Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol

Abstract

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer

“…The combined, FD and steady state intensity data can be interpreted as an evidence of collisional and static quenching of tyrosyl fluorescence by the reduced disulfide bonds. Our steady-state data for the reduced hormones extends the studies by Swadesh et al (1987) over a larger viscosities range where the collisional quenching could be distinguished from the complexational (static). For the native hormones, viscosity independent and similar quenching observed from FD and intensity data can be explained as a FRET from tyrosyl residue to the disulfide bridge.…”

“…It is also important to note that intensity decays are very similar for the native and reduced hormones. Such indistinguishable quenching of native and reduced hormones observed either from steady-state or time-resolved measurements very likely clarify the difficulties to assign the quenching mechanism of tyrosyl residue in the native hormones (Cowgill 1967;Ross et al 1986a;Swadesh et al 1987).…”

“…The conformation of these peptides has been studied using molecular mechanics (Liwo etal. 1988), X-ray crystallography (Langs et al 1986;Husain et al 1990), NMR Brewster and Hruby 1973;Krauss and Cowburn 1981), as well as steady-state (Cowgill 1964(Cowgill , 1967Chen 1967;Bodanszky et al 1981;Laws et al 1986;Ross et al 1986a;Swadesh et al 1987) and time-resolved fluorescence of the intrinsic tyrosine emission Ross et al 1986a). Ross et al (1986a) interpreted the fluorescence measurements of oxytocin in terms of structured features of the peptide conformation.…”

“…However, based on steady-state data the mechanisms and stearic requirements of disulfide quenching could not be deduced. Swadesh et al (1987) ruled out collisional quenching based on a Stern-Volmer analysis and suggested the formation of a complex between tyrosyl and cystine residues. They have not excluded other quenching mechanisms but long range energy transfer as a reason for the intramolecular quenching has not been considered.…”

Radiative Decay Engineering: Biophysical and Biomedical Applications

“…As examples, we note that flavins and FAD are fluorescent in solution, but most flavoproteins display little if any flavin emission (62)(63)(64). Tyrosine is often quenched in proteins, and frequently tryptophan residues are quenched by nearby amino acid residues such as disulfide bonds, histidine, or phenylalanine side chains (65)(66)(67). Another well-known example is DNA, nucleotides, and the individual bases.…”