Photoaffinity labeling of an acorn barnacle lectin with a photoactivatable fluorescent reagent derivative of d-galactosamine

A photoactivatable D-galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with 1-azide-5-naphthalene sulfonyl chloride (ANS-Cl). The derivative (GalN-ANS) inhibited the agglutination activity of an acorn barnacle lectin against rabbit erythrocytes to the same extent as D-galactosamine. We used GalN-ANS for photoaffinity labeling of the lectin. The photolabeled lectin was digested with pronase and the digest was separated by reversed-phase high-performance liquid chromatography by monitoring fluorescence and uv absorption to isolate the peptide labeled with GalN-ANS. Amino acid analyses of the labeled peptides revealed that GalN-ANS preferentially covalently labeled two regions in the carbohydrate recognition domain of the lectin. One of them was the highly conserved amino-acid sequence region throughout all calcium-dependent animal lectins.