A pulsed tunable dye laser has been used to obtain excitation spectra and fluorescence decay curves of solid Eu(III)- Datura innoxia and from a series of Eu(III)-containing complexes. Carboxyl and sulfate groups have been demonstrated to be the dominant functional groups for forming binding sites on the cell wall of Datura innoxia at high (≥4) and low (≤3) pH conditions, respectively. The excitation spectra associated with the 7 F0 → 5 D0 electronic transition of Eu(III) luminescence have been used to provide a measure of the electronic structure factors contributing to the interaction between Eu(III) ions and the binding sites on the cell wall of D. innoxia. The noticeably broadened and asymmetric excitation spectra obtained at high pH conditions are ascribed to multiple binding sites. The corresponding lifetime decay curves exhibited a bi-exponential decay. A pK of 4.5 was determined for the binding of Eu(III) to the cell wall at pH ≥4. Kinetic and thermodynamic studies were also undertaken.
Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.
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