Fluorescence of proteins in aqueous neutral salt solutions. II. Influence of monovalent cation chlorides, particularly cesium chloride.

The effects of varying concentrations of monovalent cation chlorides on the fluorescence of nine proteins were studied. These are discussed in terms of “direct” or “indirect” interactions with the aromatic amino acid residues. Cs⁺ is the only cation that quenches fluorescence of proteins due to “direct” interaction with aromatic amino acid residues. Quenching is due to collisional processes. An agreement with the Stern-Volmer relationship is shown and the values of [(K_Q)eff] and [(f_a)eff] are calculated. These values confirm that the fraction of fluorescence accessible to Cs⁺ belongs to the “exposed” fluorophors. The mechanism of quenching by Cs⁺ is due to the heavy-atom effect because phosphorescence enhancement is also seen at the same time. The chlorides of Na⁺, K⁺, Rb⁺, NH₄⁺, and Li⁺ do not have a similar effect on the fluorescence of all proteins. For a given protein a gradation of the same effect (i.e., quenching or dequenching) is seen. Interactions with factors that “inderectly” affect fluorescence of any protein are involved and the structural features of the protein are responsible for such “indirect” effects. The results indicate that neutral salts can act in more than one manner. The changes in fluorescence are indicative of electrostatic and lyotropic effects of ions. Only electrostatic interactions which occur in the vicinity of tryptophan in proteins are reflected. Li⁺ shows strong interactions with proteins. In 4 M LiCl, BSA, papain, and trypsin show fluorescence changes that are indicative of changes in protein structure.     

Anion activation of γ-glutamyltransferase from fruiting bodies of Lentinus edodes

Activation by different anions of γ-glutamyltransferase obtained in a. particulate form from fruiting bodies of Lentinus edodes has been studied using either L-γ-glutamyl-p-nitroanlide or lentinic acid as substrate. The mushroom transferase was activated by SCN^?, NO₃^?, Cl^?, Br^?, ClO₃^?, Bro₃^?, N₃^?, I^? and F^?, but not those alkali and earth cations previously believed to activate the animal transferase, nor by citrate, claimed to be effective for the kidney bean transferase. Among anions proved hardly to activate the transferase were ClO₄^?, NO₂?, HCO₃^?, H₂PO₄^?, SO₃^2? and SO₄^2?. A high concentration of these anions more or less impeded the halide activation. Kinetic studies revealed that halides function as activators of increasing V_max while keeping K_m constant. These observations appeared least compatible with the possibility that the anion activation might involve a non-specific effect of high solute concentration, viz. dissociation of the enzyme from the supporting structure in the particulates. The activating effect of halides described here probably extends also to the animal enzymes.     

Bacterial Perchlorate Reduction in Simulated Reverse Osmosis Rejectate

Reverse osmosis (RO) is capable of removing perchlorate (ClO₄ ^?) from contaminated groundwater and producing potable effluent; however, RO does not destroy ClO₄ ^?, but collects it in a concentrated waste stream (rejectate) that must be treated or disposed of appropriately. A packed bed bioreactor, inoculated with the pure culture perclace, was tested for its ability to remove ClO₄ ^? from a simulated RO rejectate. Perchlorate concentrations were lowered from 5 mg/L to <0.004 mg/L with a residence time of 0.8 h. In addition, this system removed 98% of ClO₄ ^? from a twice-concentrated rejectate with an influent ClO₄ ^? concentration of 8 mg/L and a residence time of 2.0 h. In both experiments, nitrate (NO₃ ^?) was removed simultaneously with ClO₄ ^? from an initial concentration as high as 900 mg/L NO₃ to below 4 mg/L. Despite the efficiency of ClO₄ ^? removal, the system suffered from clogging due to the high total dissolved solids (TDS) of the twice-concentrated rejectate.     

An OFF–ON–OFF type fluorescent probe based on a naphthalene derivative for Al3+ and F− ions and its biological application

