HX-ESI-MS and optical studies of the unfolding of thioredoxin indicate stabilization of a partially unfolded, aggregation-competent intermediate at low pH

Hydrogen exchange monitored by mass spectrometry (HX-MS), in conjunction with multiple optical probes, has been used to characterize the unfolding of thioredoxin. Equilibrium and kinetic studies have been carried out at pH 7 and 3. The HX-MS measurements are shown to be capable of distinguishing between native (N) and unfolded (U) protein molecules when both are present together, and their application in kinetic experiments allows the unfolding reaction to be delineated from the proline isomerization reaction to which it is coupled. At pH 7, equilibrium unfolding studies monitored by three optical probes, intrinsic fluorescence at 368 nm, ellipticity at 222 nm, and ellipticity at 270 nm, as well as by HX-MS, indicate that no intermediate is populated at pH 7, the unfolding reaction is slower than the proline isomerization reaction that follows it, and the three optical probes yield identical kinetics for unfolding, which occurs in a single kinetic phase. The fractional change in any of the three optical signals at any time of unfolding predicts the fraction of the molecules that have become U, as determined by HX-MS. Hence, unfolding at pH 7 appears to occur via a two-state N <==> U mechanism. In contrast at pH 3, HX-MS as well as optical measurements indicate that an unfolding intermediate is stabilized and hence accumulates in equilibrium with N and U, at concentrations of denaturant that define the transition zone of the equilibrium unfolding curve. The intermediate has lost the near-UV signal characteristic of N and possesses fewer amide hydrogen sites that are stable to exchange than does N. Kinetic experiments at pH 3, where unfolding is much faster than proline isomerization, show that more than one intermediate accumulates transiently during unfolding. Thus, the unfolding of thioredoxin occurs via an N <==> I <==> U mechanism, where I is a partially unfolded intermediate that is stabilized and hence populated at pH 3 but not at pH 7. It is shown that transient aggregation of this intermediate results in a deceleration of the kinetics of unfolding at high protein concentrations at pH 3 but not at pH 7.     

Measurements of label free protein concentration and conformational changes using a microfluidic UV-LED method

This paper presents a microchip-based system for measuring concentrations and dynamic conformational changes in proteins without any use of extrinsic fluorescent labeling. The microchannel flow of protein molecules was integrated with an ultraviolet light-emitting diode (UV-LED, lambda ex = 295 nm) and a photodetector (lambda em = 330 nm). The intrinsic fluorescence shift, arising from selectively exciting aromatic amino acid tryptophan (Trp), was monitored to quantify refolding pathways by dynamically varying the concentration of the chemical denaturant, urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls, rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. The concentrations of proteins were first measured using the UV-LED microfluidic platform, and the data showed detection limits down to 72, 128, and 250 nM for tryptophan, bovine serum albumin (BSA), and bovine carbonic anhydrase (BCA), respectively. To validate the protein assay method, folding transition experiments were performed using a well-characterized protein, BSA. The microchip protein refolding transitions using intrinsic fluorescence were well-correlated with conventional fluorometer experiments. The microfluidic platform facilitates refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material. The technique offers a real alternative to bulky microfluidic systems consisting of large and expensive laser-based designs.     

Comparative fluorescence properties of bovine,goat, human and guinea pig α lactalbumin: Characterization of the environments of individual tryptophan residues in partially folded conformers

α Lactalbumin exists as a partially folded conformer (U form) at acid pH. A second partially folded conformer (H form) is formed above 60°. Comparison of the changes in tryptophan fluorescence which occur on forming U and H for the bovine, goat, human and guinea pig proteins, as well as analysis of fluorescence properties for the bovine protein and an N bromo succinimide derivative of this protein, have made it possible to determine which tryptophan residues give rise to such changes in fluorescence, and to draw a distinction between the molecular structure of the U and H forms of the protein. Trp 28 and 109 in the native state transfer their excitation energy to trp 63 whose fluorescence is quenched by a pair of vicinal disulfide bridges. This process persists in the U form of the protein, but is absent in the H conformer. Most of the change in fluorescence seen in the N ? U conversion is due to increase in yield of trp 28, while the changes in fluorescence occurring on formation of the H form are due to exposure of trp 63 and elimination of its quenching and/or excited state transfer from 28 to 109.     

