LASER-EXCITED FLUORESCENCE OF DITYROSINE

In this research, laser-excited fluorescence was examined for sensitive detection of aqueous dityrosine. Samples were excited with a 6.3-mW, 325-nm helium-cadmium laser focused into a small volume-fluorescence cell with a 10-cm lens. The resulting fluorescence emission was collected perpendicular to the excitation and detected with two different schemes. An optical bandpass filter was used with a photomultiplier tube for sensitive quantitative measurement, while a photodiode array detector was used in conjunction with a spectrograph for qualitative characterization of fluorescence emission spectra. Dityrosine detection on the order of 2 x 10(-11) M was obtained with the use of the photomultiplier tube with bandpass optical filter. The dityrosine fluorescence yield is found to vary with the solution pH, the relative concentrations of ferric and ferrous iron, and the amount of dissolved oxygen. A maximum fluorescence yield is observed for iron-free, oxygen-free alkaline solutions. Fluorescence quenching by oxygen is a cumulative photolysis effect that diminished fluorescence yield with increased irradiation time. Flowing the solutions minimized photolysis effects in oxygenated solutions. Quenching by ferrous and ferric iron is found to be due primarily to complex formation. The ferrous iron complex appears to have a fluorescence efficiency of similar to 20% of the free dityrosine. The ferric iron dityrosine complex appears to have two ferric ions per molecule at low iron concentration. Other complexes may form at different concentrations. Solvent effects on dityrosine absorption and fluorescence spectra were also investigated. A red shift in dityrosine fluorescence maximum was observed in 1 M trichloroacetic acid and in N,N-dimethylformamide. The fluorescence emission maximum was shifted to the blue in acetonitrile and glacial acetic acid. These shifts were attributed to typical solvochromic behavior.