Premium
DETERMINATION OF THE ACRYLAMIDE QUENCHING CONSTANT FOR PROTEIN AND MODEL INDOLE TRIPLETS
Author(s) -
Ghiron Camillo,
Bazin Marc,
Santus Rene
Publication year - 1988
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1988.tb02858.x
Subject(s) - indole test , acrylamide , chemistry , quenching (fluorescence) , phosphorescence , tryptophan , triplet state , singlet state , excited state , photochemistry , acetonitrile , analytical chemistry (journal) , fluorescence , stereochemistry , molecule , organic chemistry , physics , biochemistry , amino acid , quantum mechanics , nuclear physics , copolymer , polymer
— The decay of the indole triplet of single tryptophan‐containing proteins and model compounds can be readily measured at room temperature in aqueous solution by monitoring the triplet‐triplet absorption or phosphorescence emission following a 265 nm exciting laser pulse. The quenching action of acrylamide on the triplet excited state of indole side chains was studied in an analogous fashion to that previously done at the singlet level (Eftink and Ghiron, 1977). The acrylamide triplet quenching constant ( t k q ) ranged from a high of 7.8 times 10 8 M ‐1 s ‐1 for the exterior indole of corticotropin (ACTH) to a low of 2 times 10 5 Af ‐1 s ‐1 for the interior indole of ribonuclease T, (RNase T,). The ratio (7) of these values with their respective acrylamide singlet quenching constants ( t k q ),(γ= t k q 8 K q ) ranged from a high of 0.22 for ACTH to a low of 0.001 for RNase T 1 ,. Acrylamide is also an inefficient quencher of model indoles in various solvents (i.e. it has a γ less than 1). The magnitude of γ varied from a high of 0.3 in H 2 0 to a low of 0.02 in acetonitrile, but did not correlate with viscosity, dielectric constant or polarity. The lower efficiency observed for internal indole groups can not be explained by that class of models which predict the presence of static quenching at the triplet level, since none was observed. The present results confirm the observation of Calhoun et al. of a large discrepancy between acrylamide's singlet and triplet quenching constants for buried indole side chains, but suggest that it may be largely explained by the fact that acrylamide is an inefficient quencher of the indole triplet state (1983). The magnitude of this inefficiency is probably determined by specific microenvironmental factors. Thus, unlike 8 K q , the environmentally sensitive l k H cannot be easily used to characterize the dynamics of proteins.