Resolution of Cys and Lys labeling of α-crystallin with site-sensitive fluorescent 3-hydroxyflavone dye

Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4 ′-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens α-crystallin. The labeling of SH and NH 2 groups are clearly distinguished by spectroscopic criteria. We observe that the NH 2 labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH 2-labeled species. Applying recently developed multiparametric analysis of the obtained emission spectra, we described the physicochemical properties of the sites of SH and NH 2 labeling in α-crystallin. The site of SH labeling has medium-low polarity (dielectric constant, ε=4.9 ± 0.9 ) is protic, and does not contain proximal aromatic residues (according to the obtained refractive index, n=1.41 ± 0.14 ). The site of NH 2 labeling is also of medium-low polarity. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH 2-labeling sites.