Protein fluorescence quenching by small molecules: protein penetration versus solvent exposure

Experiments were done to test the thesis that acrylamide and similar small molecules can penetrate into proteins on a nanosecond time scale. The approach taken was to measure the pattern of fluorescence quenching exhibited by quenching molecules differing in molecular character (size, polarity, charge) when these are directed against protein tryptophans that cover the whole range of tryptophan accessibility. If quenching involves protein penetration and internal quencher migration, one expects that larger quenchers and more polar quenchers should display lesser quenching. In fact, no significant dependence on quencher character was found. For proteins that display measurable quenching, the disparate quenchers studied display very similar quenching rate constants when directed against any particular protein tryptophan. For several proteins having tryptophans known to be buried, no quenching occurs. These results are not consistent with the view that the kinds of small molecules studied can quite generally penetrate into and diffuse about within proteins at near-diffusion-limited rates. Rather the results suggest that when quenching is observed, the pathway involves encounters with tryptophans that are partially exposed at the protein surface. Available crystallographic results support this conclusion.     

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide

The heterogeneous fluorescence of yeast 3-phosphoglycerate kinase, a hinge-bending enzyme with two tryptophans, has been resolved into two approximately equal components, one accessible and one inaccessible to the relatively inefficient quencher succinimide. The inaccessible component is blue-shifted and exhibits a heterogeneous fluorescence decay which has a temperature-dependence and steady-state acrylamide quenching properties typical of a single tryptophan in a buried environment. This component is therefore assigned to the buried tryptophan W333. The presence of succinimide greatly simplifies the fluorescence allowing the conformational dynamics of the buried tryptophan and its environment to be studied without interference from the other tryptophan.     

Accessibility of tryptophan residues in Na,K-ATPase

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.     

Second derivative fluorescence spectra of indole compounds

The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 1.0 nm (A) and 358.5 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70 degrees C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra.     

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.     

A spectroscopic and conformational study of pertussis toxin

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.     

Fluorescence-quenching-resolved spectroscopy of proteins

A new procedure is described for using fluorescence-quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern-Volmer quenching plot is determined at each particular emission wavelength and iterative non-linear least-squares fitting procedure allowed to resolve the steady-state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two-tryptophan-containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single-photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Multi-path quenchers: efficient quenching of common fluorophores

Fluorescence quenching groups are widely employed in biological detection, sensing, and imaging. To date, a relatively small number of such groups are in common use. Perhaps the most commonly used quencher, dabcyl, has limited efficiency with a broad range of fluorophores. Here, we describe a molecular approach to improve the efficiency of quenchers by increasing their electronic complexity. Multi-Path Quenchers (MPQ) are designed to have multiple donor or acceptor groups in their structure, allowing for a multiplicity of conjugation pathways of varied length. This has the effect of broadening the absorption spectrum, which in turn can increase quenching efficiency and versatility. Six such MPQ derivatives are synthesized and tested for quenching efficiency in a DNA hybridization context. Duplexes placing quenchers and fluorophores within contact distance or beyond this distance are used to measure quenching via contact or FRET mechanisms. Results show that several of the quenchers are considerably more efficient than dabcyl at quenching a wider range of common fluorophores, and two quench fluorescein and TAMRA as well as or better than a Black Hole Quencher.     

Quenching of alkaline phosphatase phosphorescence by O2 and NO. Evidence for inflexible regions of protein structure.

The rate constant for quenching the phosphorescence of alkaline phosphatase by molecular oxygen was measured as a function of temperature. The results disagree with previous determinations and, contrary to fluorescence quenching, show that diffusion of O2 to this region of the macromolecule is a highly hindered process. When nitric oxide is introduced as a quencher, similarly small rate constants were found. While the activation energy for this process is identical for both quenchers, it is much smaller than for structural fluctuations at the chromophore site as manifested by the intrinsic triplet-state lifetime. These findings are analyzed in terms of a mechanism that takes into account static quenching at large distances and does not require penetration of the quencher all the way to the chromophore.     

Characterization of the tryptophan environments of interleukins 1 alpha and 1 beta by fluorescence quenching and lifetime measurements

The tryptophan environments of interleukins 1 alpha and 1 beta, immunomodulatory proteins with similar biological activities but only 25% sequence homology, were characterized by steady-state and dynamic fluorescence measurements. Both proteins exhibited similar emission maxima, but the emission intensity of IL-1 beta was greatly enhanced by increasing the ionic strength of the medium, whereas that of IL-1 alpha was unaffected. The two cytokines were also similarly quenched by the polar quencher acrylamide, but differences were observed for the ionic quenchers iodide and cesium. The fluorescence intensity decays of both cytokines were characterized by two (long and short) component lifetimes. However, the average lifetime of IL-1 beta (4.4 ns) was much longer than that of IL-1 alpha (1.93 ns). Taken together with the results of steady-state measurements, we suggest that the single tryptophan of IL-1 beta is statically quenched by neighboring charged residues, whereas the tryptophan fluorescence of IL-1 alpha is unaffected by ionic strength, and that the tryptophans of the two proteins have different accessibilities to ionic quenchers. The results are discussed in terms of similarities and differences in the tryptophan environments of the two proteins.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence quenching of the buried tryptophan residue of cod parvalbumin

