Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.     

Changes in fluorescence quenching brought about by feeding dithiothreitol to illuminated leaves

When CO₂ is abruptly removed from the atmosphere surrounding an illuminated leaf, the primary electron-accepting plastoquinone of photosystem II (Q_A) (as measured by photochemical quenching, q_p) is rapidly reduced and then, after some seconds, becomes more oxidized. The reoxidation of Q_A⁻ is accompanied by an increase in ΔpH (as measured by nonphotochemical quenching, q_N) with kinetics consistent with a causal relationship. The fact that, in such circumstances, Q_A can become more oxidized in the absence of CO₂ than in its presence indicates a diminished rate of reduction of Q_A, consequent upon impaired photosystem II efficacy. Dithiothreitol (DTT) feeding, which does not affect quantum yield or the maximum rate of photosynthesis, inhibits the reoxidation of Q_A⁻ but not the increase in the proton gradient. When leaves are reilluminated in high light following a dark interval of several minutes, DTT also abolishes the separation in time between the first maximum in q_P and the first maximum in the rate of O₂ evolution. It also diminishes subsequent oscillations. These results are held to demonstrate ΔpH control of photosystem II and are consistent with DTT inhibition of the xanthophyll cycle and hydrogen peroxide reduction. They support the concept that oxygen and hydrogen peroxide are involved, as Hill oxidants, in a pH-related manner, during oscillatory behavior.     

Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan

Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K(ass.), ranging from 96 to 206 M(-1). Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.     

The quenching of tryptophan fluorescence by protonated and unprotonated imidazole

The fluorescence life-time of N-acetyl-tryptophan-amide (NATA) was measured by multifrequency phase fluorometry, in the presence of increasing concentrations of imidazole. Two pH values were tested, pH 4.5 where imidazole is fully protonated and pH 9.0 where it is fully unprotonated. At both pH values, the inverse life-time increases in a non-linear way with the imidazole concentration, showing that imidazole is not a high efficiency collisional quencher. The data can be analysed in terms of the formation of a complex with a reduced fluorescence life-time. The rate constants for association (at 25°C) are around 5 (±0.2) × 10⁹ M^–1 s^–1 and are thus diffusion controlled. The association equilibrium constant is strongly pH dependent and is much higher than the expected value of 0.4 M^–1 for a collisional complex. The intrinsic fluorescence life-time of the complex is 1.56 (±0.02) ns at pH 9.0 and 1.82 (±0.03) ns at pH 4.5, as compared to 2.37 (±0.03) ns for free NATA at pH 9.0 and 2.83 (±0.05) at pH 4.5 (all atI = 0.34). This means that at both pH values the fluorescence life-time of NATA in the complex is reduced to 61 (±0.5)% of its value in the free state. Despite this, the protonated form of imidazole is a better quencher at low concentrations, owing to a longer residence-time of the complex. At high viscosity the association equilibration is too slow and the system is described by two life-times. The quenching effect ofHis-18 on the fluorescence of the proximalTrp-94 of barnase (Locwenthal et al. 1991, Willaert et al. 1991) is discussed in terms of these findings.     

Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Lifetime and quenching of tryptophan fluorescence in whiting parvalbumin

The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites.     

Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence

Ion channels are allosteric membrane proteins that open and close an ion-permeable pore in response to various stimuli. This gating process provides the regulation that underlies electrical signaling events such as action potentials, postsynaptic potentials, and sensory receptor potentials. Recently, the molecular structures of a number of ion channels and channel domains have been solved by x-ray crystallography. These structures have highlighted a gap in our understanding of the relationship between a channel's function and its structure. Here we introduce a new technique to fill this gap by simultaneously measuring the channel function with the inside-out patch-clamp technique and the channel structure with fluorescence spectroscopy. The structure and dynamics of short-range interactions in the channel can be measured by the presence of quenching of a covalently attached bimane fluorophore by a nearby tryptophan residue in the channel. This approach was applied to study the gating rearrangements in the bovine rod cyclic nucleotide-gated ion channel CNGA1 where it was found that C481 moves towards A461 during the opening allosteric transition induced by cyclic nucleotide. The approach offers new hope for elucidating the gating rearrangements in channels of known structure.     

