FLUORESCENCE STUDIES WITH HUMAN EPIDERMAL GROWTH FACTOR

Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation.     

Oxygen fluorescence quenching studies with single tryptophan-containing proteins

The work of Lakowicz and Weber [Biochemistry 12, 4161 (1973)] demonstrated that molecular oxygen is a powerful quencher of tryptophan fluorescence in proteins. Here we report studies of the oxygen quenching of several proteins that have a single, internal tryptophan residue. Among these are apoazurin (Pseudomonas aeruginosa), asparaginase (Escherichia coli), ribonuclease T₁ (Aspergillus oryzae), and cod parvalbumin. Both fluorescence intensity and phase lifetime quenching data are reported. By comparison of these data we find that there is a significant degree of apparent static quenching in these proteins. The dynamic quenching rate constants,k _q, that we find are low compared to those for tryptophan residues in other proteins. For example, for apoazurin we find an apparentk _q of 0.59×10⁹ M ^–1 s^–1 at 25°C. This value is the lowest that has been reported for the oxygen quenching of tryptophan fluorescence.     

RAPID EQUILIBRATION OF OXYGEN IN FLUORESCENCE QUENCHING REACTIONS

A simple means is described for the rapid equilibration of oxygen in fluorescence quenching reactions. This involves bubbling the gas through experimental solutions. With this procedure, data for Stern-Volmer plots can be obtained in 1–2 h, without bubbling it takes approximately ten times as long to collect such data.     

Time-resolved fluorescence studies of ribonuclease T1 in reversed micelles

Time-resolved fluorescence intensity and anisotropy decay data were obtained for ribonuclease T₁ entrapped in bis(2-ethylhexyl) sodium sulfosuccinate/heptane reverse micelles, as a function of the size of the inner water pool at neutral pH. Data have been presented previously to show that this protein retains its native structure and undergoes reversible thermal unfolding in these reverse micelles (Shastry and Eftink,Biochemistry 36, in press). The fluorescence decay of entrapped protein is similar to that for the protein in buffer. The rotational correlation time of entrapped ribonuclease T₁ is found to be longer than that in buffer; this rotational correlation time decreases with increasing size of the water pool but is still over twice the value for the protein in buffer for the largest size of water pool investigated, indicating an increased microviscosity within the reverse micelle. Thermal unfolding of the protein results in a significant decrease in the rotational correlation time of the entrapped proteins, consistent with the protein being unfolded but not interacting with the inner surfactant wall of the reverse micelle.Nomenclature _i Amplitude of component i associated with fluorescence decay _i - _i Fractional intensity associated with fluorescence decay time _i - Rotational correlation time g_i Amplitude of component i associated with anisotropy decay _i - _max Fluorescence emission maximum - r_o Fundamental anisotropy of an immobilized fluorophore - _i Fluorescence lifetime of component i - W_o Ratio of water molecules per detergent molecules in a reverse micelle     

Stereoselective Synthesis of cis-2 and trans-2-(Indol-3-yl) Cyclopropyl Amines,Carboxylic Acids,and Esters

We report a general route for the synthesis of E and Z isomers of indol-3-yl cyclopropyl amines, carboylic acids, and esters. These cyclopropane containing molecules are of interest as conformationally constrained analogues of tryptamine and indole propionic acid, biologically active indoles. The route involves reaction of vinyl indole with ethyl diazoacetate, chromatographic separation of the E and Z stereoisomers of the resulting cyclopropane esters, hydrolysis to form the E and Z cyclopropane acids, and formation of amines by the Curtius reaction.     

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

Abstract— The bimolecular rate constant, k _q, 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 k _q vs 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).     

A COMPARISON OF FLUORESCENCE QUENCHING AND FLUORESCENCE DECAY STUDIES WITH TRYPTOPHAN

Abstract— The quenching of the fluorescence of aqueous tryptophan solutions has been studied as a function of emission wavelength using acrylamide as a collisional quencher. Our quenching studies are consistent with recent observations of the heterogeneity of tryptophan fluorescence, but they show a slight discrepancy when compared to certain analyses of the decay of tryptophan fluorescence in terms of two components.     

Synthesis of E and Z Diastereoisomers of Benzyl Methyl-2-[3-(N-Substituted)indolyl] Cyclopropane-1,1-dicarboxylates and Their Stereoselective Reductive Cleavage

A synthesis and separation procedure is described for the E and Z diastereoisomers of indolyl-cyclopropane dicarboxylates, IV and V, potential intermediates in the synthesis of the corresponding E and Z cyclopropyl analogs of the amino acid tryptophan and for other ring expansion reactions bearing the indole moiety. Also we have observed a stereoselective cleavage of the benzyl group of the E isomer, without affecting the cyclopropane ring.     

Evidence for dual La and Lb emission in 5-methylindole

The steady-state fluorescence anisotropy of 5-methylindole is shown to depend on both excitation and emission wavelengths, at room temperature in glycerol. A simulation is presented that shows that this emission wavelength dependence of the anisotropy can be explained in terms of dual emission from both the L_a and the L_b transition moments of the indole ring. For such dual emission to occur, the lowest excited-state energy level of both of these oscillators must be very similar.     

STUDIES OF THE EFFICIENCY and MECHANISM OF FLUORESCENCE QUENCHING REACTIONS USING ACRYLAMIDE and SUCCINIMIDE AS QUENCHERS

Abstract Here we report data for the quenching by acrylamide and succinimide of the fluorescence of a number of simple aromatic fluorophores in aqueous solution. Acrylamide is an efficient quencher of the fluorescence of most of these aromatic fluorophores, but succinimide is less efficient for all fluorophores and shows a very crude dependence on the ionization potential of the fluorophore. When the solvent is ethanol, the quenching efficiency by acrylamide and succinimide is found to decrease for the fluorophores, indole, naphthalene, and carbazole. These studies are consistent with an electron transfer quenching mechanism for the two quenchers. Quenching parameters (by acrylamide, succinimide, and iodide) are also reported for a number of fluorescent probes commonly used in biochemical studies. In general, the efficiency of acrylamide and succinimide quenching of these probes is low in aqueous solution.