Energy transfer photophysics from serum albumins to sequestered 3-hydroxy-2-naphthoic acid, an excited state intramolecular proton-transfer probe

The steady-state and time-resolved studies of the sensitized emission of the excited-state proton transfer (ESIPT) probe 3-hydroxy-2-naphthoic acid (3HNA) when bound to bovine serum albumin (BSA) and human serum albumin (HSA) indicate that the nonradiative dipole-dipole F?rster type energy transfer from Trp singlet state of proteins to the ESIPT singlet state of 3HNA is greater in the case of HSA. This is supported by the distance and the orientation of the donor-acceptor pair obtained from the protein-ligand docking studies. The docking studies of the complex of BSA-3HNA also indicate that Trp 134 rather than Trp 213 is involved in the energy transfer process. The local environment of Trp 134 in BSA rather than that of Trp 213 is perturbed because of interaction with 3HNA as revealed by the optical resolution of Trp 134 phosphorescence in the complex at 77 K. Docking studies support the larger rotational correlation time, thetac (approximately 50 ns), observed for Trp residue/residues in the complexes of HSA and BSA compared with that in the free proteins.     

Parametric models to compute tryptophan fluorescence wavelengths from classical protein simulations

Fluorescence spectroscopy is an important method to study protein conformational dynamics and solvation structures. Tryptophan (Trp) residues are the most important and practical intrinsic probes for protein fluorescence due to the variability of their fluorescence wavelengths: Trp residues emit in wavelengths ranging from 308 to 360 nm depending on the local molecular environment. Fluorescence involves electronic transitions, thus its computational modeling is a challenging task. We show that it is possible to predict the wavelength of emission of a Trp residue from classical molecular dynamics simulations by computing the solvent‐accessible surface area or the electrostatic interaction between the indole group and the rest of the system. Linear parametric models are obtained to predict the maximum emission wavelengths with standard errors of the order 5 nm. In a set of 19 proteins with emission wavelengths ranging from 308 to 352 nm, the best model predicts the maximum wavelength of emission with a standard error of 4.89 nm and a quadratic Pearson correlation coefficient of 0.81. These models can be used for the interpretation of fluorescence spectra of proteins with multiple Trp residues, or for which local Trp environmental variability exists and can be probed by classical molecular dynamics simulations. © 2018 Wiley Periodicals, Inc.     

Fluorescence quenching induced by conformational fluctuations in unsolvated polypeptides

Time-resolved measurements were conducted to relate the fluorescence lifetimes of dye-derivatized polypeptides to local conformational dynamics in trapped, unsolvated peptide ions. This research was performed to better understand the intramolecular interactions leading to the observed increase of fluorescence quenching with temperature and, in particular, how this quenching is related to conformational fluctuations. Dye-derivatized polyproline ions, Dye-[Pro] n -Arg (+)-Trp, are formed by electrospray ionization and trapped in a variable-temperature quadrupole ion trap where they are exposed to a pulsed laser which excites fluorescence. Lifetime data exhibit fluorescence quenching as a result of an interaction between the dye and tryptophan (Trp) side chain. This result is consistent with solution measurements performed for comparison. The lifetime temperature dependence is closely fit over the range 150-463 K by an Arrhenius model of the ensemble averaged quenching rate, k q. Model fits of the measured lifetimes yield a frequency prefactor of approximately 10 (11) s (-1) for k q characteristic of collective motions of the side chains identified in molecular dynamics (MD) simulations. The data fits also yield activation barriers of approximately 0.3 eV, which are comparable to intramolecular electrostatic interactions calculated between the unshielded charge on the Arg residue and the dye. As a result, the quenching rate appears to be determined by the rate of conformational fluctuations and not by the rate of a specific quenching mechanism. The peptide sequence of Dye-Trp-[Pro] n -Arg (+) was also studied and identified a dependence of the quenching rate on the electrostatic field in the vicinity of the dye, Trp pair. Molecular dynamics simulations were performed over the range of experimental measurements to study trajectories relevant to the quenching interaction. The MD simulations indicate that as the temperature is increased, conformational fluctuations in the presence of strong electrostatic fields of the charged Arg (+) residue can result in both (a) an increased number of dye and Trp separations <8 A and (b) increased exothermicity for electron transfer reactions between the dye and Trp. Consequently, the MD simulations are consistent with increased fluorescence quenching with temperature resulting from the occurrence of conformers having specific positions of the dye, Trp, and Arg (+). As a result, the fluorescence lifetime provides a local probe of conformational fluctuations averaged over the ion ensemble.     

