How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.     

Structure and dynamics at N- and C-terminal regions of intrinsically disordered human c-Myc PEST degron reveal a pH-induced transition

We investigated the structure and Brownian rotational motion of the PEST region (201-268) from human c-Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77-residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time-resolved fluorescence spectroscopy by labeling Cys⁹ near N-terminal with dansyl probe and inserting a Trp⁷⁰ near C-terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N-terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03-0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C-terminal also influenced the dynamics of dansyl probe near N-terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C-terminal region of PEST is implicated in this disorder-order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.     

Oxygen-17 and deuterium nuclear magnetic relaxation studies of lysozyme hydration in solution: field dispersion, concentration, pH/pD, and protein activity dependences

A comparison of 17O and 2H NMR relaxation rates of water in lysozyme solutions as a function of concentration, pH/pD, and magnetic field suggests that only 17O monitors directly the hydration of lysozyme in solution. NMR measurements are for the first time extended to 11.75 T. Lysozyme hydration data are analyzed in terms of an anisotropic, dual-motion model with fast exchange of water between the "bound" and "free" states. The analysis yields 180 mol "bound" water/mol lysozyme and two correlation times of 7.4 ns ("slow") and 29 ps ("fast") for the bound water population at 27 degrees C and pH 5.1, in the absence of salt, assuming anisotropic motions of water with an order parameter value for bound water of 0.12. Under these conditions, the value of the slow correlation time of bound water (7.4 ns) is consistent with the value of 8 ns obtained by frequency-domain fluorescence techniques for the correlation time associated with the lysozyme tumbling motion in solutions without salt. In the presence of 0.1 M NaCl the hydration number increases to 290 mol/mol lysozyme at pD 4.5 and 21 degrees C. The associated correlation times at 21 degrees C in the presence of 0.1 M NaCl are 4.7 ns and 15.5 ps, respectively. The value of the slow correlation time of 4.7 ns is consistent with the calculated value (4.9 ns) for the lysozyme monomer tumbling in solution. The systematic deviations of the relaxation rates, estimated with the single-exponential approximation, from the theoretical, multiexponential nuclear (I' + 1/2) spin relaxation are evaluated at various frequencies for 17O (I = 5/2) with the first-order, linear approximation (25). All NMR relaxation data for hydrated lysozymes are affected by protein activity and are sensitive both to the ionization of protein side chains and to the state of protein aggregation.     

Characterisation by triple-quantum filtered 17O-NMR of water molecules buried in lysozyme and trapped in a lysozyme-inhibitor complex.

Triple-quantum filtering NMR sequences were used to study the multiexponential relaxation behaviour of H2 17O in the presence of hen egg white lysozyme. By this means, the fraction and the correlation time of water were determined in slow motion, as well as the relaxation time of water in the extreme narrowing limit. The small number of water molecules in slow motion, which is between four and five per lysozyme, seems to correspond to the 'integral' water, buried or in the cleft inside the protein, whereas water in fast motion corresponds to all other water molecules, interacting or not with the macromolecules. The same experiment was performed after addition of the inhibitor tri-N-acetylglucosamine (NAG)3. For solutions of sufficient viscosity, there were approximately three supplementary water molecules in slow motion per lysozyme, probably trapped between the protein and the inhibitor. The correlation time of these water molecules was estimated at 2 ns, which should correspond to their residence time in the complex.     

Effect of lysozyme on mycobacteria

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.     

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo.     

Prevention of thermal inactivation and aggregation of lysozyme by polyamines.

Proteins tend to form inactive aggregates at high temperatures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 microM), more than 30% of 0.2 mg x mL(-1) lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggregates by heat treatment (98 degrees C for 30 min). However, in the presence of 50 mm spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 microM arginine and guanidine, while it was maintained at approximately 50% in the presence of 100 microM spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins.     

Time dependence of aggregation in crystallizing lysozyme solutions probed using NMR self-diffusion measurements

The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals.     

HEW lysozyme salting by high-concentration NaCl solutions followed by titration calorimetry

Concentration dependence of NaCl salting of 0-1.5 mM lysozyme solution in 0.1 M sodium acetate buffer, pH 4.25, was investigated for NaCl concentration varying up to 0.9 M. Calorimetric experiments demonstrated that depending on the salt concentration the estimated number of the binding sites on the lysozyme surface varied in the range of 5 up to 13, and the increase of salt concentration caused the decrease of the number of accessible sites. The small, but significant, local maximum centered at 0.63 M NaCl concentration indicated the specific salting-out of the lysozyme accompanied by binding of approximately 2-3 chloride anions. Generalized McMillan and Mayer's approach reduced to the third-order virial coefficients demonstrates the domination of lysozyme aggregation upon salt addition (a(21)-h(xxy)) and salt organization on the lysozyme surface (a(12)-h(xyy)) processes.     

