Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.     

Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.     

A comparative flash-photolysis study of electron transfer from pea and spinach plastocyanins to spinach Photosystem 1. A reaction involving a rate-limiting conformational change

Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.Abbreviations cyt- cytochrome - IPTG- isopropyl--d-thiogalactopyranoside - P,P700- reaction-center chlorophyll - Pc- plastocyanin - PS 1- Photosystem 1 - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - WT- wild-type     

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.     

Experimental and numerical study of the poplar plastocyanin isoforms using Tyr as a probe for electrostatic similarity and dissimilarity

A detailed study of the tyrosine spectral characteristics was carried out in a broad range of pHs for both isoforms of plastocyanin from poplar. It was found that Tyr 80 is always protonated while Tyr 83 can form a tirosinate at high pHs. The pK(a) of Tyr 83 is practically identical in plastocyanin a and b, but the quenching of its spectrum is different in the isoforms. This provides insights that the acidic patches surrounding Tyr 83 have different electrostatic properties in plastocyanin a and b. The protonation states and the electrostatic interactions were numerically modeled on the existing plastocyanin a structure and on a homology model of plastocyanin b. The results of numerical calculations agree with the experimental findings and identify several differences in the titration behavior of the acidic patches. The difference of the tyrosine quenching pH profiles of the isoforms is rationalized by the differences in the calculated pK(a)'s of amino acids in the neighboring acidic clusters.     

Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde

Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O₂ flash yields, pH exchange, and fluorescence induction show that the O₂ evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

pH-dependent structural change of reduced spinach plastocyanin studied by perturbed angular correlation of γ-rays and dynamic light scattering

In this study the pH-dependent structural changes of reduced spinach plastocyanin were investigated using perturbed angular correlation (PAC) of γ-rays and dynamic light scattering (DLS). PAC data of Ag-substituted plastocyanin indicated that the coordinating ligands are two histidine residues (His37, His87) and a cysteine residue (Cys84) in a planar configuration, whereas the methionine (Met92) found perpendicular to this plane is not a coordinating ligand at neutral pH. Two slightly different conformations with differences in the Cys–metal ion–His angles could be observed with PAC spectroscopy. At pH 5.3 a third coordination geometry appears which can be explained as the absence of the His87 residue and the coordination of Met92 as a ligand. With DLS the aggregation of reduced plastocyanin could be observed below pH 5.3, indicating that not only the metal binding site but also the aggregation properties of the protein change upon pH reduction. Both the structural changes at the metal binding site and the aggregation are shown to be reversible. These results support the hypothesis that the pH of the thylakoid lumen has to remain moderate during steady-state photosynthesis and indicate that low pH induced aggregation of plastocyanin might serve as a regulatory switch for photosynthesis.     

钙调神经磷酸酶在胍变性过程中活力及构象变化的比较

钙调神经磷酸酶（ＣａＮ）在盐酸胍溶液中的内源荧光、远紫外ＣＤ谱及剩余活力的变化提示：ＣａＮ的酶活力在胍浓度为０．５ｍｏｌ／Ｌ左右可完全丧失,同时伴有内源荧光强度的下降,３３３ｎｍ最大发射峰的红移（提示了色氨酸和酪氨酸残基的暴露）。比较不同胍浓度下牛脑ＣａＮ的失活与整体构象变化,表明酶的失活先于整体构象变化。在０．６ｍｏｌ／Ｌ胍溶液中,内源荧光变化的动力学过程只能测出一相,而酶失活的动力学过程为快、慢两相,快相动力学速度常数比整体构象变化速度常数大１－２个数量级,慢相失活速度常数与整体构象变化速度常数相近。提示低浓度胍可引起该酶的完全失活,活性部位的空间构象比整个酶分子的构象更易受到变性剂的扰乱。     

Crystal structure of spinach plastocyanin at 1.7 A resolution.

The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site-directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild-type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (approximately 4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59-61 loop where the largest shift is 3.0 A for the C(alpha) atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.     

Intraprotein electron transfer in a ruthenium-modified Tyr83-His plastocyanin mutant: evidence for strong electronic coupling

