ACRYLAMIDE QUENCHING OF TRYPTOPHAN PHOTOCHEMISTRY AND PHOTOPHYSICS

Abstract

Abstract— Studies of acrylamide quenching of tryptophan (Trp) fluorescence, photochemistry, and photoionization have been conducted. Quenching of Trp fluorescence in aqueous solution by addition of acrylamide in the concentration range 0.0–0.5 M was measured and resulted in a Stern‐Volmer quenching constant of K sv = 21 ± 3 M ‐1 . Photolysis experiments were performed in which Trp was photolyzed at 295 nm in the presence of varying concentrations of acrylamide. The loss of Trp was monitored using reverse‐phase high performance liquid chromatography (RP‐HPLC) and was observed to follow first order kinetics. Production of N‐formylkynurenine (NFK) was observed by RP‐HPLC in irradiated Trp samples both in the presence and absence of added acrylamide. In addition, no new photochemical product was detected. This was taken as evidence that acrylamide did not alter the photochemical pathway but just reduced the reaction rate as expected for a physical quenching mechanism. Plotting the reciprocal of photolysis rate constant versus acrylamide concentration produced a Stern‐Volmer constant for quenching of Trp photochemistry of K sv = 6 ± 2 M ‐1 . The K sv values for both fluorescence quenching and photolysis quenching were thus large, implying efficient quenching of both processes by acrylamide. Assuming an excited singlet state lifetime of 2.8 ns, the calculated second‐order quenching rate constants for fluorescence and photolysis were k q = 7.5 × 10 9 and 2.1 × 10 9 M ‐1 S ‐1 respectively. The possible involvement of photoionization in the photolysis mechanism was investigated by studies of acrylamide quenching of voltage transients produced by xenon flash lamp excitation of Trp at aqueous/teflon or aqueous/mica interfaces. Plots of reciprocal photovoltage amplitude versus acrylamide concentration yielded linear Stern‐Volmer plots with K sv = 5 ± 1 M ‐1 . This number may not be directly comparable to those obtained above for photolysis and fluorescence quenching, because quenching of photovoltage involved an excited Trp adsorbed on a teflon surface, while the former processes occurred in bulk solution. Nonetheless, the value of K sv observed in the photoionization experiments showed that this process was also efficiently quenched by acrylamide.