A novel fluorescent probe‐based naphthalene Schiff, 1‐(C2‐glucosyl‐ylimino‐methyl)‐naphthalene‐2‐ol (L) was synthesized by coupling d ‐glucosamine hydrochloride with 2‐hydroxy‐1‐naphthaldehyde. It exhibited excellent selectivity and highly sensitivity for Al³⁺ in ethanol with a strong fluorescence response, while other common metal ions such as Pb²⁺, Mg²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Fe²⁺, Mn²⁺, Hg²⁺, Li⁺, Na⁺, K⁺, Fe³⁺, Cr³⁺, Zn²⁺, Ag⁺, Ba²⁺ and Ca²⁺ did not cause the same fluorescence response. The probe selectively bound Al³⁺ with a binding constant (K_a) of 5.748 × 10³ M^?1 and a lowest detection limit (LOD) of 4.08 nM. Moreover, the study found that the fluorescence of the L ? Al³⁺ complex could be quenched after addition of F^? in the same medium, while other anions, including Cl^?, Br^?, I^?, NO₂^?, NO₃^?, ClO₄^?, CO₃^2?, HCO₃^?, SO₄^2?, HSO₄^?, CH₃COO^?, PO₄^3?, HPO₄^2?, S^2? and S₂O₃^2? had nearly no influence on probe behaviour. Binding of the [L ? Al³⁺] complex to a F^? anion was established by different fluorescence titration studies, with a detection limit of 3.2 nM in ethanol. The fluorescent probe was also successfully applied in the imaging detection of Al³⁺ and F^? in living cells.     

Partial reactivation of impaired immune competence in aged mice by synthetic thymus factors

The stability of mouse uterine cytosol receptor-[³H]estradiol complex was evaluated in the presence of neutral salts of the Hoffmeister series. Marked increases in the rate of dissociation of the complex were observed with the more chaotropic anions (SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$, $\text{NO}{}_3{}^{\mathrm{?}}$, Br^?), and the effects of these ions were greater at lower temperatures, where water assumes a more rigid structure. At higher temperatures F^? and CH₃COO^?, which tend to stabilize water structure, led to retardation of the rate of dissociation of the hormone-receptor complex. There was essentially no change in steroid specificity in the presence of the markedly chaotropic salts. The perturbation of water structure adjacent to the steroid binding site is a factor to be considered in the isolation of steroid receptor complexes.     

New Screening Method for the Rapid Identification of Blocked Mutants and Their Products: Application to Erythromycin and Oleandomycin Producing Strains

γ-Glutamyltransferase from fruiting bodies of Lentinus edodes was further tested for its activation by chaotropic ions such as SCN^?, NO₃^?, Cl^?, Br^?, I^?, F^? and C1O₄^?. The thiocyanate ion increased the Km value for γ-glutamyl-p-nitroanilide without affecting the V_max value of the reaction, whereas other anions as represented by NO₃^? and Br^? increased the V_max without affecting the Km. Jhe inactivation of the enzyme by the SH group-orienting reagents, iodoacetamide and hydrogen peroxide, was stimulated by SCN^? but not by the other anions.

The activator anions protected the enzyme against its inactivation by chemical modification with 2,3-butanedione in borate. Their efficiency was parallel to the activator potency of the respective anions, except for SCN^? which provided less protection than expected from its activation potency. These dissociable effects of activator anions might be explained by two different mechanisms; binding of SCN^? to a basic group to bring about a significant change in protein conformation and binding of other anions by electrostatic and hydrophobic forces to an arginyl residue located near the active site of the enzyme.     

Dioxouranium(VI) complexes of L-arginine

Complexes of dioxouranium(VI) with the amino acid L-arginine have been prepared and studied by ir and pmr measurements. The results indicate the formation of UO₂L XnH₂O (L = Arg^?; X = NO₃^?, CH₃COO^?, or ClO₄^?; n = 2 or 3). The bonding involves carboxylato-, amino-, and probably guanido groups of the ligand. The coordination sphere of dioxouranium(VI) also includes nitrate or acetate, but not the perchlorate group. Uranyl(VI) may reach the coordination number of 5 in the equatorial plane by the coordination of moleclues of H₂O.     

Effect of Single-Charged Anions of Different Nature on the DNA Molecule Conformation in Water–Salt Solutions

Methods of intrinsic viscosity () and beam flow birefringence were used to study the effects of some single-charged ions (F^–, Cl^–, Br^–, I^–, NO^– ₂, NO^– ₃, ClO^– ₄, SCN^–, CH₃COO^–) on the size and thermodynamic rigidity of a DNA molecule in aqueous solutions of sodium salts in a broad interval of ionic strength when temperature T is changed. It has been shown that the close interactions in a macromolecule and the resulting DNA persistent length a are independent of the type of the salt anion over the whole interval of . On the contrary, the specific volume of the DNA molecule in solution, proportional to the value, is quite sensitive to the anionic composition of the solvent, which is due to the effect of anions and their hydration on the long-range interactions in the macromolecule. The presence of polyatomic and halide anions is manifested differently in the value of DNA. Possible factors responsible for the observed effect and the role of structural alterations of water upon anion hydration are discussed.     

