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.     

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.     

A practical synthesis of 2,4(5)-diarylimidazoles from simple building blocks

A simple and efficient approach to selectively obtain 2,4(5)-diarylimidazoles suppressing formation of 2-aroyl-4(5)-arylimidazoles is described. The yield of each of the two products strongly depends on the reaction conditions employed. This reaction provides a simple method to prepare small libraries of biologically active compounds by parallel synthesis.     

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.     

TRIPLET-TRIPLET ENERGY TRANSFER IN P-TRYPSIN 1 THE ROLE OF THE INDOLE TRIPLET IN THE ULTRAVIOLET-INDUCED PHOTOLYSIS OF β-TRYPSIN

Detection of triplet-triplet energy transfer in an aqueous solution of P-trypsin is reported. This conclusion is based on the observation that a light excited phenolate side chain can sensitize the destruction of an adjacent indole side chain. The role that the indole triplet might play in the UV-induced photolysis of /l-trypsin is also investi-gated. The results suggest that the UV (309 nm)-induced inactivation of P-trypsin is not caused by indole ring destruction but by the disruption of disulfide bonds without thiol formation.     

Alkene epoxidation with urea-hydrogen peroxide complex and PS-DVB supported phthalic anhydride

A new PS-DVB supported phthalic anhydride and UHP (urea-hydrogen peroxide complex) have been used for metal-free alkene epoxidation reactions. The resin was prepared by MW mediated ‘PEGylation’ of Merrifield resin followed by esterification with trimellitic anhydride chloride. Epoxidation of several alkenes was carried out with this resin and UHP.