Camillo A. Ghiron

A Hydrophobic Quencher of Protein Fluorescence: 2,2,2-Trichloroethanol

Previously the neutral fluorescence quenching probe, acrylamide, was employed to determine the degree of exposure of tryptophan residues in proteins. A less polar neutral quencher 2,2,2-trichloroethanol (trichloroethanol) was used in the present work to investigate whether it would preferentially interact with apolar regions of proteins. For most proteins studied, the degree of quenching by trichloroethanol is found to be about the same as with acrylamide. However, for human and bovine serum albumin hydrophobic interactions between trichloroethanol and these proteins occur, leading to an exalted quenching. The fluorescence quencher thus senses the presence of a hydrophobic domain in the vicinity of the tryptophan residues in these proteins. Trichloroethanol is shown to induce conformational changes in certain proteins and to be a potentially useful quencher for proteins having predominantly tyrosine emission.

Photosensitization of aqueous model systems by hypericin

Absorption and fluorescence measurements of purified hypericin (HY) were made in various media. Photosensitization of two aqueous systems was investigated: resealed red blood cell membranes (ghosts) and hen lysozyme (Lys). Solubilization of HY by ghost membranes was shown by means of diffuse reflectance spectroscopy. Visible light irradiation of the ghosts incorporating HY led to lipid peroxidation with evidence of singlet oxygen involvement. A binding model applicable for insoluble ligands is indicative of strong HY binding to HSA. The HY-HSA complex photosensitized inactivation of Lys. The pseudo-first-order reaction kinetics with protection by azide ion are consistent with a Type II mechanism mediated by singlet oxygen. The results are discussed in the context of the HY photodynamic and antiretroviral activities.

Does the fluorescence quencher acrylamide bind to proteins

We have studied the protein concentration dependence of the acrylamide quenching of the fluorescence of the proteins, human serum albumin and monellin, and we have found no such dependence for the concentration range of 0.5-20 mg/ml. These quenching studies were performed by fluorescence lifetime measurements using phase/modulation fluorometry. We have also performed equilibrium dialysis studies, which show no large degree of association of acrylamide with serum albumin, and we have found that acrylamide has only a small effect on the activity of selected enzymes. These various studies do not indicate the existence of strong acrylamide-protein interactions and are in discord with a recent report by Blatt et al. in this journal (Blatt, E., Husain, A. and Sawyer, W.H. (1986) Biochim. Biophys. Acta 871, 6-13).

FLASH PHOTOLYSIS OF TRYPTOPHAN AND N‐ACETYL‐L‐TRYPTOPHANAMIDE; THE EFFECT OF BROMIDE ON TRANSIENT YIELDS

Abstract— The initial yields of the cation and neutral radicals and the triplet are greatly enhanced when high concentrations of Br‐ are present during the flash photolysis of aqueous solutions containing either tryptophan or N‐acetyl‐L‐trytophanamide. The present study is an attempt to elucidate the mechanism by which Br‐ induces these effects. The results obtained indicate that the initial event involves an interaction between the fluorescent state and Br‐ to promote the formation of a long‐lived radical precursor that may be the triplet. It is shown that all of the Br‐‐induced neutral and cation radical formation originate from this long‐lived state. Furthermore, it was found that the mechanism of radical production from the Br‐‐induced long‐lived precursor does not involve hydrated electron formation.

Luminescence studies with trp repressor and its single-tryptophan mutants

Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli. Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F. Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed. Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein. Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom. Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively. ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue. The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

Flash photolysis of N-acetyl-L-tryptophanamide; acid-base equilibrium of the radical transients

Abstract The transient with a λ max at 570 m observed in the flash photolysis of N-acetyl-L-tryptophanamide is confirmed to be the positive radical. This species is in rapid equilibrium with its deprotonated form (λ max =510 mm) with a pK of 4.3.

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.

THE DESTRUCTION OF TRYPTOPHANYL RESIDUES IN TRYPSIN BY 280‐nm RADIATION

Abstract— The destruction of tryptophanyl residues in trypsin by 280‐nm radiation was studied in relation to enzyme inactivation. Quantum yields for destruction of this residue (determined using the pDAB reagent) and for the inactivation of trypsin were measured when the enzyme was exposed to different environmental perturbations. The conformational modifications of trypsin induced in 6 M guanidine‐HCl did not alter the rates of tryptophan destruction and enzyme inactivation. However, an enhanced destruction of the tryptophanyl residues was observed when trypsin solutions were irradiated at 60°C in the presence of air. The increased rate of tryptophan destruction at this temperature was not accompanied by a corresponding increase in the inactivation quantum yield. It was concluded that any photochemically induced reactions of this chromophore that are sensitive to conformational modifications or that result in the destruction of the indole ring are not important in the inactivation mechanism of trypsin.

FLASH PHOTOLYSIS OF N-ACETYL-L-TRYPTOPHANAMIDE: ANALYSIS OF TRANSIENTS EMPLOYING VIDICON TUBE DETECTION*

Abstract— An improved method for recording transient absorption spectra obtained by flash photolysis is described. The detection system, comprising a vidicon tube coupled to a multichannel analyzer, is shown to permit the rapid acquisition of accurate and precise spectra spanning any 360 nm interval within the 200 to 1100 nm wavelength range. The successful dissection of the complex spectrum obtained when N‐acetyl‐L‐tryptophanamide is flashed under nitrogen‐saturated conditions is utilized to highlight the capabilities of the detection system. This analysis suggests that the observed radicals must originate, in part, from an oxygen sensitive long‐lived precursor which is not the triplet state.

TEMPERATURE AND VISCOSITY DEPENDENCE OF FLUORESCENCE QUENCHING BY OXYGEN IN MODEL SYSTEMS

Abstract— The bimolecular rate constant, kq, for the quenching of the fluorescence of pyrenebutyric acid and naphthaleneacetic acid by molecular oxygen has been studied as a function of temperature and viscosity, η. Fluorescence lifetime measurements were used to monitor the degree of quenching. Glycerol‐water and sucrose‐water mixtures were used to increase the bulk viscosity. Plots of log kqvs log η show deviations from Stokes‐Einstein behavior, but the data are consistent with the patterns that have been observed for other diffusion limited reactions. This work provides background information, regarding the viscosity dependence of oxygen quenching reactions, which is essential for the correct interpretation of oxygen quenching/viscosity dependence studies with more complex systems (i.e. quenching of tryptophan residues in proteins).