Cod parvalbumin (isotype III) is a single tryptophan-containing protein. The fluorescence characteristics of this tryptophan residue (lambda em approximately 315 nm) suggest that it is buried from solvent and that it is located in an apolar core of the protein. Solute quenching studies of the tryptophan fluorescence of parvalbumin reveal dynamic quenching rate constants, kq, of 1.1 X 10(8) and 2.3 X 10(9) M-1 s-1 (at 25 degrees C) with acrylamide and oxygen, respectively, as quenchers. From temperature dependence studies, activation energies of 6.5 +/- 1.5 and 6.0 +/- 0.5 kcal/mol are found for acrylamide and oxygen quenching. The kq for acrylamide quenching is found to be relatively unchanged (+/- 10%) by an 8-fold increase in the bulk viscosity (glycerol/water mixture). These temperature and viscosity studies argue that the acrylamide quenching process involves a dynamic penetration of the quencher, facilitated by fluctuations in the protein's structure.     

Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays

We used 2-GHz frequency-domain fluorometry to examine the intensity decays of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease in the presence and absence of quenching by oxygen or acrylamide. When analyzed with a multiexponential model, the decays of NATA and nuclease both become more heterogeneous in the presence of quenching. We attribute the increased complexity to transient effects in quenching or equivalently a time-dependent rate constant for quenching. The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t)) and the radiation model, which is known to correct some flaws in the more approximate Smoluchowski model. The radiation model provides improved fits to the data, as evidenced by average 10-fold decreases in chi R2. The radiation model also provides an estimate of the sum of the diffusion coefficients and the specific rate constant for quenching. The apparent diffusion coefficients for acrylamide and oxygen in nuclease, as seen by its single tryptophan (residue 140) are 15- and 11-fold lower than in water, respectively. The apparent values of the oxygen diffusion coefficient in water, as seen by NATA, are 2- to 3-fold larger than expected from earlier steady-state measurements. The ability to recover the detailed form of the intensity decays by the frequency-domain method should allow comparison of experimental results with calculated trajectories of quenchers in proteins.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.Abbreviations CRP cyclic AMP receptor protein - NATA N-acetyltryptophanamide - FQRS fluorescence-quenching-resolved spectra - FDCD fluorescence-detected circular dichroism - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - FPLC fast protein liquid chromatography     

Fluorescence spectroscopic studies on tryptophan at the saccharide-binding site of castor bean hemagglutinin

The environment of tryptophan in castor bean hemagglutinin (CBH) was analyzed by fluorescence spectroscopy with regard to saccharide binding. Upon binding of specific saccharides, the fluorescence maximum of 333 nm of CBH shifted to a wavelength 2 nm shorter, owing to the change in the environment of tryptophan at the saccharide-binding site. By analyzing the change in the fluorescence intensity at 320 nm as a function of concentration of saccharides, the association constants for binding of saccharides to CBH were determined. The results suggest that the saccharide-binding site on each B-chain is actually composed of a subsite with which the saccharide residue linked to galactopyranoside at the non-reducing end can interact, and another site which recognizes the galactopyranoside moiety. Quenching data indicated that five out of 22 tryptophans in CBH are surface-localized and are available for quenching with both KI and acrylamide, and three other tryptophans are buried and are available only to acrylamide. Binding of raffinose to CBH decreased by 2 the number of tryptophan residues accessible to quenchers in the CBH molecule. We speculate that raffinose binds to CBH in such a manner as to shield the tryptophan located at the subsite from quenching by KI and acrylamide. The results also suggest that the tryptophan residue at the saccharide-binding site on each B-chain is localized near the surface, and present in the positively charged environment.     

Some regularities of dynamic accessibility of buried fluorescent residues to external quenchers in proteins

The temperature and viscosity dependences of quenching of buried tryptophan residues of several proteins by external ionic (iodide) and neutral (acrylamide) quenchers have been studied. The effective quenching rate constant is shown to be proportional to the diffusion coefficient of the free solvent (water or 20 vol% glycerol). This fact supports the idea that the accessibility of buried chromophores of proteins to quenchers is based on the dynamic perturbations in protein structure (the dynamic accessibility). These structural perturbations are assumed to be governed by some diffusion-limited processes in the solvent surrounding the protein molecule.     

Fluorescence quenching due to mercuric ion interaction with aromatic amino acids and proteins

Mercuric ion interacts with indoles, including tryptophan, to produce complexes whose absorption spectra are broader, less structured, and red-shifted as compared with those of the parent compound. Fluorescence and phosphorescence are totally quenched. In a survey of the effect of transition metal ions on tryptophan fluorescence, the strong quenching by Hg²⁺ was unique among the uncolored ions. Mercuric nitrate quenched the fluorescence of practically every protein tested, but the sensitivity to quenching varied with the protein. Ovalbumin was the most sensitive to quenching by Hg²⁺, over 70% of the intrinsic fluorescence being quenched by 2 moles of mercuric ion. Difference absorption spectra show that sulfhydryl groups are attacked by these reagents and Hg²⁺ is, in addition, perturbing the environment near some tryptophans. In contrast to Hg²⁺, Zn²⁺ had negligible effect on protein fluorescence. The emission spectra of proteins which were partly quenched by mercuric ion showed shifts in their maxima to higher or lower wavelengths. This suggests that mercuric ion quenched certain tryptophans more than others, and supports the idea that protein fluorescence is heterogeneous and arises from tryptophans in different microenvironments.