Origin of tryptophan fluorescence lifetimes part 1. Fluorescence lifetimes origin of tryptophan free in solution.

Fluorescence intensity decays of L-tryptophan free in polar, hydrophobic and mixture of polar-hydrophobic solvents were recorded along the emission spectrum (310-410 nm). Analysis of the data show that emission of tryptophan occurs with two lifetimes in 100% polar and hydrophobic environments. The values of the two lifetimes are not the same in both environments while their populations (pre-exponentials values) are identical. Fluorescence lifetimes and pre-exponentials values do not change with the excitation wavelength and thus are independent of excitation energy. Our results indicate that tryptophan emission occurs from two specific sub-structures existing in the excited state. These sub-structures differ from those present in the ground states and characterize an internal property and/or organization of the tryptophan structure in the excited state. By sub-substructure, we mean here tryptophan backbone and its electronic cloud. In ethanol, three fluorescence lifetimes were measured; two lifetimes are very close to those observed in water (0.4-0.5 ns and 2-4 ns). Presence of a third lifetime for tryptophan in ethanol results from the interaction of both hydrophobic and hydrophilic dipoles or chemical functions of ethanol with the fluorophore.

Origin of tryptophan fluorescence lifetimes. Part 2: fluorescence lifetimes origin of tryptophan in proteins.

Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310-410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.

Fluorescence origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) bound to proteins.

6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.

Fluorescence characterization of the hydrophobic pocket of cyclophilin B.

Human cyclophilin B is a monomeric protein that contains two tryptophan residues, Trp104 and 128. Trp128-residue belongs to the binding site of cyclosporin A and is the homologous of Trp 121 in CyPA, while Trp104 residue belongs to the hydrophobic pocket. In the present work, we studied the dynamics of Trp residue(s) of cyclophilin B and of the CyPB(w128A) mutant and of TNS-mutant complex. Our results showed that Trp-104 and TNS show restricted motions within their environments and that energy transfer between the two fluorophores is occurring.

New insights in the interpretation of tryptophan fluorescence : origin of the fluorescence lifetime and characterization of a new fluorescence parameter in proteins: the emission to excitation ratio.

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore-environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.

Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

Fluorescence fingerprints of Eisenia fetida and Eisenia andrei.

We describe a fluorescent method that allows to differentiate the worms Eisenia fetida and Eisenia andrei. In fact, the coelomic fluid of E. andrei displays specific fluorescence absent in that of E. fetida. The two species do not metabolize the same types of molecules and thus can be differentiated at the molecular level. Each species has specific fluorescence fingerprints.

The oxidative metabolism of polymorphonuclear neutrophils in pediatric liver graft recipients.

BACKGROUND: Data on the oxidative metabolism of polymorphonuclear neutrophils (PMN) after solid organ transplantation are very limited. We hypothesized that immunosuppressive agents reduce the capacity of PMN to produce reactive oxygen species, such as O2(-), H2O2, OH, and OCL(-) leading to an increased susceptibility to infectious complications after liver transplantation. METHODS: A lucigenin-enhanced chemiluminescence (CL) assay was used with soluble and particulate stimuli to study the oxidative metabolism of PMN in pediatric liver graft recipients. Sixteen patients (median age: 2.4 yr) were enrolled in a prospective study and integrated CL response was compared with the CL activity of 29 healthy controls. RESULTS: In the second week post-transplant, we found a significantly reduced CL activity. Pre-operatively, and after lowering steroids and cyclosporin A (CsA) the oxidative burst was normal. CONCLUSIONS: Our data suggest that CsA and steroids may not only influence T and B cells but also PMN, which may be a relevant factor for the incidence of infectious complications in pediatric liver graft recipients.

Urethral catheter removal 3 days after radical retropubic prostatectomy is feasible and desirable.

The purpose of this work was to assess the feasibility of urethral catheter removal 3 days after radical retropubic prostatectomy (RRP). Twenty-two patients who underwent RRP with a watertight eight-suture vesicourethral anastomosis had their urethral catheter removed usually on postoperative day (POD) 3. The average day of urethral catheter removal was POD 3.2. At 3 months, 56% of patients required no or one protective pad to stay dry and 68.4% of patients 'never leaked' or 'leaked occasionally'. Following RRP, the urethral catheter can be removed as early as POD 3 if the intraoperative anastomosis is watertight without compromising urinary continence.

Th2 cytokine profile in infants predisposes to improved graft acceptance after liver transplantation.

