To detect the local polarity such as the N-terminal domain of a protein molecule, 3-(4-chloro-6-hydrazino-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine has been designed and synthesized as a polarity-sensitive fluorescent probe by using an s-triazine ring as a backbone, neutral red and hydrazine as a polarity-sensitive fluorophore, and a labeling group, respectively. The fluorescence properties of the probe have been characterized. The probe has the following features: (1) stable in various solvents; (2) the long-wavelength emission of >550 nm that can avoid the interferences of the background fluorescence shorter than 500 nm from common biomacromolecules; and (3) the maximum emission wavelength (lambda(em)) sensitive to solvent polarity only but not to pH and temperature. The hydrazino group in such a probe reacts readily with an active carbonyl produced by transamination of a protein molecule, leading to N-terminal specific attachment of the fluorophore and thereby allowing the monitoring of local polarity. With this probe, the polarity of the N-terminal domain in both native and heat-denatured alpha-lactalbumin has been first determined, which corresponds to that with a dielectric constant of about 16, and the hydrophobic core near the N-terminus is found to be conservative for heating. The present strategy may provide a general method to study the local environmental changes of a protein molecule under different denaturation conditions.

Fluorescence resonance energy transfer (FRET) is a powerful technique for the determination of distances between two fluorophores. The overall geometry of protein structures [1][2][3][4] and the conformational changes of a molecule under different conditions can be studied by this method if appropriate sites of the molecule are labeled with fluorescence donor and acceptor probes. Nevertheless, it is rather difficult to specifically introduce two different fluorophore groups into one molecule, [2] especially into a homodimeric biomacromolecule that has two identical reactive sites. Different from the conventional FRET technique, donor-donor energy migration (DDEM) takes advantage of certain fluorescence probes that display an overlap of their absorption and emission spectra and are therefore able to transfer energy between themselves.[2-4] Energy transfer in this case is a reversible process and can be measured through analysis of the timeresolved depolarization of the fluorescence emission (as donor-donor energy migration results in additional depolarization). As only one type of probe is required, DDEM simplifies greatly not only the labeling operation but also the theoretical analysis and the time-resolved measurements and has been widely used to study the steady-state conformational changes of biomacromolecules.DsbC (1), a member of the Dsb family in the periplasm of Gram-negative bacteria, is a thiol-protein oxidoreductase that displays molecular chaperone activity. [5][6][7] The DsbC molecule is a V-shaped homodimer consisting of two 23.4-kDa subunits.[8] Each subunit is composed of a C-terminal thiore-