Fluorescence characterization of type I collagen from normal and silicotic rats and its quenching dynamics induced by hypocrellin B

In this paper, we studied the quenching mechanism of intrinsic fluorescence of type I collagen by a new type photosensitizer and fluorescence quencher, hypocrellin B (HB). It was indicated that type I collagen can emit Tyr-intrinsic fluorescence with the excitation wavelength of Tyr (λ_ex = 269 nm). Its fluorescence decay conform to the triexponential rule of the fluorescence lifetime. The intrinsic fluorescence of type I collagen can be effectively quenched by HB through a process of charge and energy transference, which is involved in the collisional quenching, the dipolar inducement, and the formation of exciplex between HB and excited fluorophores of collagen. The fluorescence quenching would be weakened by higher ionic environments. The fluorescence emission and its quenching rate of abnormal silicotic collagen show falling trends, implying its much weakened potential of charge and energy transference, and its lessen bioelectric activities. In conclusion, the bioelectric properties of collagen depends on the perfect order of its molecular structure and orderly intramolecular and intermolecular interactions, which is important in its performing normal physiological functions. It is also demonstrated that the fluorescence quenching technique, using HB as a quencher, is truly an effectively method for biomolecular studies. © 1997 John Wiley & Sons, Inc. Biopoly 42: 219–226, 1997     

Effects of Quenching Mechanism and Type of Quencher Association on Stern-Volmer Plots in Compartmentalized Systems

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.     

Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry study.

Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and the model for the interpretation of protein quenching studies is emphasized.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

A model of dynamic quenching of fluorescence in globular proteins

A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the quencher concentration. Simulations performed for typical fluorescent probes in proteins showed good agreement with experiments.     

Adenine nucleotide translocase greatly increases the partition of trinitrophenyl-ATP into reduced Triton X-100 micelles.

The presence of adenine nucleotide translocase (ANT) was found to greatly enhance the partitioning of the ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) into reduced Triton X-100 micelles. The protein's effect was studied through the quenching of fluorescence of purified ANT, irreversibly inhibited by carboxyatractyloside (CAT), solubilized in reduced Triton X-100 micelles. The dependence of quenching of the protein's time-resolved tryptophan fluorescence on TNP-ATP concentration was measured and found to follow a Stern-Volmer mechanism. However, the calculated quenching constant was too large to be accounted for by the aqueous TNP-ATP concentration. Experiments were therefore conducted to determine the partitioning of the quencher between the three phases present: aqueous, protein-free micelle, and protein micelle; a system also described by the equation of Omann, G. M., and M. Glaser (1985. Biophys. J. 47:623-627.). By measuring the dependence of the apparent quenching rate constant on the protein concentration and protein/micelle ratios, this equation was used to calculate both the quencher partition coefficient into protein-free micelles (Pm) and into protein-micelles (Ppm), as well as the bimolecular quenching rate constant (kpm) in protein micelles. From the quenching experiments, kpm = 5.0 x 10(8)M-1s-1,Pm = 290 and pyrene quenching experiment to be 325, and by a rapid filtration experiment to be 450. Clearly, the presence of the integral membrane protein ANT-CAT in reduced Triton X-100 micelles greatly increases the partition of TNP-ATP into the micelle. ANT alters the properties and thus, the structure of the detergent micelle, which has direct implications for the use of detergent micelles as a model system for membrane proteins and may indicate that analogous effects occur in the mitochondrial membrane.     

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.     

Fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin

The effect of bacteriorhodopsin (BR) on the percolation properties of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine bilayers was examined by studying the quenching of a lipid-bound fluorophore by a lipid-bound quencher, and by spin-spin interactions of a nitroxide-labeled lipid using electron spin resonance (ESR). At the low concentrations of BR used, differential scanning calorimetry showed that although the transition enthalpy was reduced in a concentration-dependent manner by incorporation of BR, the solidus and fluidus phase boundaries and overall shape of the heat capacity profiles were essentially unchanged. However, fluorescence quenching and spin-label ESR data showed that the domain topology, as reflected in the percolation properties, is strongly affected by the protein. In contrast to our previous fluorescence data for the pure lipid mixtures, quenching in the coexistence region is independent of the fluid phase fraction when BR is present. In addition, the percolation threshold estimated by spin-label ESR is shifted in the presence of BR to a higher gel phase fraction at a given lipid composition. Both the fluorescence quenching and spin-label ESR data, together with the results of earlier simulations, strongly suggest that the fluid phase domains are substantially larger and/or less ramified in the presence of BR than in its absence. We have previously reported a similar effect of a transmembrane peptide, pOmpA (Escherichia coli outer membrane protein A signal peptide), on fluid domain connectivity in binary phosphatidylcholine mixtures.     