A site-directed mutant of spinach plastocyanin, Pc(Tyr83-His), has been modified by covalent attachment of a photoactive [Ru(bpy)₂(im)]²⁺ complex to the His83 residue. The residue is surface exposed and located about 10–12?Å from the copper ion at the entrance of a proposed natural electron transfer pathway from cytochrome f. Electron transfer within the Ru-Pc complex has been studied with time-resolved optical spectroscopy using two different approaches. In the first, the fully reduced [Cu(I), Ru(II)] protein was photoexcited and subsequently oxidized by an external quencher, forming the [Cu(I), Ru(III)] protein. This was followed by an electron transfer from reduced Cu(I) to Ru(III). In the second method, the initially oxidized Cu(II) ion acted as an internal quencher for excited Ru(II) and the photoinduced reduction of the Cu(II) ion was followed by a thermal recombination with the Ru(III) ion. The reoxidation of the Cu ion, which has an estimated driving force of 0.56?eV, occured with a rate constant k _et?=?(9.5±1.0)×10⁶?s^–1, observed with both methods. The results suggest a strong electronic coupling (H _DA>0.3?cm^–1) along the Ru-His(83)-Cys(84)-Cu pathway.     

pH-induced conformational changes in spinach ferredoxin: steady-state and time-resolved fluorescence studies.

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.     

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

Characterization of Plastocyanin Isolated From Brazilian Elodea

Plastocyanin obtained from an aquatic higher plant, Brazilianelodea, was characterized by electronic absorption, CD, andEPR spectroscopy. The blue copper center of Brazilian elodeaplastocyanin is electronically and geometrically similar tothat of terrestrial higher-plant (cucumber, spinach, poplar,etc.) and algal plastocyanins as well as that of another aquatichigher-plant (hornwort) plastocyanin. The midpoint redox potentialof the copper in the plastocyanin was estimated to be +364 mVat pH 7.2, which is in the range of midpoint potentials from+347 to +395 mV reported for plastocyanins. The ratio of Phe/Tyrresidue was determined to be 3 by amino acid analysis. Valuesof 2–3.5 have been commonly observed in many higher-plantplastocyanins; a conspicuous exception to this is hornwort plastocyaninwith a Phe/Tyr value of 9. (Received January 16, 1986; Accepted April 18, 1987)     

Human glycolipid transfer protein: probing conformation using fluorescence spectroscopy

Glycolipid transfer protein (GLTP) is a soluble 24 kDa protein that selectively accelerates the intermembrane transfer of glycolipids in vitro. Little is known about the GLTP structure and dynamics. Here, we report the cloning of human GLTP and characterize the environment of the three tryptophans (Trps) of the protein using fluorescence spectroscopy. Excitation at 295 nm yielded an emission maximum (lambda(max)) near 347 nm, indicating a relatively polar average environment for emitting Trps. Quenching with acrylamide at physiological ionic strength or with potassium iodide resulted in linear Stern-Volmer plots, suggesting accessibility of emitting Trps to soluble quenchers. Insights into reversible conformational changes accompanying changes in GLTP activity were provided by addition and rapid dilution of urea while monitoring changes in Trp or 1-anilinonaphthalene-8-sulfonic acid fluorescence. Incubation of GLTP with glycolipid liposomes caused a blue shift in the Trp emission maximum but diminished the fluorescence intensity. The blue-shifted emission maximum, centered near 335 nm, persisted after separation of glycolipid liposomes from GLTP, consistent with formation of a GLTP-glycolipid complex at a glycolipid-liganding site containing Trp. The results provide the first insights into human GLTP structural dynamics by fluorescence spectroscopy, including global conformational changes that accompany GLTP folding into an active conformational state as well as more subtle conformational changes that play a role in GLTP-mediated transfer of glycolipids between membranes, and establish a foundation for future studies of membrane rafts using GLTP.     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).     

Fluorescence spectra,fluorescence quantum yield and dissociation constant of sarafloxacin

In this study, the fluorescence spectra of sarafloxacin (SAR) under different pH conditions were investigated to determine the structural changes due to protonation that result from change in pH. At pH < 1.02, SAR exists in the H₃L²⁺ form for which the maximum fluorescence emission wavelength was about 455 nm. At pH 1.87–4.94, SAR exists in the H₂L⁺ form in which H₃L²⁺ loses one proton in the nitrogen molecule at the 1‐position in the quinoline ring. Fluorescence intensity was strong and steady and the maximum emission wavelength was 458 nm. At pH 7.14–9.30, the maximum emission wavelengths were gradually blue shifted to 430 nm with increase in pH, here SAR exists in the form of a bipolar ion HL in which H₂L⁺ loses a carboxyl group proton. At pH > 11.6, HL transforms into anionic L^? in which HL loses one proton from the piperazine ring, leading to a decrease in fluorescence intensity, and the maximum emission wavelength was red shifted to approximately 466 nm. The two‐step dissociation constant pKa for SAR was calculated, pK _a1 was 6.06 ± 0.37 and pK _a2 for SAR was 10.53 ± 0.19. In a pH 3.62 buffer solution with quinine sulfate as the reference, the fluorescence quantum yield of SAR at the maximum excitation wavelength of 276 nm was 0.09.