Time-resolved fluorescence of glucagon studied by global compartmental analysis

Abstract

The photophysics of the polypeptide hormone glucagon in aqueous phosphate buffer (lambda(ex) = 295 nm, pH 6.7, 17.2 degrees C) in the presence of the added quencher sodium iodide is investigated using standard simultaneous biexponential and global compartmental analyses of its fluorescence decay surface. The use of the scanning procedure in global compartmental analysis allows distinction between reversible and irreversible transformation processes in the excited state (J. Phys. Chem. 1995, 99, 8959-8971). Furthermore, by comparison of the species-associated with the decay-associated emission spectra, a unidirectional process in the excited state can be distinguished from the case where no interconversion occurs between the two excited-state species. Scanning different rate constants and comparing the species-associated with the decay-associated emission spectra indicate that the dual exponential fluorescence decays of the single tryptophan-containing polypeptide glucagon are due to two non-interconverting excited-state species resulting from excitation of their respective ground states. On the time scale of fluorescence there is no interconversion between the two excited-state species. The composite deactivation rate constant values for monomeric glucagon in phosphate buffer (k(01) = 1.01 x 10(9) s(-1) and k(02) = 2.7 x 10(8) s(-1)) are separated into contributions via fluorescence (k(F1) = 0.05 x 10(9) s(-1) and k(F2) = 1.6 x 10(8) s(-1)) and nonradiative processes (k(NR1) = 0.96 x 10(9) s(-1) and k(NR2) = 1.1 x 10(8) s(-1)). The fluorescence quantum yields in the absence of added quencher are phi(F1)(0) = 0.05 and phi(F2)(0) = 0.58. The relatively high values (> 1 x 10(9) M-1 s(-1)) of the rate constants of quenching are indicative of a tryptophyl residue exposed to the surrounding aqueous environment. The obtained values of the rate constants may indicate that the decay component of 1 ns is associated with a tryptophyl residue in a flexible extended random coil conformation of the polypeptide chain while its 3.7 ns counterpart corresponds to a tryptophan in a compact, quite rigid backbone conformation. The method used to elucidate the origin of the biexponential fluorescence decays of glucagon is generally applicable to any intramolecular two-state excited-state process and will be useful in the study of peptides and proteins with dual exponential fluorescence decay kinetics.