The temperature and pH-dependent transition of hen lysozyme. Characterization of two temperature-defined domains and of an N-acetylglucosamine (inhibitor)-insensitive form

The previously described temperature and pH-dependent transition in the solid state of hen lysozyme was studied in solution. Experiment concerning the velocity of lysis ofM. luteus by lysozyme and its behavior in presence of an inhibitor (GlcNAc) as well as a reinvestigation of the Arrhenius curves over a large range of pH, demonstrated the existence of two temperature-induced domains. An inhibitor-insensitive lysozyme form was characterized at 40° (physiological temperature).112th communication on lysozymes. Inquiries should be addressed to P. Jollès.     

Evidence for a novel growth factor in Xenopus oocytes

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.     

Effect of salt and membrane fluidity on fluorophore motions of a gramicidin C derivative.

We have used fluorescence spectroscopy to investigate the effect of salt and membrane fluidity on the rotational motion of a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) derivative of gramicidin C (dansyl-gC) in dimyristoylphosphatidylcholine bilayers, under conditions where the peptide is a formyl-NH to formyl-NH terminal dimer, and in octyl glucoside micelles, where the peptide is an intertwined helical dimer. Energy-transfer experiments showed no changes in either conformation or dimer aggregation under the conditions explored here (15-40 degrees C, 1-350 bar, 0-0.33 M Mg2+, and 0-1 M Na+). The addition of permeable (Na+) or nonpermeable (Mg2+) ions did not affect the temperature or pressure behavior of dansyl rotation. However, fluorescence lifetime measurements indicated an increase in solvent accessibility in the presence of sodium. In bilayers, the temperature dependence of the fluorescence polarization and lifetime shows strong interactions between the dansyl residue and the peptide, and at no time did the dansyl motions become solvent controlled as has been observed for aqueous solvent peptides [Scarlata, S. F., Rholam, M., & Weber, G. (1984) Biochemistry 23, 6789]. In micelles, the change in rotational motion with temperature followed solvent expansion, showing that in this case the dansyl residue does not associate extensively with the peptide. Our results indicate that because of the extensive coupling between the dansyl residue and the rest of the peptide, membrane fluidity does not play a major role in controlling side-chain motions.     

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.     

Role of melanin in fungal biosorption of tributyltin chloride

Summary Intact biomass of an albino and a melanic strain of Aureobacidium pullulans, as well as purified melanin from the latter strain, was capable of tributyltin chloride (TBTC) removal from solution. Melanized biomass had a greater biosorptive capacity than albino biomass, this difference being attributable to the presence of melanin. Purified melanin had a large capacity for TBTC biosorption, the calculated maximum uptake capacity, q _e, being approximately 35 mmol (g dry wt)^–1. TBTC biosorption by intact biomass and melanin obeyed the Langmuir adsorption isotherm over the concentration range used, and was relatively unaffected by external pH between pH 3.5 and 6.5: an approximate 20% decrease in TBTC biosorption resulted at external pH 2.5. A TBTC concentration of 0.3 M in growth medium resulted in a lag period which was longer with the albino strain (approximately 50 h) than with the pigmented strain (approximately 25 h). The addition of melanin to TBTC-containing growth media resulted in a reduction in toxicity and attainment of higher cell yields. The applied and environmental significance of these interactions are discussed. Offprint requests to: G. M. Gadd     

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Sorbitol prevents the self-aggregation of unfolded lysozyme leading to and up to 13 degrees C stabilisation of the folded form

We present a calorimetric investigation of stabilisation of hen egg-white lysozyme with sorbitol in the pH range 3.8-10.5. Differential scanning calorimetry and steady-state fluorescence were used to determine the denaturation temperatures of lysozyme as a function of sorbitol concentration. The fluorescence data were collected in the presence of 2M urea to lower the melting point of the protein to an observable range of the instrument. The effect of sorbitol on the activation energy of unfolding was investigated by scanrate studies. The effect of sorbitol lysozyme interaction was investigated using isothermal titration calorimetry. The titration experiments were performed with folded as well as unfolded lysozyme to investigate in more detail the nature of the interaction. The data obtained in those experiments show a remarkable stabilisation effect of sorbitol. We observed a 4.0 degrees C increase in the Tm for 1 M sorbitol in the pH range 3.8-8.5 by scanning calorimetry. The effect increases dramatically at pH 9.5 where we observe a 9.5 degrees C stabilisation. An increase in the sorbitol concentration to 2 M stabilises lysozyme by 11.3-13.4 degrees C in the pH range 9.5-10.5. In the absence of urea, no significant effects of sorbitol were observed on the activation energy for unfolding for lysozyme at pH 4.5. This indicates together with the results from the titration experiments that sorbitol may stabilise the folded form of lysozyme by destabilising the unfolded form of lysozyme. At pH values at and above lysozyme's pI (approximately 9.3), the unfolding of the protein is accompanied with a substantial amount of self-aggregation seen in the calorimetry experiments in the ratio of DeltaH(cal)/DeltaH(vH). In the presence of sorbitol, the self-aggregation was counterbalanced by higher sorbitol concentrations. These results strongly suggest a negative influence of sorbitol on the unfolded form of lysozyme and thereby stabilising the native form.     