Studies of the effect of halide ions on the fluorescence of quinine sulfate

The effect of halide ions (Cl^?, Br^? and I^?) on the fluorescence of quinine sulfate in dilute sulfuric acid solution was studied by fluorescence spectra, ultraviolet‐visible (UV‐visible) absorption spectra and fluorescence decay technique. The results exhibited that halide ions with heavier atomic mass could significantly reduce the fluorescence intensity of quinine sulfate, as a result, the order of fluorescence quenching caused by halide ions is Cl^? < Br^? < I^?. Therefore, halide ions with high concentration could seriously quench the fluorescence of quinine sulfate. The UV‐visible absorption spectra and fluorescence decay technique revealed that the fluorescence quenching of quinine sulfate caused by halide ions was attributed to dynamic quenching, static quenching process, self‐quenching fluorescence effect and electronic transfer.     

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol^?1 and 1.9·10⁹ 1·mol^?1·s^?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol^?1 and 092·10⁹ 1·mol^?1·^?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

Interaction of β‐cyclodextrin‐capped CdSe quantum dots with inorganic anions and cations

A facile method was developed for the preparation of water soluble β‐Cyclodextrin (β‐CD)‐modified CdSe quantum dots (QDs) (β‐CD‐QDs) by directly replacing the oleic acid ligands on the QDs surface with β‐CD in an alkaline aqueous solution. The as‐prepared QDs show good stability in aqueous solution for several months. Oxoanions, including phosphoric acid ion, sulphite acid ion and carbonic acid ion, affect the fluorescence of β‐CD‐QDs. Among them, H₂PO₄^– exhibited the largest quenching effect. For the polyprotic acids (HO)₃AO, the effect of acidic anions on the fluorescence of β‐CD‐QDs was in the order: monoanion (HO)₂AO₂^– > dianion (HO)AO₃^2– >> trianion AO₄^3–. After photoactivation for several days in the presence of anions at alkaline pH, the β‐CD‐QDs exhibited strong fluorescence emission. The effect of various heavy and transition metal ions on the fluorescence properties of the β‐CD‐QDs was investigated further. It was found that Ag⁺, Hg²⁺ and Co²⁺ have significant quenching effect on the fluorescence of the β‐CD‐QDs. The Stern–Volmer quenching constants increased in the order: Hg²⁺ < Co²⁺ <Ag⁺. The adsorption model of metal ions on β‐CD‐QDs was explored. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of salts on C-phycocyanin

The effect of a number of inorganic anions on the quaternary structure of C-phycocyanin has been investigated by fluorescence polarization. Dissociation to monomer occurred in the order: SCN⁻ > ClO₄⁻ > NO₃⁻ > Br⁻ > Cl⁻. These results suggest that hydrophobic interactions are important in the hexamer-monomer equilibrium of C-phycocyanin.     

RAPID AMMONIUM‐ AND NITRATE‐INDUCED PERTURBATIONS TO CHL a FLUORESCENCE IN NITROGEN‐STRESSED DUNALIELLA TERTIOLECTA (CHLOROPHYTA)1

When NH₄ ⁺ or NO₃ ^? was supplied to NO₃ ^? ‐stressed cells of the microalga Dunaliella tertiolecta Butcher, immediate transient changes in chl a fluorescence were observed over several minutes that were not seen in N‐replete cells. These changes were predominantly due to nonphotochemical fluorescence quenching. Fluorescence changes were accompanied by changes in photosynthetic oxygen evolution, indicating interactions between photosynthesis and N assimilation. The magnitude of the fluorescence change showed a Michaelis‐Menten relationship with half‐saturation concentration of 0.5 μM for NO₃ ^? and 10 μM for NH₄ ⁺ . Changes in fluorescence responses were characterized in D. tertiolecta both over 5 days of N starvation and in cells cultured at a range of NO₃ ^? ‐limited growth rates. Variation in responses was more marked in starved than in limited cells. During N starvation, the timing and onset of the fluorescence responses were different for NO₃ ^? versus NH₄ ⁺ and were correlated with changes in maximum N uptake rate during N starvation. In severely N‐starved cells, the major fluorescence response to NO₃ ^? disappeared, whereas the response to NH₄ ⁺ persisted. N‐starved cells previously grown with NH₄ ⁺ alone showed fluorescence responses with NH₄ ⁺ but not NO₃ ^? additions. The distinct responses to NO₃ ^? and NH₄ ⁺ may be due to the differences between regulation of the uptake mechanisms for the two N sources during N starvation. This method offers potential for assessing the importance of NO₃ ^? or NH₄ ⁺ as an N source to phytoplankton populations and as a diagnostic tool for N limitation.     

