Purification and characterization of sea squirt α- N-acetylgalactosaminidase

Abstract

Sea squirt α- N-acetylgalactosaminidase was purified to homogeneity. Its molecular weight was estimated to be approximately 160,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition. The chromatographic and electrophoretic behaviors indicated that the enzyme was composed of four subunits. The optimum pH of the enzyme reaction was about 4.0 at 37°C, while the enzyme was stable in the range of pH 5.0 to 6.0 during 4 h preincubation at 37°C. Although the enzyme (0.1 unit) was stable at 0°C for 30 min in the presence of 7.5 mM metal ions (Al 3+, Ba 2+, Ca 2+, K +, Mn 2+, Pb 2+, Sr 2+, and Zn 2+), almost 40% of the enzyme activity was lost in the presence of Cu 2+, Hg 2+, monoiodoacetic acid, and EDTA. The enzyme hydrolyzed aryl N-acetyl-α- d-galactosaminide as well as GalNAcα1(→4GalNAcα1→) n 4GalNAc- p-aminobenzoic acid ethyl ester (ABEE) (n=1−4), but GalNAcα1→4GalNAc-ABEE only scarcely. Furthermore, an allergenic pentasaccharitol ABEE derivative, GalNAcα1→2Fucα1→3(GalNAcβ1→4) GlcNAcβ1→2(3-acetoamido-3-deoxy) l-threose-ABEE, the minimum structural unit for the sea squirt allergenicity was hydrolyzed to 95 mol% for 72 h incubation with the enzyme. The enzyme could be utilized as a powerful tool for the structural analyses of the carbohydrate epitopes of the sea squirt allergen molecules.