Daily and Seasonal Variation in the Spectral Composition of Light Exposure in Humans

Light is considered the most potent synchronizer of the human circadian system and exerts many other non-image-forming effects, including those that affect brain function. These effects are mediated in part by intrinsically photosensitive retinal ganglion cells that express the photopigment melanopsin. The spectral sensitivity of melanopsin is greatest for blue light at approximately 480 nm. At present, there is little information on how the spectral composition of light to which people are exposed varies over the 24 h period and across seasons. Twenty-two subjects, aged 22±4 yrs (mean±SD) participated during the winter months (November–February), and 12 subjects aged 25±3 yrs participated during the summer months (April–August). Subjects wore Actiwatch-RGB monitors, as well as Actiwatch-L monitors, for seven consecutive days while living in England. These monitors measured activity and light exposure in the red, green, and blue spectral regions, in addition to broad-spectrum white light, with a 2 min resolution. Light exposure during the day was analyzed for the interval between 09:00 and 21:00 h. The time course of white-light exposure differed significantly between seasons (p?=?0.0022), with light exposure increasing in the morning hours and declining in the afternoon hours, and with a more prominent decline in the winter. Overall light exposure was significantly higher in summer than winter (p?=?0.0002). Seasonal differences in the relative contribution of blue-light exposure to overall light exposure were also observed (p?=?0.0006), in particular during the evening hours. During the summer evenings (17:00–21:00 h), the relative contribution of blue light was significantly higher (p?<?0.0001) (40.2±1.1%) than during winter evenings (26.6±0.9%). The present data show that in addition to overall light exposure, the spectral composition of light exposure varies over the day and with season.     

Tetrodotoxin Desensitization in Aggregates of Embryonic Chick Heart Cells

Spontaneous beating of heart-cell aggregates from 4-day chick embryos was initially blocked by 10^-5 g/ml tetrodotoxin (TTX). With continued exposure to the drug, the fraction of blocked aggregates decreased from about 80% at 15 min to about 25% at 2–3 h, at which time, beating aggregates had become desensitized to the toxin, showing no response to a fresh dose. Aggregates from 5-day hearts were more sensitive to TTX, but fewer became desensitized in its presence. Desensitization to TTX was not seen in 6- and 7-day aggregates. Inhibition of protein synthesis by cycloheximide did not affect beating or initial sensitivity to TTX of 4-day aggregates, but desensitization failed to occur. Before TTX, the mean value of maximal upstroke velocity (V_max) of the action potentials in 4-day aggregates was 33 V/s. After desensitization V_max was 12 V/s. Activity of desensitized aggregates in the presence of TTX was augmented by elevated calcium levels, and suppressed by presumed inhibitors of slow inward current (manganese, D600). Desensitization was reversible; upon removal of TTX 10^-5 g/ml, aggregates regained their responsiveness to a fresh dose of the drug with a 2–3 h time-course similar to that of desensitization. This was prevented by continued exposure to TTX at concentrations as low as 10^-8 g/ml. These data suggest that (a) desensitization involves a change in the mode of action-potential generating from one involving Na-specific, TTX-sensitive channels to one utilizing slower Mn-sensitive channels; (b) the process of desensitization occurs over a period of 2–3 h and is dependent upon the products of protein synthesis; and (c) desensitization is reversible after removal of TTX over a 2–3 h time-course similar to its onset.     

Tryptophan fluorescence lifetimes in lysozyme.

Tryptophan fluorescence lifetimes at pH 2 and pH 8 have been obtained for lysozyme and for lysozyme derivatives in which tryptophan-62 or tryptophan-108 or both are nonfluorescent. The lifetimes range from about 0.5 ns to 2.8 ns for the various emitting tryptophans. The tryptophan lifetimes appear to increase with exposure of tryptophan to solvent, but intramolecular contacts, probably with cystine residues, can considerably shorten the lifetime. Intertryptophanyl interactions can also affect fluorescence lifetimes. The trytophan-108 lifetime in lysozyme is shorter than in the derivative in which tryptophan-62 is oxidized; this is ascribed to energy transfer from tryptophan-108 to tryptophan-62. From the lifetime results the relative intensities emitted by specific tryptophans can be estimated, and these values also support the existence of intertryptophanyl energy transfer. The emission intensity from tryptophan-62 is greater in the presence of tryptophan-108, and the emission intensity of tryptophan-108 appears to be greater in the absence of tryptophan-62. Conformational effects accompanying chemical modification of tryptophan cannot be completely ruled out, however. The tryptophan-62 lifetime at pH 8 in lysozyme is shorter than in the derivatives, which might indicate a subtle conformational effect. Studies with tri-(N-acetyl-glucosamine)-protein complexes indicate that both the tryptophan lifetimes and the number of emitting tryptophans may be changing upon complexation. The results illustrate the usefulness and the limitations of lifetime measurements in understanding protein fluorescence.