Two-dimensional diffusion of amphiphiles in phospholipid monolayers at the air-water interface.

Steady-state and time-resolved fluorescence spectroscopy has been used to examine lateral diffusion in dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and dimyristoyl-L-alpha-phosphatidylcholine (DMPC) monolayers at the air-water interface, by studying the fluorescence quenching of a pyrene-labeled phospholipid (pyrene-DPPE) by two amphiphilic quenchers. Steady-state fluorescence measurements revealed pyrene-DPPE to be homogeneously distributed in the DMPC lipid matrix for all measured surface pressures and only in the liquid-expanded (LE) phase of the DPPC monolayer. Time-resolved fluorescence decays for pyrene-DPPE in DMPC and DPPC (LE phase) in the absence of quencher were best described by a single-exponential function, also suggesting a homogeneous distribution of pyrene-DPPE within the monolayer films. Addition of quencher to the monolayer film produced nonexponential decay behavior, which is adequately described by the continuum theory of diffusion-controlled quenching in a two-dimensional environment. Steady-state fluorescence measurements yielded lateral diffusion coefficients significantly larger than those obtained from time-resolved data. The difference in these values was ascribed to the influence of static quenching in the case of the steady-state measurements. The lateral diffusion coefficients obtained in the DMPC monolayers were found to decrease with increasing surface pressure, reflecting a decrease in monolayer fluidity with compression.     

Chlorophyll a fluorescence induction: a personal perspective of the thermal phase, the J–I–P rise

The fast (up to 1?s) chlorophyll (Chl) a fluorescence induction (FI) curve, measured under saturating continuous light, has a photochemical phase, the O-J rise, related mainly to the reduction of Q(A), the primary electron acceptor plastoquinone of Photosystem II (PSII); here, the fluorescence rise depends strongly on the number of photons absorbed. This is followed by a thermal phase, the J-I-P rise, which disappears at subfreezing temperatures. According to the mainstream interpretation of the fast FI, the variable fluorescence originates from PSII antenna, and the oxidized Q(A) is the most important quencher influencing the O-J-I-P curve. As the reaction centers of PSII are gradually closed by the photochemical reduction of Q(A), Chl fluorescence, F, rises from the O level (the minimal level) to the P level (the peak); yet, the relationship between F and [Q(A) (-)] is not linear, due to the presence of other quenchers and modifiers. Several alternative theories have been proposed, which give different interpretations of the O-J-I-P transient. The main idea in these alternative theories is that in saturating light, Q(A) is almost completely reduced already at the end of the photochemical phase O-J, but the fluorescence yield is lower than its maximum value due to the presence of either a second quencher besides Q(A), or there is an another process quenching the fluorescence; in the second quencher hypothesis, this quencher is consumed (or the process of quenching the fluorescence is reversed) during the thermal phase J-I-P. In this review, we discuss these theories. Based on our critical examination, that includes pros and cons of each theory, as well mathematical modeling, we conclude that the mainstream interpretation of the O-J-I-P transient is the most credible one, as none of the alternative ideas provide adequate explanation or experimental proof for the almost complete reduction of Q(A) at the end of the O-J phase, and for the origin of the fluorescence rise during the thermal phase. However, we suggest that some of the factors influencing the fluorescence yield that have been proposed in these newer theories, as e.g., the membrane potential ΔΨ, as suggested by Vredenberg and his associates, can potentially contribute to modulate the O-J-I-P transient in parallel with the reduction of Q(A), through changes at the PSII antenna and/or at the reaction center, or, possibly, through the control of the oxidation-reduction of the PQ-pool, including proton transfer into the lumen, as suggested by Rubin and his associates. We present in this review our personal perspective mainly on our understanding of the thermal phase, the J-I-P rise during Chl a FI in plants and algae.     

