Detection of Local Polarity of α-Lactalbumin by N-Terminal Specific Labeling with a New Tailor-Made Fluorescent Probe

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 (λ<INF>em</INF>) 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 α-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. Keywords: analytical methods • local polarity detection • fluorescent probe • N-terminal specific labeling • α-lactalbumin