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Fluorescence Study of the Conformational Properties of Recombinant Tick Anticoagulant Peptide ( Ornithodorus moubata ) Using Multifrequency Phase Fluorometry
Author(s) -
Sillen Alain,
Vos Rita,
Engelborghs Yves
Publication year - 1996
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1996.tb01835.x
Subject(s) - chemistry , fluorescence , tryptophan , fluorescence anisotropy , fluorescence spectroscopy , time resolved spectroscopy , peptide , analytical chemistry (journal) , biophysics , chromatography , amino acid , biochemistry , biology , membrane , physics , quantum mechanics
— Steady‐state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide ( Ornithodorus moubata ) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pK a of 6.01 ± 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the his‐tidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52,1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N ‐bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trpll, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.