Metal chelates of heterocyclic nitrogen-containing ketones. XIV. Metal ion,anion and substituent effects on the enolization of mono-keto compounds

A number of new complexes of iron(II), cobakt(II), nickel(II), copper(II) and palladium(II) containing 2-picolyl-p-nitrophenyl- or 2-picolyl-p-tolyl ketone, L and L′, respectively, and various anions (Cl^?, Br^?, NSC^?, BF₄^? or ClO₄^?) have been synthesized and characterized by elemental analyses, magnetic susceptibility, ESR, IR and reflectance spectral measurements. The stereochemistry and the nature of the complexes are markedly dependent upon the molar of the reactants, the anions and the ligand substituents. In all complexes the ligands are cheated to the metal ion via the pryridine nitrogen and the carbonyl oxygen atoms, whereby in the case of [ML₂]X₂, M = iron(II) and [ML₃]X₂, M = cobalt(II) or nickel(II) and X = ClO₄^? or BF₄^?, the 2-picolyl-p-nitrophenyl ketone exists in its enol form which is only deprotonated in the presence of palladium(II). The ligand field parameters (Dq, B′, λ and β) are calculated and related to the electronic environment and the basicity of the ligands.     

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, GdnH⁺, and Ca²⁺), identity. The Q* decreased in the order F⁻ > Cl⁻ > Br⁻ > NO₃⁻ ∼ I⁻ > SCN⁻ > ClO₄⁻ ≫ SO₄²⁻, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO₄²⁻ anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

Major Ion Chemistry of Soil Solution of Mountainous Soils,Alvand, Hamedan,Western Iran

Soil solution chemistry reflects the most dynamic processes occurring in soils and is responsible for their current status. This study was undertaken to determine the soil solution status in 25 mountainous soils. The major cations in the studied soil solutions are in the decreasing order of Ca²⁺ > Mg²⁺ > Na⁺ > K⁺. The anions are also arranged in decreasing order as HCO^? ₃ > Cl^? > NO^? ₃ > SO ^2? ₄ . Concentrations of NO^? ₃ , P, and K⁺ in soil solutions were in the range of 12–364 mg l^?1, 1.75–34.8 mg l^?1, and 0.78– 198 mg l^?1, respectively. Results suggest that the concentration of P in the soil solutions could be primarily controlled by of the solubility of octacalcium phosphate and ß-tricalcium phosphate. In general, the greater the dissolved P concentration in the soil solution, the closer the solution was to equilibrium with respect to the more soluble Ca²⁺ phosphate minerals. Surface soil accumulations of P, NO^? ₃ , and K⁺ have occurred in these soils to such an extent that loss of these nutrients in surface runoff and the high risk for nutrient transfer into groundwater in concentrations exceeding the groundwater quality standard has become a priority management concern.     

A comparison of regulatory effects of chloride on nitrate uptake, and of nitrate on chloride uptake into Pisum sativum seedlings

Net nitrate uptake, ³⁶ClO^?₃/NO^?₃ influx and ³⁶Cl^? influx into Pisum sativum L. cv. Feltham First seedlings have been examined following growth in culture medium containing different combinations of chloride and nitrate. When young (6 days old) seedlings, that had been grown in the absence of N were used, nitrate accumulation stimulated net nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.99) while chloride accumulation inhibited nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.65). When nitrate was provided during growth there was no effect of chloride pretreatment on net nitrate uptake and there was little effect of total [NO^?₃+ Cl^?]_i on ³⁶ClO^?₃/NO^?₃ influx (r²= 0.26). A direct effect of Cl^? on ³⁶ClO^?₃/NO^?₃ influx was only found when seedlings had been starved of N for more prolonged periods (14 days). When moderate chloride was supplied during growth, ³⁶Cl^? influx was insensitive to nitrate or chloride accumulated, but significantly correlated with log_e [NO^?₃+ Cl^?]_i (r²= 0.75). When trace amounts of Cl^? were supplied during growth ³⁶Cl^? influx was inhibited by (a) NO^?₃ in the external medium and (b) Cl^? pretreatment, but was insensitive to NO^?₃ pretreatment. The sensitivity of ³⁶Cl^? influx to external nitrate was not found following Cl^? pretreatment in the absence of nitrate. The possibility that there are two populations of chloride carriers which differ in their sensitivity to external nitrate is discussed. Tentative schematic models to account for the regulation of nitrate and chloride uptake are proposed in the context of current hypotheses for regulation of ion transport and control systems theory.     

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.