BACKGROUND: The T helper cell type 1 (Th1) cytokines interleukin (IL)-2 and interferon (IFN)-gamma are mediators of acute graft rejection after liver transplantation and Th2 cytokines, such as IL-4 and IL-10, may have a protective role and correlate with graft acceptance. To test the hypothesis that infants aged <1 year have an immunological advantage with regard to graft acceptance because of a partially immature immune system with a physiological balance toward a Th2 cytokine profile, we conducted the present study. METHODS: We compared the T helper serum cytokine profiles in 105 infants and children after liver transplantation with or without acute graft rejection and analyzed the normal age-distributed concentrations of T helper cytokines in 51 healthy controls. RESULTS: The incidence of acute graft rejection was as follows: 0 to 12 months, 26.8%; 1 to 3 years, 40.0%; and >3 years, 71.8%. There was a significantly lower incidence of acute rejection in infants 0 to 12 months of age compared with children >1 year (11/41 vs. 38/64; P=0.001). In healthy infants, significant increasing Th1 cytokine concentrations and decreasing Th2 cytokine concentrations were found with increasing age. Patients with acute rejection had significantly higher values of Th1 cytokines compared with nonrejecting subjects, who had significantly higher concentrations of Th2 cytokines. A longitudinal analysis of serum cytokines from patients showed that changes of the cytokine patterns in the follow-up did not differ significantly from preoperative values, except in the 4 weeks posttransplant. CONCLUSIONS: We conclude from the data that the physiological balance toward a Th2 cytokine profile of infants in the first months of life predisposes to improved graft acceptance. Transplantation of children with biliary atresia as early as possible, avoiding Th1 stimulation by recurrent infections and vaccinations, may have a positive impact on overall tolerance.

Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study.

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.

Prostate cancer gene therapy: comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B7-1 for in situ gene therapy and gene-modified, cell-based vaccines.

We have documented previously that adenovirus-mediated interleukin 12 (IL-12) gene therapy is effective for orthotopic tumor control and suppression of pre-established metastases in a preclinical prostate cancer model (Y. Nasu et al., Gene Ther., 6: 338-349, 1999). In this report, we directly compare the effectiveness of an adenovirus that expresses both IL-12 and the costimulatory molecule B7-1 (AdmIL12/B7) with one that expresses IL-12 alone (AdmIL-12) using the poorly immunogenic RM-9 orthotopic murine model of prostate cancer. We document AdmIL-12/B7-mediated secretion of IL-12 and increased surface expression of B7-1 in infected RM-9 tumor cells. A significant reduction in orthotopic tumor size and increased survival was demonstrated in mice treated with a single orthotopic injection of AdmIL-12/B7 compared with AdmIL-12 or controls. Six of 19 animals treated with AdmIL-12/B7 survived long term with apparent eradication of the primary tumor in contrast to one of 38 animals in the AdmIL-12-treated group. Orthotopic treatment of tumors with both vectors led to an infiltration of both CD4+ and CD8+ immunoreactive cells, with AdmIL-12/B7 treatment having a more prolonged infiltration of CD8+ cells. AdmIL-12/B7 was also more effective than AdmIL-12 or controls at suppression of pre-established metastases. We further developed a vaccine model based on s.c. injection of infected, irradiated RM-9 cells and found that both AdmIL-12 and AdmIL-12/B7 are effective at suppressing the development and growth of challenge orthotopic tumors using this protocol.

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.

Race is not an independent predictor of positive surgical margins after radical prostatectomy.

OBJECTIVES: To determine whether race is an independent predictor of positive surgical margins in patients who undergo radical prostatectomy. METHODS: Radical prostatectomies were performed on 750 patients at five Veterans Affairs hospitals: Shreveport, Louisiana (n = 451), Houston, Texas (n = 92), Jackson, Mississippi (n = 83), New Orleans, Louisiana (n = 69), and Little Rock, Arkansas (n = 55). All men who did not receive neoadjuvant hormonal therapy and for whom complete follow-up data were available were included in the analysis (of 607, 260 were African-American and 347 were white). Multiple logistic regression analysis was used to determine the significance of race as an independent predictor of surgical margin status after radical prostatectomy for clinically localized prostate cancer. RESULTS: After controlling for clinical stage, Gleason grade, and preoperative prostate-specific antigen (PSA), multivariable logistic regression analysis revealed that race was not an independent predictor of positive surgical margins (P = 0.9). Of the variables evaluated, both preoperative PSA (P = 0.0005) and biopsy Gleason grade (P = 0.047) were significant predictors of an increased risk of a positive surgical margin. CONCLUSIONS: Positive surgical margins are a widely accepted surrogate marker of increased biologic potential in patients with prostate cancer. In our study population, race was not an independent predictor of surgical margin status. Coupled with observations that survival is not related to race, this finding suggests that the biology of prostate cancer in African-American and white men is similar and that observed racial differences more likely are due to ethnic factors that influence tumorigenicity.

