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Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.

This article discusses several strategies for the use steady-state and time-resolved fluorescence methods to monitor unfolding transitions in proteins. The assumptions and limitations of several methods are discussed. Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species. Several examples are given, involving the unfolding of Staphylococcal aureus nuclease A, in which thermodynamic information is obtained for the temperature and denaturant induced transitions in this protein.

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-