No abstract

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.

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-

The acrylamide quenching reaction is shown to be very discriminating in sensing the exposure of fluorescing tryptophanyl residues in globular proteins. The quenching rate constants for some proteins, such as aldolase and human serum albumin, are reported to be independent of the solvent viscosity, indicating that the reaction is limited by penetration of the quencher through the protein matrix. Temperature-dependent studies are performed to determine the activation energy and entropy for the penetration of acrylamide into these proteins. The tryptophanyl residues in aldolase are shown to be shielded by a large activation energy barrier, while the single residue in human serum albumin is shielded by a large activation entropy barrier. These parameters characterize the nature of the protein matrix enveloping the fluorophors.