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.

Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White.

Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.

Dynamics of the Lens culinaris agglutinin-lactotransferrin and serotransferrin complexes, followed by fluorescence intensity quenching of fluorescein (FITC) with iodide and temperature.

Dynamics of the fluorescent Lens culinaris agglutinin-fluorescein complex (LCA-FITC) are studied in absence and in presence of two glycoproteins, lactotransferrin (LTF) and serotransferrin (STF). Glycans of the serotransferrin are not fucosylated, while those of the lactotransferrin have an alpha-1,6-fucose bound to the N-acetylglucosamine residue linked to the peptide chain, and an alpha-1,3-fucose bound to the N-acetyllactosamine residues. Interaction between the lectin and the two glycoproteins occurs via the carbohydrate residues. Affinity between LCA and LTF is 50 times higher than that between LCA and STF, as a result of the alpha-1, 6-fucose-LCA linkage. In the present work, we studied the effect of the tight bond between the alpha-1,6-fucose and LCA on the dynamics of the amino acids of the lectin, by fluorescence intensity quenching with iodide and by thermal intensity quenching. Fluorescence intensity quenching with iodide indicates that the bimolecular diffusion constant of iodide is 2.402+/-0.068x109 and 1. 160+/-0.090x109 M-1 s-1, when the interaction occurs with free fluorescein and with fluorescein bound to LCA, respectively. Binding of STF or LTF to the LCA-FITC complex yields a bimolecular diffusion constant of 1.675+/-0.06x109 and 1.155+/-0.087x109 M-1 s-1, respectively. Thermal intensity quenching does not occur for fluorescein free in solution while it is linear with temperature with a relative change of 0.656%, 0.889% and 0.488% for FITC-LCA, FITC-LCA-LTF and FITC-LCA-STF complexes, respectively. Fluorescence intensity quenching with iodide and thermal quenching experiments indicate that the dynamics of LCA increase as the result of the flexibility of the carbohydrate residues (case of STF-LCA complex), and the presence of the alpha-1,6-fucose inhibits the effect of the other carbohydrate residues as the result of the tight bond that exists between the fucose and the lectin (case of LTF-LCA complex).

Correlation between dynamics, structure and spectral properties of human alpha 1-acid glycoprotein (orosomucoid): a fluorescence approach.

Dynamics of proteins and membranes are usually investigated by red-edge excitation spectra and fluorescence anisotropy. In a viscous or rigid medium, the fluorescence maximum position changes with the excitation wavelength upon red-edge excitation. In addition to the shift in the emission maximum on red edge excitation, fluorescence anisotropy is also known to be dependent on the excitation and emission wavelengths in viscous media. However, this dependence has always been explained by the fact that the fluorophore is rigid, i.e. it does not display any residual motions. The aim of the present work was to check the validity of this latest assumption and to explain the possible origin of the dependence of the anisotropy on both the excitation and emission wavelengths. Therefore, we compared the results obtained from the fluorescence of the Trp residues of two alpha 1-acid glycoproteins (orosomucoid). One protein was purified by chromatographic methods (orosomucoid(c)) and the other was obtained with ammonium sulfate precipitation (orosomucoid(s)). Trp residues of orosomucoidc display free motions while those of orosomucoids are rigid. The general qualitative feature of the excitation anisotropy spectra recorded on both types of preparation is identical and resembles that obtained for other proteins containing tryptophan residue in protein. The fluorescence anisotropy measured across the emission spectra decreases for both preparations, indicating that this phenomenon is characteristic for fluorophores surrounded by a rigid microenvironment or by a microenvironment that displays motions. The fluorescence anisotropy variation across the emission and the excitation spectra is more important when the fluorophore possesses constrained motions than when it displays a high degree of freedom. Our results clearly demonstrate that the tertiary structure of the protein and the structure and dynamics of the microenvironments of the Trp residues are the origin of the dependence of anisotropy on the excitation and emission wavelengths.