Solvent‐Dependent Fluorescence Lifetimes of Estrone, 17β‐Estradiol and 17α‐Ethinylestradiol

The fluorescence lifetimes of the estrogens, estrone, 17β‐estradiol and 17α‐ethinylestradiol, were studied in various solvents. The fluorescence lifetimes of 17β‐estradiol and 17α‐ethinylestradiol decreased from 4.7 to 0.9 ns as the solvent hydrogen bond accepting ability increased, in good agreement with other phenolic molecules. Estrone's two fluorescence bands had distinct lifetimes, with the 304 nm band having a lifetime shorter than 200 ps, reflecting efficient energy transfer to the carbonyl group, which had lifetimes ranging from 4.4 to 4.9 ns depending on the solvent. Solvent effects on the ¹ππ*, ¹πσ* and ¹nπ* states that are relevant to estrogen photophysics can adequately explain these trends. The solvent dependence on the excited states of these potent endocrine disruptors has significant implications for their photochemistry.     

Fluorescence Study of the Conformational Properties of Recombinant Tick Anticoagulant Peptide (Ornithodorus moubata) Using Multifrequency Phase Fluorometry

Abstract— Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide ( Ornithodorus moubata ) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pK_a of 6.01 ± 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the his-tidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52,1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N -bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trpll, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.     

A tryptophan rotamer located in a polar environment probes pH-dependent conformational changes in bovine beta-lactoglobulin A

Bovine beta-lactoglobulin A (BLGA) is a well characterized globular protein whose tertiary structure has been investigated in detail. BLGA undergoes a pH-dependent conformational change which X-ray data described as involving mostly the loop connecting strands E and F and the deprotonation of a glutamic acid residue (Glu89). These structural changes have been investigated using, among other techniques, fluorescence spectroscopy. The intrinsic fluorescence of BLGA is dominated by two Trp residues. These residues are located far from the EF loop and would not be expected to probe the pH-induced conformational change of the protein. Trp19 is located at the bottom of the interior beta-barrel, whereas Trp61 is located at the aperture of the barrel near the CD loop and is "silent" in the emission of native BLGA because of the proximity of a disulfide moiety. Our study suggests that, surprisingly, the fluorescence of Trp19 has the characteristic of a more polar environment than structural models from X-ray data would suggest and that at least two distinct conformations (or rotamers) of Trp19 contribute to the fluorescence of the protein. The less populated rotamer (relative amplitude (alpha) approximately 20%, tau approximately 3 ns) probes a more polar environment and a pH-dependent conformational change of BLGA in the region of Trp19 which X-ray data do not detect. Finally, our study provides the estimate of the fluorescence lifetime of Trp61 in the "unquenched" form.     

Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin

Bovine serum albumin (BSA) is a soft globular protein that undergoes conformational changes through several identified transition steps in the pH range 2–13.5. The ability to change conformation makes BSA capable of complexing different ligands from fatty acids to cations or drugs and carries them in the bloodstream. Microcalorimetric titration of BSA with NaOH solution was performed to measure the enthalpy of conformational changes. Two exothermic enthalpy changes were found in the course of the titration between pH 3 and 9.5, which can be identified with the E–F, and the F–N transitions. The enthalpy change at pH 3.5 (transition from the E to the F form of BSA, folding of intra-domain helices in domain I) is independent of the protein concentration. The second transition (F–N, folding of domain III) was observed at pH 4.8 for the 0.1% BSA solution, but it shifted to higher pH values as the protein concentration increased to 0.2% and 0.3%. The tightening of the protein structure with increasing pH was verified measuring intrinsic fluorescence of tryptophan residues. At even higher pH value, pH 10.5, fluorescence measurements revealed protein expansion. The BSA conformational changes were also measured by dynamic light scattering. The hydrodynamic diameter was smaller at the i.e.p. of BSA (5–7 nm at pH ~5) and larger at the two ends of the pH range (17.5 nm at pH 2 and 8.3 nm at pH 10).     

