A Close Look at Fluorescence Quenching of Organic Dyes by Tryptophan

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Abstract
Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in‐vitro and in‐vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady‐state and time‐resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion‐limited dynamic quenching with a bimolecular quenching rate constant kd of 4.0×109 s−1 M−1, 2) static quenching with a bimolecular association constant Ks of 61 M−1, and 3) a sphere‐of‐action contribution to static quenching described by an exponential factor with a quenching constant λ of 22 M−1. The latter two are characterized as nonfluorescent complexes, formed with ≈30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20–30 kJmol−1 is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub‐nanometer length and nanosecond time‐scales.