Fluorescence quenching of indole and model micelle systems

Abstract

Acrylamide quenching of indole fluorescence proceeds via both a dynamic and a static process. The rate constant for the dynamic process has a diffusion limited value of about 7 X lo9 M-l s-l. The static quenching component can be described by the expression exp(V[Q]) with V values being about 2.0 M-l. The possible physical interpretations of the static parameter, V , are discussed, particularly as they relate to the local distribution of quencher molecules in ordered systems. To demonstrate the potential utility of acrylamide, along with other quenchers, in providing topographical information about ordered systems, quenching studies are presented for an indole-micelle complex, a crude model for a protein. Both the collisional and static quenching components furnish insight as to the positioning of the indole ring in the micelles. Whereas the action of ionic and hydrophobic quenchers is exaggerated in the micelle study, acrylamide appears to be a perfectly neutral quenching probe.dole ring in a globular protein will determine the ease with which its excited state will be deactivated by the bombardment of quencher molecules from the solvent. For this reason, the quenching rate constant should provide a kinetic measurement of the residue's exposure to the solution.This experimental strategy would hold if the exposure of IntroductionFluorescence quenching reactions2-8 have been recently applied to studies with highly ordered biological macromolecules, such as proteins.9-13 An important class of fluorophors in proteins are the tryptophan residues, which have indole as their side chain. The position assumed by an in-

Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog

“…3 B), the maximal fluorescence intensities were normalized to 100. ment. Micelles are dynamic entities that allow some polar molecules to penetrate their interior [56] and this may account for the higher λ max of TOE in comparison with the Ca 2ϩ -ATPase, in which Trp accessibility is better protected by the protein structure. Additionally the λ max value obtained for TOE in DodMal is similar to that of TOE in unilamellar lipid vesicles at the same pH [44,45].…”

Systematic Peptide Engineering and Structural Characterization to Search for the Shortest Antimicrobial Peptide Analogue of Gaegurin 5

“…This large blue shift, which was not observed for NATA (an indole derivative used as a control; Table III), means that the indole side chain of the tryptophan was partitioned into a more hydrophobic environment, such as the core of the detergent micelles, probably by interacting with the hydrophobic acyl chains (21,28,29,43). The extent to which the Trp residues were sequestered in the hydrophobic micelle interior was evaluated by a fluorescence-quenching experiment using acrylamide (29). The experimental accuracy was validated by checking the agreement of the quenching parameters for NATA between the experimental data (Table III; with a concomitant reduction in the static quenching constant, V (Fig.…”

“…Both peptides showed a large blue shift of max by more than 10 nm (Table III) when the solvent was changed from water to detergent micelles. This large blue shift, which was not observed for NATA (an indole derivative used as a control; Table III), means that the indole side chain of the tryptophan was partitioned into a more hydrophobic environment, such as the core of the detergent micelles, probably by interacting with the hydrophobic acyl chains (21,28,29,43). The extent to which the Trp residues were sequestered in the hydrophobic micelle interior was evaluated by a fluorescence-quenching experiment using acrylamide (29).…”

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

“…At pH4.0 we are dealing with an enzyme that, if assayed at that pH, is inactive and cannot bind its substrates. However, its activity can be restored completely upon careful increase of the pH to 8.0. On the other hand, upon oxidation by SucNBr of the Trp fluorescing at 335 nm the enzymatic activity cannot be restored by increasing the pH to 8.0.…”

“…Because only small changes in fluorescence are involved, no further investigations were pursued. DISCUSS I 0 N Eftink and Ghiron [8,9] first introduced the use of acrylamide as Trp fluorescence quencher to sense the degree of exposure of Trp residues in proteins and to monitor conformational changes. Their technique along with pH variation and SucNBr oxidation experiments was applied to GTP :AMP phosphotransferase from beef heart mitochondria.…”