Chemical Modification of Tryptophan Residues in Superoxide Dismutase from Camellia Pollen and Its Fluorescence Spectrum

The amino acid composition of the superoxide dismutase（SOD） from camellia pollen was measured and the tryptophan（Trp） residues were modified by using N-bromosuccinimide（NBS）. The results show that there are 21 Trp residues in an SOD molecule and seven of which are located on the surface of the enzyme. By researching the fluorescence spectra of the native SOD and the modified SOD, we have found that the emission wavelength of Trp is at 335 nm and the fluorescence intensity will decrease when the enzyme is modified. The results also show that potassium iodide（KI） can significantly quench the fluorescence of the native SOD, but it has a less pronounced effect on the modified enzyme. Glycerin as a surface activation reagent can stabilize the fluorescence of the modified enzyme.     

Energy migration in novel pH-triggered self-assembled beta-sheet ribbons

Energy migration between tryptophan residues has been experimentally demonstrated in self-assembled peptide tapes. Each peptide contains 11 amino acids with a Trp at position 6. The peptide self-assembly is pH-sensitive and forms amphiphilic tapes, which further stack in ribbons (double tapes) and fibrils in water depending on the concentration. Fluorescence spectra, quenching, and anisotropy experiments showed that when the pH is lowered from 9 to 2, the peptide self-assembly buries the tryptophan in a hydrophobic and restricted environment in the interior of stable ribbons as expected on the basis of the peptide design. These fluorescence data support directly and for the first time the presence of such ribbons which are characterized by a highly packed and stable hydrophobic interior. In common with Trp in many proteins, fluorescence lifetimes are nonexponential, but the average lifetime is shorter at low pH, possibly due to quenching with neighboring Phe residues. Unexpectedly, time-resolved fluorescence anisotropy does not change significantly with self-assembly when in water. In highly viscous sucrose-water mixtures, the anisotropy decay at low pH was largely unchanged compared to that in water, whereas at high pH, the anisotropy decay increased significantly. We concluded that depolarization at low pH was not due to rotational diffusion but mainly due to energy migration between adjacent tryptophan residues. This was supported by a master equation kinetic model of Trp-Trp energy migration, which showed that the simulated and experimental results are in good agreement, although on average only three Trp residues were visited before emission.     

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.     

Circular dichroism and fluorescence spectroscopy studies of the effect of cyclodextrins on the thermal stability of chicken egg white lysozyme in aqueous solution

Circular dichroism spectroscopy revealed that the thermal stability of chicken egg white lysozyme in an aqueous buffer solution is significantly lowered by the addition of 6-O-α-d-glucosyl-β-cyclodextrin (G1-β-CD), whereas it is raised by the addition of methyl α-d-glucopyranoside. The α- and γ-cyclodextrin also lowered the thermal stability, although the effects were less prominent than that of G1-β-CD. Fluorescence spectroscopy suggested that cyclodextrins include the side chains of tryptophan residues within their internal cavities to lower the thermal stability of lysozyme. The fluorescence intensity of a sample, re-cooled to 25 °C after thermal denaturation at 75 °C in the presence of G1-β-CD, was stronger than that observed for native lysozyme. The fact that the fluorescence intensity of the re-cooling sample was stronger than that of the native one indicates that G1-β-CD persists in binding to the side chains of tryptophan residues of the re-cooled lysozyme.     

Picosecond dynamics of a peptide from the acetylcholine receptor interacting with a neurotoxin probed by tailored tryptophan fluorescence

A tryptophan analog, dehydro-N-acetyl-L-tryptophanamide (delta-NATA), which is produced enzymatically via L-tryptophan 2',3'-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of delta-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide-water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of delta-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin alpha (dehydrotryptophan-toxin alpha) permitted its investigation in complex with a synthetic 15-amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide alpha-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15-amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp187 of the peptide has a fast motion (taur1 = 140 ps and r(0)1 = 0.14) relative to the overall rotation of the complex (taur2 = 3.4 ns and r(0)2 = 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding.     

Two-dimensional fluorescence correlation spectroscopy IV: resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.     

Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin

Fluorescence quenching and time-resolved fluorescence studies have been performed on the galactose-specific lectin purified from snake gourd (Trichosanthes anguina) seeds, in order to investigate the tryptophan accessibility and environment in the native protein and in the presence of bound ligand. Estimation of the tryptophan content by N-bromosuccinimide modification in the presence of 8 M urea yields four residues per dimeric molecule. The emission spectrum of native lectin in the absence as well as in the presence of 50 mM methyl--d-galatopyranoside (MeGal) shows a maximum around 331 nm, which shifts to 361.8 nm upon reduction of the disulfide bonds and denaturation with 8 M urea, indicating that all four tryptophan residues in the native state of this protein are in a hydrophobic environment. The extent of quenching that is observed is highest with acrylamide, intermediate with succinimide, and low with Cs⁺ and I⁻, further supporting the idea that the tryptophan residues are predominantly buried in the hydrophobic core of the protein. The presence of MeGal (50 mM) affects the quenching only marginally. Time-resolved fluorescence measurements yield bi-exponential decay curves with lifetimes of 1.45 and 4.99 ns in the absence of sugar, and 1.36 and 4.8 ns in its presence. These results suggest that the tryptophan residues are not directly involved in the saccharide binding activity of the T. anguina lectin. Of the four quenchers employed in this study, the cationic quencher, Cs⁺, is found to be a very sensitive probe for the tryptophan environment of this lectin and may be useful in investigating the environment of partially buried tryptophan residues and unfolding processes in other proteins as well.     

Sensitization of lanthanides by nonnatural amino acids

The sensitization of Eu(III) and Tb(III) by ethylenediaminetetraaceticacid (EDTA)-derivatized tryptophan (Trp), 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) has been examined. These Trp analogs were utilized in the present study because they can be incorporated into proteins in place of native Trp residues and because they absorb strongly beyond 305 nm (where Trp absorbance goes to zero), allowing selective excitation of such species in the presence of other Trp-containing proteins. All three indole derivatives were able to sensitize Tb(III) luminescence, with the relative sensitization being in the order Trp > 5HW > 7AW. On the other hand, only the 7AW-EDTA complex was able to sensitize Eu(III) luminescence, likely owing to a better spectral overlap between 7AW emission and Eu(III) absorbance. The sensitized emission of Tb(III) and Eu(II) displayed the expected long emission lifetimes at 545 nm [for Tb(III)] and 617 nm [for Eu(III)], indicating that long-lifetime lanthanide emission could be produced using nonnatural amino-acid donors. Thus, 7AW- and 5HW-sensitized lanthanide emissions should prove to be useful in biophysical studies, such as the use of fluorescence energy transfer to probe biomolecular interactions in vivo.     

聚半乳糖醛酸酶（PG2）中色氨酸残基的化学修饰

用化学修饰法以Ｎ－溴代琥珀酰亚胺作修饰剂，对聚半乳糖醛酸酶（ＰＧ２）中色氨酸（Ｔｒｐ）残基与酶活性的关系进行研究，发现１个ＰＧ２分子中有６个Ｔｒｐ残基，其中４个位于酶分子内部，２个位于酶分子表面，该发现通过表面荧光猝灭实验得到进一步证明，酶分子表面的２个Ｔｒｐ残基中，有１个为活性必需的氨基酸，它的修饰导致大部分酶活力丧失，底物保护实验进一步证明该活性必需的Ｔｒｐ可能位于酶的活性部位，酶被修饰后其圆     

Environment of tryptophan 57 in porcine fructose-1,6-bisphosphatase studied by time-resolved fluorescence and site-directed mutagenesis.

The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

Tryptophan Modification and Fluorescence Spectrum of Hyaluronidase

Tryptophan residues in hyaluronidase （HAase） were modified by N-bromosuccinimide （NBS）, the results indicated that there were eleven tryptophan residues in HAase, one of which was exposed and essential for the activity of the enzyme. The study on fluorescence quenching showed that KI could not quench all of the fluorescence from Trp residues in HAase, while acrylamide （Acr） could quench almost all of the fluorescence from Trp residues in HAase. The collisional quenching constants （KD） of HAase at different concentrations of Acr were calculated in terms of Stern-Volmer equation. The results implied that some of the Trp residues were buried in the interior of HAase and the Trp residue on the surface of HAase was not located in the hydrophobic pocket.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins

Residual structures in denatured proteins have acquired importance in recent years owing to their role as protein-folding initiation sites. Locating these structures in proteins has proved quite formidable, requiring techniques like NMR. Here in this report, we take advantage of the ubiquitous presence of tryptophan residues in residual structures to hunt for their presence using steady-state fluorescence spectroscopy. The surface accessibility and rotational dynamics of tryptophan in putative residual structures among ten different proteins, namely glucagon, melittin, subtilisin carlsberg, myelin basic protein, ribonuclease T₁, human serum albumin, barstar mutant, bovine serum albumin, lysozyme and Trp-Met-Asp-Phe-NH₂ peptide, was studied using steady state fluorescence quenching and anisotropy, respectively. Five proteins, namely ribonuclease T₁, bovine serum albumin, melittin, barstar and hen egg white lysozyme appear likely to possess tryptophan(s) in hydrophobic clusters based on their reduced bimolecular quenching rates and higher steady-state anisotropy in proportion to their chain length. We also show that the fluorescence emission maximum of tryptophan is insensitive to the presence of residual structures.     

