Anomalous “unquenching” of the fluorescence decay times of β-lactoglobulin induced by the known quencher acrylamide

Picosecond time-resolved fluorescence, together with the addition of quenching agents, was employed to discriminate the fluorescence contributions of the two tryptophans of β-lactoglobulin (Trp19 and Trp61) to the fluorescence decays of the protein. The fluorescence decays of β-lactoglobulin at pH 3, 5 and 8 are best fitted using sums of three exponentials and show a dominant contribution (98%) of the components associated with the buried Trp19, which decays according to a double exponential function. The addition of acrylamide (0.05 M) causes an increase of the decay times associated with Trp19. This effect is observed at all pH values studied, but the effect is stronger at pH 3 and pH 5, than at pH 8. The unexpected increase of the decay times of Trp19 and the variation of the respective amplitudes were rationalized in terms of alterations of Trp19 mobility. The hindrance of Trp19 upon acrylamide binding was also monitored and supported by fluorescence anisotropy measurements.