Interaction Study between ESIPT Fluorescent Lipophile-Based Benzazoles and BSA

In this study, the interactions of ESIPT fluorescent lipophile-based benzazoles with bovine serum albumin (BSA) were studied and their binding affinity was evaluated. In phosphate-buffered saline (PBS) solution these compounds produce absorption maxima in the UV region and a main fluorescence emission with a large Stokes shift in the blue–green regions due to a proton transfer process in the excited state. The interactions of the benzazoles with BSA were studied using UV-Vis absorption and steady-state fluorescence spectroscopy. The observed spectral quenching of BSA indicates that these compounds could bind to BSA through a strong binding affinity afforded by a static quenching mechanism (K_q~10¹² L·mol⁻¹·s⁻¹). The docking simulations indicate that compounds 13 and 16 bind closely to Trp134 in domain I, adopting similar binding poses and interactions. On the other hand, compounds 12, 14, 15, and 17 were bound between domains I and III and did not directly interact with Trp134.     

pH-Induced conformational changes of BSA in fluorescent AuNCs@BSA and its effects on NCs emission

We investigated the pH-induced fluorescence changes of BSA-protected gold nanoclusters, Au₁₆NCs@BSA, and the corresponding conformational changes of ligand protein by fluorescence, circular dichrosim (CD) and IR spectral measurements. The studies presented here demonstrated that BSA in AuNCs@BSA underwent identifiable conformational changes on both the secondary and the tertiary structure levels. The results of CD and IR interpreted the significant change of second structures at extreme acidity and alkaline, where more unordered structures were gained. Of note was that the extreme alkaline (pH = 11.43) induced the changes from exposed to buried α-helices, which was different from the pH-induced structural changes of BSA. In addition, the large fluorescence intensity gap of tryptophan between AuNCs@BSA and native BSA indicated efficient energy transfer took place between BSA and AuNCs, implying that the gold core resided near tryptophan in BSA.     

Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence

Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.     

Time-resolved fluorescence study on the photomerocyanine form of spiropyran and its derivative with azobenzene

The excited state dynamics of the photomerocyanine (PMC) form originating from spirobenzopyran and the bi-functional photochromic compound spirobenzopyran–azobenzene (SpAz), containing typical photochromic molecules of spirobenzopyran (Sp) and azobenzene (Az), were investigated using picosecond time-resolved fluorescence measurements in solution at 200 and 285 K and in a PMMA polymer film at 298 K. While the fluorescence lifetimes of PMC were about twice as long as those of SpAz under all experimental conditions, both lifetimes showed similar strong dependence on viscosity rather than temperature. These results suggest that non-radiative decay to an intermediate state could be accompanied by a significant conformational change. The effect of the Az moiety in this relaxation process is also discussed. It is unlikely that an energy transfer from the PMC moiety to the Az moiety occurs. It was concluded that the PMC moiety in the bi-functional SpAz is independent from the Az moiety.     

Aggregation of Poly(γ‐benzyl L‐glutamate)s Followed by Time‐Resolved Emission Anisotropy

Syntheses of poly(γ‐benzyl L ‐glutamate)s (PBLGs) labeled with various fluorophores (tryptophan, dansyl, and anthracene) having different molecular weights are reported. Association of PBLG chains was studied by time‐resolved emission anisotropy in the solvents supporting the aggregation process (1,4‐dioxane and tetrahydrofuran) and in N,N‐dimethylformamide, where the aggregates were not formed. The influence of molecular weight and polymer concentration on PBLG association was studied as well. The limiting emission anisotropy (r_∞) and rotational correlation times (ϕ) were determined. The chain relaxation dynamics were compared with the fluorescence lifetimes of the fluorophores and spectroscopically suitable labels were selected. Tryptophan was found to be an inconvenient fluorophore for the association study of PBLGs because of its short excited‐state lifetime. Dansyl and anthracene fluorophores, however, proved to be suitable labels for the chain dynamics study of PBLGs in solution. The mobilities of PBLG chains in 1,4‐dioxane were slower than those in tetrahydrofuran and N,N‐dimethylformamide because of PBLG association in this solvent.     