Various kinetic and spectral-fluorescent properties of NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm

Malate dehydrogenase from bovine adrenal cortex has been purified to homogeneity, using affinity chromatography on 2',5'-ADP-Sepharose 4B. The kinetic data do not contradict the consecutive mechanism of the reaction with the ordered addition of substrates: NADP binds first, then malate. The enzyme conformation initiated by NADP and malate binding is less thermostable. Malate dehydrogenase has intrinsic tryptophan fluorescence with the spectrum maximum at 335 +/- 1 nm, half-width of 50 +/- 1 nm and quantum yield of 0.08. The tryptophan residues belonging to class 1 (75%) and class 2 (25%) make the main contribution to the intrinsic fluorescence of malate. The binding of cofactors and substrates results in the quenching of enzyme fluorescence. The values of dissociation constants for malate dehydrogenase complexes with NADP (4 microM), with NADP . H (8 microM) and with pyruvate (2.5 mM) correlate with the corresponding values of Km. The shifts in pH of the medium induce changes in the fluorescence parameters which are probably related to conformational changes in the enzyme molecule. The changes in the fluorescence parameters correlate with the alterations of the malate dehydrogenase enzymatic activity.     

Folding and stability of trp aporepressor from Escherichia coli

Equilibrium and kinetic studies of the urea-induced unfolding of trp aporepressor from Escherichia coli were performed to probe the folding mechanism of this intertwined, dimeric protein. The equilibrium unfolding transitions at pH 7.6 and 25 degrees C monitored by difference absorbance, fluorescence, and circular dichroism spectroscopy are coincident within experimental error. All three transitions are well described by a two-state model involving the native dimer and the unfolded monomer; the free energy of folding in the absence of denaturant and under standard-state conditions is estimated to be 23.3 +/- 0.9 kcal/mol of dimer. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration in the manner expected from the law of mass action for the two-state model. We find no evidence for stable folding intermediates. Kinetic studies reveal that unfolding is governed by a single first-order reaction whose relaxation time decreases exponentially with increasing urea concentration and also decreases with increasing protein concentration in the transition zone. Refolding involves at least three phases that depend on both the protein concentration and the final urea concentration in a complex manner. The relaxation time of the slowest of these refolding phases is identical with that for the single phase in unfolding in the transition zone, consistent with the results expected for a reaction that is kinetically reversible. The two faster refolding phases are presumed to arise from slow isomerization reactions in the unfolded form and reflect parallel folding channels.     

Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models.

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.     

The binding of aurovertin to isolated F1 (mitochondrial ATPase).

1. Isolated F1 contains 14.9% N, indicating the presence of at least 8% non-protein material. The Lowry method, standardized with bovine serum albumin, correctly measures the protein content. 2. An extinction coefficient of 28.5 mM-1.cm-1 at 367.5 nm was found for aurovertin D in ethanol. 3. The fluorescence enhancement of aurovertin bound to F1 at pH 7.5 was found to be more than 100-fold. 4. Binding parameters calculated from the fluorescence enhancement with fixed F1 and variable aurovertin concentrations, and vice versa, indicate two binding sites per F1 molecule. 5. The fluorescence data are not readily interpreted on the basis of successive binding of aurovertin by 3-component binding reactions of the form E + A in equilibrium EA, but fit closely a model of two non-interacting sites binding aurovertin in a 4-component reaction, EF + A in equilibrium EA + F, with an equilibrium constant of about 2.     

Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions.

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.     

