Quenching of Alexa Dyes by Amino Acids

Abstract

Alexa dyes, rhodamine-derived fluorophores, are popular choices for labeling proteins due to their superior photo-physical properties. They are often used in quantitative fluorescence measurements like Forster Resonance Energy Transfer (FRET) or fluorescence lifetime imaging (FLIM). Consequently, it is important to consider the effects of nearby amino acid residues on the brightness of fluorophores that may influence quantitative measurements of fluorescence intensities or lifetimes. We report on the quenching of Alexa dyes (488, 555 and 594) by various natural amino acids. We observed quenching of Alexa488 by Tryptophan, Histidine, Methionine and Tyrosine. Lifetime measurements indicate that with the exception of Tyrosine, the quenching by the amino acids occur through both static and dynamic processes. Additionally, cyclic voltammetry experiments suggest that photo-induced electron transfer (PET) is a possible mechanism for the quenching of Alexa488.