Relating Trp‐Glu dipeptide fluorescence to molecular conformation: The role of the discrete chi 1 and chi 2 angles

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp‐Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time‐resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp‐Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles—60°, 180°, and 300°—play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH₃ cation‐π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp‐Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence. © 2013 Wiley Periodicals, Inc.     

Fluorescence of cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid: a single tryptophan chi(1) rotamer model

A constrained derivative, cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid, cis-W3, was designed to test the rotamer model of tryptophan photophysics. The conformational constraint enforces a single chi(1) conformation, analogous to the chi(1) = 60 degrees rotamer of tryptophan. The side-chain torsion angles in the X-ray structure of cis-W3 were chi(1) = 58.5 degrees and chi(2) = -88.7 degrees. Molecular mechanics calculations suggested two chi(2) rotamers for cis-W3 in solution, -100 degrees and 80 degrees, analogous to the chi(2) = +/-90 degrees rotamers of tryptophan. The fluorescence decay of the cis-W3 zwitterion was biexponential with lifetimes of 3.1 and 0.3 ns at 25 degrees C. The relative amplitudes of the lifetime components match the chi(2) rotamer populations predicted by molecular mechanics. The longer lifetime represents the major chi(2) = -100 degrees rotamer. The shorter lifetime represents the minor chi(2) = 80 degrees rotamer having the ammonium group closer to C4 of the indole ring (labeled C5 in the cis-W3 X-ray structure). Intramolecular excited-state proton transfer occurs at indole C4 in the tryptophan zwitterion (Saito, I.; Sugiyama, H.; Yamamoto, A.; Muramatsu, S.; Matsuura,T. J. Am. Chem. Soc. 1984, 106, 4286-4287). Photochemical isotope exchange experiments showed that H-D exchange occurs exclusively at C5 in the cis-W3 zwitterion, consistent with the presence of the chi(2) = 80 degrees rotamer in solution. The rates of two nonradiative processes, excited-state proton and electron transfer, were measured for individual chi(2) rotamers. The excited-state proton-transfer rate was determined from H-D exchange and fluorescence lifetime data. The excited-state electron-transfer rate was determined from the temperature dependence of the fluorescence lifetime. The major quenching process in the -100 degrees rotamer is electron transfer from the excited indole to carboxylate. Electron transfer also occurs in the 80 degrees rotamer, but the major quenching process is intramolecular proton transfer. Both quenching processes are suppressed by deprotonation of the amino group. The results for cis-W3 provide compelling evidence that the complex fluorescence decay of the tryptophan zwitterion originates in ground-state heterogeneity with the different lifetimes primarily reflecting different intramolecular excited-state proton- and electron-transfer rates in various rotamers.     

Structural Transformation of Bovine Serum Albumin Induced by Dimethyl Sulfoxide and Probed by Fluorescence Correlation Spectroscopy and Additional Methods

Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous–organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)‐labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40 % DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self‐quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 μs in its native state to 50 μs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near‐UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1‐anilinonaphthalene‐8‐sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40 % DMSO in the ensemble measurements.     