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

表面活性剂增敏——牛血清白蛋白荧光猝灭法测定槐花中芦丁含量

目的：应用牛血清白蛋白荧光猝灭法建立一种测定槐花中芦丁含量的新方法。方法：牛血清白蛋白（BSA）具有很强的内源荧光性,而芦丁溶液本身不产生荧光。当芦丁与BSA结合后,会导致其荧光强度下降,表面活性剂吐温-80（T-80）对体系有促进荧光猝灭作用。BSA在λex=338nm处的荧光猝灭程度与芦丁的量在一定浓度范围内呈良好的线性关系,据此建立测定槐花中芦丁含量的新方法。结果：该结合物的最大发射波长为λmax=338nm,与芦丁摩尔浓度在6×10-7-3.0×10-5mol.L-1范围内线性关系良好。其线性回归方程为ΔF=136.36CRu（×10-5mol.L-1）-0.5454,相关系数r=0.9976,检出限为1.58×10-7mol.L-1,RSD为2.8%-4.3%,加标回收率为97.6%~101.2%。结论：本方法操作简便、快速,用于实际样本的测定,结果满意。     

Non-random conformation of a mouse IgG2a monoclonal antibody at low pH

The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.     

Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.

In the present work, we have characterized the protein--protein interactions in the trp repressor (TR) from Escherichia coli using fluorescence spectroscopy. The steady-state and time-resolved fluorescence anisotropy of repressor labeled with 5-(dimethylamino)naphthalene-1-sulfonamide (DNS) was used to monitor subunit equilibria in the absence and presence of corepressor. In the absence of tryptophan, the repressor is in equilibrium between tetramers and dimers in the concentration range studied (approximately 0.04-40 microM in dimer). Binding of corepressor resulted in a marked destabilization of the tetramer. The beginning of a dimer-monomer dissociation transition was observed by monitoring the decrease in the intrinsic tryptophan emission energy upon dilution below 0.1 microM in dimer, indicating an upper limit for the dimer-dissociation constant near 1 nM. DNA titrations with a 26 base pair sequence containing the trp EDCBA operator performed in the absence and presence of the corepressor are consistent with a 1:1 dimer/operator stoichiometry in the presence of tryptophan, while the aporepressor binds with TR dimer/DNA stoichiometries greater than one and which depend upon both the concentration of protein and that of the operator. Using the multiple observable parameters available in fluorescence, we have thus carried out a thorough investigation of the coupled equilibria in this bacterial repressor. Our results are consistent with a physiologically relevant thermodynamic role for tetramerization in the regulatory function of the trp repressor. The present results which have brought to light novel protein--protein interactions in the trp repressor system indicate that fluorescence spectroscopic methods could prove quite useful in the study of the role of protein--protein interactions in eukaryotic systems as well.     

Biochemical evaluation of a synthesized isoflavone-selenium complex by molecular spectra

A coordination compound of 5, 7-dihydrox-4'-methoxyisoflavone and selenium was synthesized and its structure was identified by IR, LC-MS and (1)H-NMR. Its biochemical effects were investigated using bovine serum albumin (BSA) as a target protein molecule, in which process three-dimensional (3D) fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra and fluorescence probe techniques were employed. The interaction of SEIF and BSA was discussed by fluorescence quenching method and F?rster non-radiation energy transfer theory. The thermodynamic parameters ΔH (θ), ΔG (θ), ΔS (θ) at different temperatures were calculated according to Van't Hoff isobaric equation and the results indicated the interaction was an exothermic as well as a spontaneous process. The binding site was explored by fluorescence probe method using warfarin and ibuprofen as markers. Intramolecular forces which are responsible for maintaining the binding were mainly hydrogen bond and van der Waals power. The average distance from the tryptophan residue in domain II of BSA (donor) to SEIF (acceptor) is 3.57 nm at body temperature. The conformation changes of BSA were investigated by 3D fluorescence and CD spectra.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).     