Assessment of the role of tryptophan residues in phospholipase A2 by fluorescence quenching

Tryptophan (Trp) fluorescence of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis snake venoms was quenched by acrylamide and iodide. Trp residues in N. naja atra PLA2 were equally accessible to acrylamide and iodide. Iodide quenching studies indicate that there are two classes of Trp fluorophores in N. nigricollis CMS-9. The accessible class consists of Trp-18 and Trp-19. Removal of the N-terminal octapeptide caused a perturbation of the micro-environment of the Trp residues in the PLA2 enzymes. The presence of a substrate lowers the susceptibility of the Trp residues to iodide quenching in N. naja atra PLA2, suggesting that all three Trp residues are at the substrate binding site, but in N. nigricollis CMS-9 Trp-18 and Trp-19 are related to substrate binding.     

Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

Pyruvate kinase acts as an allosteric enzyme, playing a crucial role in the catalysis of the final step of the glycolytic pathway. In this study, site-specific mutagenesis and tryptophan fluorescence quenching were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Movement of the B domain was found to be essential for the catalytic reaction. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft. Trp-157 accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481 and Trp-514 are brought into an aqueous environment after phenylalanine binding.     

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.     

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.     

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.     

Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

Intrinsic tryptophan fluorescence of membranes of GH3 pituitary cells: quenching by thyrotropin-releasing hormone.

The intrinsic tryptophan fluorescence of membranes prepared from the GH₃ strain of hormone-producing pituitary cells was monitored by spectrofluorometry. Membranes of GH₃ cells have specific receptors which bind thyrotropin-releasing hormone (TRH). When TRH binds to GH₃ membranes there is quenching of tryptophan fluorescence. The kinetics of the change in fluorescence of GH₃ membranes and of TRH binding are similar. In addition, the concentration of TRH required to produce a half-maximum change in fluorescence is 10 nM, and that required for half-maximum binding of TRH to receptors is 11 nM. Inactive TRH analogs which do not bind to TRH receptors likewise do not alter GH₃ membrane fluorescence, and a pituitary cell strain which lacks TRH receptors does not change membrane fluorescence on incubation with TRH. We conclude that the TRH-receptor interaction in GH₃ membranes is associated with a change in membrane conformation that is readily measured by differential spectrofluorometry.     

Study of reactivity of tryptophan residues in serum albumins and lysozyme by N-bromosuccinamide fluorescence quenching

The kinetics of quenching by N-bromosuccinamide of the fluorescence of tryptophan residues in various proteins and peptides were studied using the stopped-flow technique. Human serum albumin, which has a single tryptophan residue located in a hydrophobic fold, showed biphasic kinetics; one component was second order and the other first order, the rate for the latter component being independent of the concentration of quencher. Bovine serum albumin, which has in addition a tryptophan residue at the surface of the molecule, showed triphasic kinetics; two components corresponded to those for human serum albumin, and there was a faster second-order component resulting from the surface tryptophan. It is concluded that reaction with the buried tryptophan involves the initial second-order formation of a complex in which the quencher is located at the mouth of a hydrophobic fold, and that this is followed by a first-order conformational change which allows interaction to occur between the quencher and the tryptophan. The kinetics of lysozyme fluorescence quenching at pH 5.4 showed two relaxations whose rates were proportional to the N-bromosuccinamide concentration. The results of kinetic and titration experiments suggest that a molecule of lysozyme contains at least two groups of tryptophan residues of significantly different reactivities. The faster component probably reflects the bromination of the tryptophan residues at the active site. An octapeptide consisting of residues 22 to 29 of glucagon showed essentially the same quenching kinetics as glucagon itself, and Leu-Trp-Met showed the same behavior as Gly-Trp-Gly. The results indicate that quenching by N-bromosuccinamide provides a useful indication of the accessibility of tryptophan residues, and that side reactions do not significantly affect the kinetics.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.