization dominated by cyanobacteria associated with heterotrophic bacteria and archaea. Using the λ-scan confocal laser scanning microscopy (CLSM) option, this study examines the autofluorescence emission spectra produced by single cyanobacterial cells found inside halite rocks and by their photosynthetic pigments. Photosynthetic pigments could be identified according to the shapes of the emission spectra and wavelengths of fluorescence peaks. According to their fluorescence fingerprints, three groups of cyanobacterial cells were identified within this natural extreme microhabitat: (i) cells producing a single fluorescence peak corresponding to the emission range of phycobiliproteins and chlorophyll a, (ii) cells producing two fluorescence peaks within the red and green signal ranges, and (iii) cells emitting only low-intensity fluorescence within the nonspecific green fluorescence signal range. Photosynthetic pigment fingerprints emerged as indicators of the preservation state or viability of the cells. These observations were supported by a cell plasma membrane integrity test based on Sytox Green DNA staining and by transmission electron microscopy ultrastructural observations of cyanobacterial cells.     

Second derivative fluorescence spectroscopy of tryptophan in proteins

The second derivatives of N-acetyl- -tryptophan amide (AcTrpNH₂) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH₂ fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase–PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.     

Chlorophyll fluorescence emission spectroscopy of oxygenic organisms at 77 K

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).     

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。