Heat perturbation of bovine eye lens α‐crystallin probed by covalently attached ratiometric fluorescent eye 4′‐diethylamino‐3‐hydroxyflavone

Abstract Bovine eye lens α‐crystallin was covalently labeled with 6‐bromomethyl‐4′‐diethylamino‐3‐hydroxyflavone and studied under native‐like conditions and at the elevated temperature (60°C) that is known to facilitate α‐crystallin chaperone‐like activity. This novel SH‐reactive two‐band ratiometric fluorescent probe is characterized by two highly emissive N*‐ and T*‐bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the α‐crystallin–dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH 2 groups in α‐crystallin, a recently developed procedure allowed us to distinguish between the heat‐induced spectral changes of the dye molecules attached to SH and NH 2 groups. We observed that at elevated temperature the environment of the SH‐attached dye becomes more polar and flexible. The number of H‐bond acceptor groups in the vicinity of the dye decreases. Since α‐crystallin contains a single Cys residue within the C‐terminal domain of its αA subunit (the αB subunit contains none), we can attribute the observed effects to temperature‐induced changes in the C‐terminal domain of this protein. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 340–348, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com