Interaction of a Rhodococcus sp. trehalose lipid biosurfactant with model proteins: thermodynamic and structural changes

One major application of surfactants is to prevent aggregation during various processes of protein manipulation. In this work, a bacterial trehalose lipid (TL) with biosurfactant activity, secreted by Rhodococcus sp., has been identified and purified. The interactions of this glycolipid with selected model proteins have been studied by using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) have been chosen because of their quite different secondary structures: BSA contains essentially no β-sheets and an average 66% α-helix, whereas Cyt-c possesses up to 25% β-sheets and up to 45% α-helical structure. Differential scanning calorimetry shows that addition of TL to BSA at concentrations below the critical micelle concentration (cmc) shifts the thermal unfolding temperature to higher values. FTIR indicates that TL does not alter the secondary structure of native BSA, but the presence of TL protects the protein toward thermal denaturation, mainly by avoiding formation of β-aggregates. Studies on the intrinsic Trp fluorescence of BSA show that addition of TL to the native protein results in conformational changes. BSA unfolding upon thermal denaturation in the absence of TL makes the Trp residues less accessible to the quencher, as shown by a decrease in the value of Stern-Volmer dynamic quenching constant, whereas denaturation in the presence of the biosurfactant prevents unfolding, in agreement with FTIR results. In the case of Cyt-c, interaction with TL gives rise to a new thermal denaturation transition, as observed by DSC, at temperatures below that of the native protein, therefore facilitating thermal unfolding. Binding of TL to native BSA and Cyt-c, as determined by ITC, suggests a rather nonspecific interaction of the biosurfactant with both proteins. FTIR indicates that TL slightly modifies the secondary structure of native Cyt-c, but protein denaturation in the presence of TL results in a higher proportion of β-aggregates than in its absence (20% vs 3.9%). The study of Trp fluorescence upon TL addition to Cyt-c results in a completely opposite scenario to that described above for BSA. In this case, addition of TL considerably increases the value of the dynamic quenching constant, both in native and denatured protein; that is, the interaction with the glycolipid induces conformational changes which facilitate the exposure of Trp residues to the quencher. Considering the structures of both proteins, it could be derived that the characteristics of TL interactions, either promoting or avoiding thermal unfolding, are highly dependent on the protein secondary structure. Our results also suggest the rather unspecific nature of these interactions. These might well involve protein hydrophobic domains which, being buried into the protein native structures, become exposed upon thermal unfolding.     

Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions

Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.     

Temperature dependence of photoinduced electron transfer within self-associated porphyrin: guanine monophosphate complexes

In the current report, the temperature dependence of photoinduced electron transfer between tetrakis-(4-tetramethylpyridyl)porphine (T4MPyP) and guanine monophosphate (GMP) has been examined. In the presence of GMP the fluorescence lifetime analysis reveals a Lorentzian distribution of lifetimes centered at 0.7 ns with a width of 0.9 ns displaying significant temperature dependence. Fitting temperature dependent data to the Marcus equation gives a reorganizational energy (λ) for the electron transfer reaction of 0.6 eV and an electronic coupling factor (H_AB) of 3×10⁻³ eV. These results suggest conformational regulation of electron transfer within the non-covalent porphyrin:nucleotide complex.     

Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides

Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide c[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D- and 2D-(1)H NMR techniques. The peptide backbone has a single predominant conformation. The tryptophan side chain has three chi(1) rotamers: a major chi(1) = -60 degrees rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the chi(1) rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the chi(1) = -60 degrees rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to peptide bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8- and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the chi(1) = 180 degrees rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the epsilon-ammonium does not occur in these peptides.     

New diode laser-excitable green fluorescent label and its application to detection of bovine serum albumin via microchip electrophoresis

A novel amino-reactive fluorescent label is presented that is based on a yellow daylight chromophore and fluorophore. Its absorption band is wide and peaks at 431 nm in water solution, thus well matching the lines of either the 375-nm and the 431-nm diode lasers and of many frequency-variable dye lasers. When conjugated to bovine serum albumin (BSA), the fluorescence peaks at 501 nm with a quantum yield of 0.21. Its large Stokes' shift of 70 nm facilitates the discrimination of undesired excitation light which is particularly important for sensitive detection in miniaturized separation techniques such as microchip capillary electrophoresis (MCE). Unlike several other fluorophores, the fluorescence intensity of the new label is independent of pH over a broad range (3 to 9). The applicability of the label is demonstrated by labeling the amino acid lysine and the 66 kD protein BSA, and by separating BSA from the free label via MCE within 90 s. The limit of detection is in the order of 12 nM at an optically active path length of 20 µm.