LASER-EXCITED FLUORESCENCE OF DITYROSINE

In this research, laser-excited fluorescence was examined for sensitiv e 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 collec ted perpendicular to the excitation and detected with two different sc hemes. An optical bandpass filter was used with a photomultiplier tube for sensitive quantitative measurement, while a photodiode array dete ctor was used in conjunction with a spectrograph for qualitative chara cterization of fluorescence emission spectra. Dityrosine detection on the order of 2 x 10(-11) M was obtained with the use of the photomulti plier tube with bandpass optical filter. The dityrosine fluorescence y ield is found to vary with the solution pH, the relative concentration s of ferric and ferrous iron, and the amount of dissolved oxygen. A ma ximum fluorescence yield is observed for iron-free, oxygen-free alkali ne solutions. Fluorescence quenching by oxygen is a cumulative photoly sis effect that diminished fluorescence yield with increased irradiati on time. Flowing the solutions minimized photolysis effects in oxygena ted solutions. Quenching by ferrous and ferric iron is found to be due primarily to complex formation. The ferrous iron complex appears to h ave a fluorescence efficiency of similar to 20% of the free dityrosine . The ferric iron dityrosine complex appears to have two ferric ions p er molecule at low iron concentration. Other complexes may form at dif ferent concentrations. Solvent effects on dityrosine absorption and fl uorescence spectra were also investigated. A red shift in dityrosine f luorescence 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 at tributed to typical solvochromic behavior.