Compartmental analysis in photophysics: kinetics and identifiability of models for quenching of fluorescent probes in micelles

The parameters describing the kinetics of excited-state processes can possibly be recovered by analysis of the fluorescence decay surface measured as a function of the experimental variables. The identifiability analysis of a photophysical model assuming errorless time-resolved fluorescence data can verify whether the model parameters can be determined. In this work, we have used the methods of similarity transformation and Taylor series to investigate the identifiability of two models utilized to describe the time-resolved fluorescence quenching of stationary probes in micelles. The first model assumes that exchange of the quencher between micelles is much slower than the fluorescence decay of the unquenched probe (the 'immobile' quencher model). The second model assumes that quenchers exchange between the aqueous and micellar phases (the 'mobile' quencher model). For the 'immobile' quencher model, the rate constants for deactivation (k(0)) and quenching (k(q)) of the excited probe are uniquely identified together with the average number of quencher molecules per micelle. For the 'mobile' quencher model, the rate constants k(0) and k(q) are uniquely identified, as are the rate constants for entry (k(+)) and exit (k(-)) of one quencher molecule into and from a micelle, and the micellar aggregation number. The concomitant rate equations describing the time-resolved fluorescence are solved using z-transforms.     

Penetration of an emulsion surface by cholesteryl ester transfer protein

Quenching of the intrinsic fluorescence of cholesteryl ester transfer protein (CETP) by spin labelled fatty acids (5-NS and 16-NS) was investigated to determine the degree to which the protein penetrated the phospholipid monolayer surface of a lipid emulsion. When bound to the phospholipid surface approximately 50% of the fluorophores of the transfer protein were accessible to quenching by 5-NS whose nitroxy group locates near the monolayer surface. On the other hand, only 22% of the fluorophores of CETP were accessible to quenching by 16-NS whose nitroxy group locates deeper in the surface monolayer. Quenching of the CETP fluorescence by an aqueous phase quencher (acrylamide) shows that the protein undergoes a conformational change on binding which increases the proportion of the tryptophan residues exposed to the aqueous phase. The results indicate that CETP does not penetrate the lipid surface to a significant degree. Received: 29 March 1996 / Accepted: 30 May 1996     

Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions.

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.     

Kinetics of lipid-membrane binding and conformational change of L-BABP

We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group. We fitted simultaneously the experimental curves to a three-state kinetic model. We conclude that in a first step, the binding of L-BABP to the interfacial region of the anionic lipid polar head groups occurred simultaneously with a conformational change to the partly unfolded state. In a second slower step, Trp6 buried within the polar head group region, releasing contacts with the aqueous phase.     

Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers-phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ(av) were quantified for a range of quencher concentrations. Applying the Stern-Volmer formalism, apparent quenching rate constants k(q) were determined from the dependencies on quencher concentration of the ratio τ(0)(av)/τ(av), where τ(0)(av) is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k(q)'/k(q), i.e., the apparent quenching rate constants determined in aggregates (or membranes), k(q)', and in detergent-solubilised LHCII trimers, k(q), respectively. In LHCII macroaggregates, the resulting values for the domain size were 15-30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12-24 LHCII trimers, corresponding to 500-1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment-protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.     

Determination of the aggregation number of detergent micelles using steady-state fluorescence quenching.

The development of a simple, reliable method for determination of detergent micelle aggregation number that relies solely on measurement of steady-state fluorescence quenching is presented. The degree of steady-state fluorescence quenching of a micelle-solubilized fluorophore (pyrene) by a quencher that partitions greatly into the micelles (coumarin 153) is dependent on the micelle concentration, which can therefore be determined. The aggregation number is calculated as the micelle concentration/detergent monomer concentration (the total detergent concentration above the critical micelle concentration). For the determination to be accurate, the partition coefficient of the quencher into the micelle phase is determined and used to calculate the micellar concentration of quencher. Also, the quenching of pyrene by a coumarin 153 molecule within the same micelle must be complete, and this was confirmed by time-resolved fluorescence measurements. Aggregation numbers were determined for one cationic and several nonionic detergents and were found to be consistent with literature values. The approach presented is an improvement on a previous luminescence quenching technique (Turro, N.J., and A. Yekta. 1978. J. Am. Chem. Soc. 100:5951-5952) and can be used on cationic, anionic, and nonionic detergents with micelles ranging greatly in size and under varying conditions, such as detergent concentration, ionic strength, or temperature.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I⁻, Cs⁺ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I⁻, was proven to be an ineffective quencher with Stern-Volmer constants, K_sv, of 0.60 and 0.63 M⁻¹, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs⁺, showed about a 2-fold difference in the K_sv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25–37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs⁺ showed more than 40% increase in the K_sv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.