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.     

pH-induced conformational states of bovine growth hormone

The folding behavior of bovine growth hormone (bGH) is examined by chemical and pH denaturation using several spectroscopic probes of protein secondary and tertiary structure. Partially denaturing concentrations of urea eliminate the native-state quenching of intrinsic tryptophan fluorescence, from the single protein tryptophan, but the fluorescence emission spectrum is not red-shifted like the unfolded state, and the protein retains substantial secondary structure. A neutral-to-acid pH shift also eliminates tryptophan quenching; however, the loss of quenching is not accompanied by an emission red-shift. In addition, the protein undergoes a pH-dependent UV absorbance transition; the changes in absorptivity have the same midpoint as the transition associated with the change in intrinsic tryptophan fluorescence. The magnitude of the absorption transition is similar to that observed previously for urea denaturation of the protein. In a similar fashion, a pH-dependent CD transition is also observed; however, the transition occurs at a higher pH. The behavior of the various optical probes indicates that the pH-induced conformational transition produces a highly populated species in which the microenvironment surrounding the single protein tryptophan residue resembles that observed during the urea-induced unfolding/refolding transition. The pH-induced changes in tertiary structure occur at a lower pH than the changes associated with a portion of the secondary structure. Proton NMR of the low-pH intermediate indicates that the three His and six Tyr resonances are indistinguishable from the unfolded state. The intermediate(s) observed by either chemical or pH-induced denaturation resemble(s) a molten globule state which contains significant secondary structure. The residual secondary structure present in the intermediate could be nonnative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes of disulfide isomerase C induced by guanidine hydrochloride

Unfolding--refolding of Escherichia coli disulfide isomerase C (DsbC) induced by GdnHCl was studied by intrinsic fluorescence. Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure. It is shown that although Cys 141 is the next neighbour of a single tryptophan residue Trp 140, sulfur atoms of the disulfide bond Cys 141--Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170. It has been revealed that, though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38 and Tyr 52) contribute to the bulk fluorescence of the molecule. The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data), the character of parametric dependencies between fluorescence intensity recorded at 320 and 365 nm, and the existence of an isosbestic point of protein fluorescence spectra in solutions with different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation. The reversibility of this process is also shown.     

Evidence for dynamically determined conformational states in rhodanese catalysis

Physical and kinetic studies have been used to explore hysteretic effects that are observed in rhodanese catalysis at pH 5 and also at neutral pH when the ionic strength of the medium is high. Experiments that involve observation of changes in intrinsic protein fluorescence of the enzyme and kinetic investigation of its interactions with product thiocyanate anion at pH 5 have implicated enzyme isomerization as the cause of hysteresis. Taken all together, the data indicate that the conformations of enzyme forms in the catalytic cycle are dynamically determined, depending on the relative rates of conformational relaxation and catalysis as influenced by the concentrations of substrates and products.     

Physicochemical Studies on the Interaction between N-Decanoyl-N-methylglucamide and Bovine Serum Albumin

The protein-surfactant system constituted by bovine serum albumin (BSA) and N-decanoyl-N-methylglucamide (MEGA-10) has been studied by using surface tension, steady-state fluorescence, and dynamic light scattering measurements. It was found that the presence of protein delays the surfactant aggregation, which was interpreted as a sign of binding between surfactant and protein. Binding studies were carried out by two different methods. First, a treatment based on surface tension measurements was used to obtain information on the number of surfactant molecules bound per protein molecule under saturation conditions. Second, the binding curve for the BSA/MEGA-10 system was determined by examining the behavior of the intrinsic BSA fluorescence upon the surfactant addition. Both approaches indicate that the binding process is essentially cooperative in nature. The results of the aggregation numbers of MEGA-10 micelles, as well as those of resonance energy transfer from tryptophan residues to 8-anilinonaphthalene-1-sulfonate, corroborate the formation of micelle-like aggregates of surfactants, smaller than the free micelles, adsorbed on the protein surface. The dynamic light scattering results were not conclusive, in the sense that it was not possible to discriminate between protein-surfactant complexes and free micelles. However, the overall results suggest the formation of "pearl necklace" complexes in equilibrium with the free micelles of the surfactant.     

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.