Protein phosphorescence quenching: distinction between quencher penetration and external quenching mechanisms.
The accessibility of quenching solutes, Q, of various molecular sizes to buried Trp residues in proteins, as attested by dynamic quenching of their phosphorescence emission, is instrumental for probing structural fluctuations in these macromolecules. However, interpretation of quenching rates in terms of Q migration through the globular fold requires that alternative reaction pathways, such as long-range interactions with Q in the solvent, be ruled out. In theory, the external quenching rate can be estimated from the distance dependence of the through-space interaction by assuming compliance with the rapid diffusion limit regime. To validate the applicability of theoretical predictions to external quenching of protein phosphorescence, we compared the rate of quenching of the buried Trp residues of RNase T1 and parvalbumin by acrylamide and the bigger double-headed derivative bisacrylamide. The results showed that larger bisacrylamide is twice as efficient a quencher as acrylamide, implying that for these superficially buried residues the reaction is dominated by long-range interactions with acrylamide in the aqueous phase. To test the dependence of the quenching rate constant, k(q), on solvent viscosity, quenching studies were extended to glycerol-water solutions ranging in bulk viscosity from 1 to 120 cP. Apart from an initial about 2-fold increase, k(q) was found to be independent of solvent viscosity, thus demonstrating that external quenching rigorously complies with the rapid diffusion limit regime. Experiments were extended to larger acrylamide derivatives to evaluate the impact of Q size on the external quenching rate.
Strambini
Giovanni B
GB
Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy. strambini@pi.ibf.cnr.it
Gonnelli
Margherita
M
D000178:Acrylamides / Q000737:chemistry; D007700:Kinetics; D010320:Parvalbumins / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014783:Viscosity
D000178:Acrylamides; D010320:Parvalbumins; D012997:Solvents; D014364:Tryptophan; D006163:Ribonuclease T1