An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

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.     

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.     

The dynamics of the relay loop tryptophan residue in the Dictyostelium myosin motor domain and the origin of spectroscopic signals

Steady-state and time-resolved fluorescence measurements were performed on a Dictyostelium discoideum myosin II motor domain construct retaining a single tryptophan residue at position 501, located on the relay loop. Other tryptophan residues were mutated to phenylalanine. The Trp-501 residue showed a large enhancement in fluorescence in the presence of ATP and a small quench in the presence of ADP as a result of perturbing both the ground and excited state processes. Fluorescence lifetime and quantum yield measurements indicated that at least three microstates of Trp-501 were present in all nucleotide states examined, and these could not be assigned to a particular gross conformation of the motor domain. Enhancement in emission intensity was associated with a reduction of the contribution from a statically quenched component and an increase in a component with a 5-ns lifetime, with little change in the contribution from a 1-ns lifetime component. Anisotropy measurements indicated that the Trp-501 side chain was relatively immobile in all nucleotide states, and the fluorescence was effectively depolarized by rotation of the whole motor domain with a correlation time on 50-70 ns. Overall these data suggest that the backbone of the relay loop remains structured throughout the myosin ATPase cycle but that the Trp-501 side chain experiences a different weighting in local environments provided by surrounding residues as the adjacent converter domain rolls around the relay loop.     

Influence of phosphate ion on the fluorescence of 3-fluorotyrosine

Summary 3-Fluorotyrosine fluorescence is quenched effectively by phosphate ions not only by a dynamic but also by a static mechanism owing to H-bond complex formation in ground state. 3-Fluorotyrosine pK_a values both in the ground and first excited state (8.3 and 4, respectively) are appreciably lower than those of tyrosine, thus promoting 3-fluorotyrosinate ion formation in the excited state. Additional emission owing to 3-fluorotyrosinate ion (near 350 nm) may be taken erroneously for tryptophan fluorescence.     

Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition

7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence of a nitrogen atom at position 7 in the indolyl nucleus of AW results in a red shift of the absorption maximum and fluorescence emission by 10 and 46 nm, respectively, compared to W. In the present work, we report the chemical synthesis and the conformational and functional characterization of an analog (denoted as Y3AW) of the N-terminal domain 1-47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW. The results obtained were compared with those of the corresponding Y3W analog. We found that the replacement W --> AW reduces affinity for thrombin by 10-fold, likely because of the lower hydrophobicity of AW compared with that of W. Measurements of the resonance energy transfer effect, which was observed between Tyr13 and the amino acid at position 3 upon disulfide-coupled folding, demonstrate that AW behaves as a better energy acceptor than W for studying protein renaturation. The interaction of Y3AW with thrombin was studied by exciting the sample at 320 nm and recording the change in fluorescence of Y3AW on binding to the enzyme. Our results indicate that the fluorescence of AW of hirudin 1-47 in the Y3AW-thrombin complex is strongly quenched, possibly because of the presence of two structural water molecules at the hirudin-thrombin interface that can promote the nonradiative decay of AW in the excited state. The data herein reported demonstrate that the incorporation of AW can be of broad applicability in the study of protein folding and protein-protein interaction.     

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.     

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase.

The fluorescence signal of the single tryptophan residue (Trp69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp69 in cutinase is caused by the breaking of the disulfide bond between Cys31 and Cys109 upon irradiation, while the amide-aromatic hydrogen bond between Ala32 and Trp69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.     

Fluorescence quenching in the DsbA protein from Escherichia coli: complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.     

Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.     

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.     

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.     

Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation

The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.     

Cytochrome c oxidase exhibits a rapid conformational change upon reduction of CuA: a tryptophan fluorescence study

When cytochrome c oxidase is reduced, it undergoes a conformational change that shifts its tryptophan fluorescence maximum from 329 to 345 nm. Studies of ligand-bound, mixed-valence forms of the enzyme show that this conformational change is dependent on the redox state of the low-potential metal centers, cytochrome a and CuA. The intrinsic fluorescence of oxidized cytochrome c oxidase is not effectively quenched by Cs+; however, marked quenching is observed for the reduced enzyme with a Stern-Volmer constant of 0.69. These observations, together with the significant red shift of the emission maximum, suggest that the emitting tryptophan residues are becoming more solvent accessible in the reduced enzyme. Stopped-flow spectra show that this conformational transition occurs rapidly upon reduction of the low-potential sites with a pseudo-first-order rate constant of 4.07 +/- 0.40 s-1. The conformational change monitored by tryptophan fluorescence is suggested to be related to the previously proposed "open-closed" transition of cytochrome c oxidase. Reductive titration of the cyanide-inhibited enzyme with ferrocytochrome c shows a nonlinear response of the fluorescence shift to added electron equivalents. A theoretical treatment of the reduction of the two interacting sites of the cyanide-inhibited enzyme has been developed that gives the population of each redox state as a function of the total number of electrons accepted by the enzyme. This treatment depends on two parameters: the difference in redox potential between the two metals and the redox interaction between the redox centers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen quenching of sensitized terbium luminescence in complexes of terbium with small organic ligands and proteins

Oxygen does not quench the luminescence of either free Tb or of Tb bound to dipicolinate. However, sensitized Tb luminescence in complexes of that ion with elastase, thermolysin, and alpha-amylase is quenched by oxygen at rates that far exceed that with which the intrinsic fluorescence of the proteins is quenched. We infer that this more rapid quenching of Tb luminescence indicates a major role for energy transfer from tryptophan moieties in a triplet excited state.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide-induced conformational change

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit. The fluorescence properties of the enzyme are strongly influenced by halide binding. To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine. All mutations, except for W238F, influenced the enzymatic properties. Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers. In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme. Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol. Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity. Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower. The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide. These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well. Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60 degrees C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol(-1) for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7 degrees C and the activation energy increases to 10.2 kcal mol(-1). This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60 degrees C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60 degrees C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.     

Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies.

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.