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.

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.

Motions of tryptophan residues in asialylated human alpha 1-acid glycoprotein.

The fluorescence of the tryptophan residues of asialylated human alpha 1-acid glycoprotein (orosomucoid) was investigated. Red excitation spectra did not lead to a shift of the fluorescence emission maximum of the fluorophore, i.e., motions of the Trp residues depend on their microenvironment. This result was confirmed by anisotropy studies as a function of temperature in the range of 7 to 35 degrees C (Perrin plot). In order to determine the frictional resistance to the local rotations of the tryptophan residues the protein was dissolved in a mixture of 80% glycerol buffer, and the fluorescence anisotropy was measured in the temperature range of -45 to + 20 degrees C. The Y-plot analysis of the anisotropy indicated that the mean motion of the two Trp residues buried in the protein core was different from that of the Trp residue of the surface. The average angles of rotations for buried and surface residues were 10 and 14 degrees C of arc, respectively.

Binding effect of progesterone on the dynamics of alpha1-acid glycoprotein.

The fluorescence of the tryptophan residues of asialylated human alpha1-acid glycoprotein (orosomucoid) was investigated in presence of progesterone. Red-edge excitation spectra did not lead to a shift of the fluorescence emission maximum of the fluorophore, i.e., motions of the Trp residues depend on their microenvironment. This was confirmed by anisotropy studies as a function of temperature in the range of 7-35 degrees C (Perrin plot). These two results identical to those obtained in absence of progesterone [J. Albani, Biochim. Biophys. Acta 1291 (1996) 215-220] indicate that binding of progesterone to orosomucoid does not modify the mean residual motion of the Trp residues. Measurement of the anisotropy in a temperature range of -45 degrees to +6 degrees C in a mixture of 80% glycerol-buffer, allows us to determine the frictional resistance to the local rotations of the tryptophan residues [G. Weber, S.F. Scarlata, M. Rholam, Biochemistry 23 (1984) 6785-6788]. The Y-plot analysis of the anisotropy reveals that the mean motion of the two Trp residues buried in the protein core was different from that of the Trp residue of the surface. The average angles of rotations for buried and surface residues were 16 degrees and 21.5 degrees of arc, respectively, instead of 10 degrees and 14 degrees of arc observed in absence of progesterone [J. Albani, Biochim. Biophys. Acta 1291 (1996) 215-220]. Thus, binding of progesterone to orosomucoid increases the free space of rotation of the two classes of Trp residues.

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).

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.

A fluorescence decay time study of tryptophan in isolated hemoglobin subunits.

The time-resolved fluorescence behavior of tryptophan residues in isolated human hemoglobin subunits was determined using a sync-pumped dye laser system and time-correlated single photon counting detection. Two decay components having values near 80 ps and 2 ns were found in the fluorescence decay of the alpha-subunit. The data for the beta-chains were best fitted with 3 decay components of 90 ps, 2.5 ns and 6.4 ns. We propose that the decay times correspond to conformations of the proteins in which the disposition of the tryptophan to the heme residue differs.

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.

Effects of sialic acids and the beta-drug adrenergic blocker, propranolol, on the dynamics of human alpha 1-acid glycoprotein: a fluorescence study.

The effect of propranolol on the dynamics of human alpha 1-acid glycoprotein (orosomucoid) (sialylated and asialylated) was studied. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS) bound to the protein was used as a probe. The results were identical for all samples. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino acid dipole has time to occur. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies. Sialic acid residues and propranolol do not affect the rigidity of the binding site of TNS.

Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.

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.

Interaction between cytochrome b2 core and flavodehydrogenase from the yeast Hansenula anomala.

The binding of cytochrome b2 core (a monomer) to flavodehydrogenase (a tetramer), both purified from Hansenula anomala flavocytochrome b2, has been studied in the presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS). The association constant of the TNS-flavodehydrogenase complex was found to be equal to 0.64 microM-1 with a stoichiometry of one TNS per tetramer. Binding of cytochrome b2 core to flavodehydrogenase was followed by monitoring changes in the TNS fluorescence. Our results indicated that the binding is cooperative, with a stoichiometry of four cytochrome b2 cores per tetramer of flavodehydrogenase.

Interaction between human alpha1-acid glycoprotein (orosomucoid) and 2-p-toluidinylnaphthalene-6-sulfonate.

The interaction between human alpha1-acid glycoprotein (orosomucoid) and the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) has been studied. An association constant of 16.7 (+/- 3) x 10(3) M-1 was obtained for the complex at 20 degrees C with a stoichiometry of 1:1. From the effect of temperature on the binding process, the standard enthalpy change for the binding is calculated to be delta H0 = -18 +/- 3 kJ mol-1 and the standard entropy change delta S0 = 19 +/- 12 J K-1 mol-1. The tryptophan fluorescence of the protein can be described by a sum of three exponentials. Upon TNS binding, the average fluorescence lifetime of the protein in the complex changes much less than the fluorescence intensity. The bound TNS is therefore a very efficient acceptor for the protein fluorescence. The TNS bound to orosomucoid present two fluorescence lifetimes 11 and 4.3 ns. The possible origins of the two lifetimes are discussed.

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.

A study of the interaction between two proteins, one containing a flavin mononucleotide.

Interaction between cytochrome c and flavocytochrome b2 has been studied in presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Affinity of the probe to flavocytochrome b2 increases when the complex between the two proteins is obtained. Binding of TNS increases the fluorescence of flavocytochrome b2 FMN. When the stoichiometry of the complex between the two proteins is reached, TNS looses its affinity and stops binding on the flavocytochrome b2; consequently, FMN fluorescence increase is no more observed. The dissociation constant of the complex was found equal to 0.1 microM. A similar result was obtained for the interaction between cytochrome c and flavodehydrogenase domain. The latter was obtained by proteolysis of flavocytochrome b2.

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.

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.

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.