Characterization of local polarity and hydrophobic binding sites of beta-lactoglobulin by using N-terminal specific fluorescence labeling.

Because of wide ligand-binding ability and significant industrial interest of beta-lactoglobulin (beta-LG), its binding properties have been extensively studied. However, there still exists a controversy as to where a ligand binds, since at least two potential hydrophobic binding sites in beta-LG have been postulated for ligand binding: an internal one (calyx) and an external one (near the N-terminus). In this work, the local polarity and hydrophobic binding sites of beta-LG have been characterized by using N-terminal specific fluorescence labeling combined with a polarity-sensitive fluorescent probe 3-(4-chloro-6-hydrazino- 1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CHTDP).

Detection of local polarity of alpha-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 (lambda(em)) sensitive to solvent polarity only but not to pH and temperature.