透明质酸酶的色氨酸残基修饰与荧光光谱研究

北五味子[Schisandra chinensis（Turcz．）Baill．]属广义木兰科植物,主产于我国东北,故又称“辽五味”,中药五味子的主要药材为北五味子的干燥果实,作为一种传统中药,五味子具有收敛固涩,益气生津,补肾宁心的功效,用于肺喘虚咳,心悸失眠诸病。     

在不同激发波长下的单壁碳纳米管的拉曼光谱研究

采用直流电弧法制备单壁碳纳米管样品,用457.5和632.8nm两种不同的激发光分别测得单壁碳纳米管的正常拉曼光谱和共振拉曼光谱.通过理论分析得到了单壁碳纳米管的直径分布,进一步推测了其类型及结构参数;对单壁碳纳米管的正切拉伸模的成分进行了归属.在632.8nm激发波长下得到了IG/ID值随激光功率变化的曲线,认为在2.5mW时,单壁碳纳米管缺陷的结构可能发生了改变.在用457.5nm波长激发的单壁碳纳米管的拉曼光谱中,首次发现了1421cm-1的拉曼谱峰.     

Thermostability of glucose oxidase in silica gel obtained by sol-gel method and in solution studied by fluorimetric method

The thermostability of glucose oxidase entrapped in silica gel obtained by sol-gel method was studied by thermostimulated fluorescence of FAD at pH 5 and 7 and compared with that of the native enzyme in the solution and at the presence of ethanol. The unfolding temperatures were found to be lower for the enzyme immobilised in gel as compared with the native enzyme but higher as for the enzyme at the presence of ethanol. In gel, the thermal denaturation of glucose oxidase is independent on pH while in solution the enzyme is more stable at pH 5. The investigation the enzyme in different environment by steady-state fluorescence of FAD and tryptophan, synchronous fluorescence and time-resolved fluorescence of tryptophan indicates that the state of the molecule (tertiary structure and molecular dynamics) is different in gel and in solution. The ethanol produced during gel precursor hydrolysis is not the main factor influencing the thermostability of the enzyme but more important are interactions of the protein with the gel lattice.     

Differentiation of the local structure around tryptophan 51 and 64 in recombinant human erythropoietin by tryptophan phosphorescence

Abstract Recombinant human erythropoietin is a 4-helix bundle, glycosylated cytokine containing three tryptophan residues at positions 51, 64 and 88 whose phosphorescence emission may represent a sensitive probe of the structure at multiple sites near or at the protein surface. This report characterizes the phosphorescence properties (spectral energy, thermal spectral relaxation and phosphorescence lifetime), from low temperature glasses to ambient temperature, of the native protein plus that of three single point mutation analogs where each Trp was replaced by Phe. The structural information inferred from the phosphorescence parameters was essentially in good agreement with the structure of the Escherichia coli-produced nonglycosylated protein determined by nuclear magnetic resonance (Cheetham et al., Nat Struct Biol [1998] 5:861). The results showed that the fluorescence and phosphorescence spectra of the native protein were entirely due to independent contributions of Trp51 and Trp64 and that Trp88 was quenched under all conditions. The phosphorescence emissions of Trp51 and Trp64 were differentiated by their unique spectra at 77 K with Trp64 exhibiting an unusually blueshifted spectrum likely due to the attractive interaction of Arg110 and Lys116 with the ground state dipole of Trp64. In the native protein the room temperature phosphorescence lifetime of Trp64 was relatively short with a time of 1.62 ms whereas the lifetime of Trp51 was five-fold longer. Characterization of the single point mutation analogs showed that each lifetime was composed of multiple components revealing the presence of multiple stable conformations of the protein at these surface sites.     

Two-photon excitation of proteins

Fluorescence emission after two-photon excitation at 532 nm by means of a Nd : YAG laser is observed in apohemoglobin, hemoglobin, albumin and tryptophan at room temperature. The experimental results show that the fluorescence of these proteins originates from tryptophan residues. No fluorescence of a biphotonic nature could be detected from lysozyme and tyrosine.     

Conformational changes of lysozyme during photodynamic inactivation

Abstract— Egg white lysozyme was inactivated by photodynamic treatment in sodium phosphate buffer at pH8 using methylene blue, eosin Y and FMN as sensitizers. Measurements sensitive to changes in protein conformation, in particular, tryptophyl fluorescence and protease digestibility, were made during the course of inactivation. The rate of change of lysozyme tertiary structure as measured in these ways correlated closely with the rate of loss of enzyme activity during photodynamic treatment. Further, forms of lysozyme which were enzymatically active, but which were more sensitive to high temperature than native enzyme were produced by photodynamic treatment. It is concluded that the photodynamic inactivation of lysozyme under the conditions used results largely from the photooxidation of amino acid residues essential for the maintenance of the catalytically active conformation of the enzyme.