Acrylamide quenching studies with azurin B

Abstract

The acrylamide quenching of holoazurin B was studied as a function of emission wavelength in order to investigate discrepancies in interpretation of previous fluorescence measurements. A red-fluorescing acrylamide-quenchable component, which may be an impurity, is observed, suggesting that prior studies need to be interpreted with great caution. The presence of this component is not detectable when the 3-fold more fluorescent apo form is studied. Significant acrylamide quenching of apoazurin B is observed. The quenching constant of 5 · 10⁷ M⁻¹ · s⁻¹ at 20°C and the activation energy of 17 kcal/mol obtained are the most extreme values yet reported for a single tryptophan-containing protein. Since azurin B's indole is definitively known to be buried in the hydrophobic interior of the molecule, these results provide additional support for the contention that light-excited proteins undergo structural fluctuations in the nanosecond time range.