Purification and characterization of sea squirt alpha-N-acetylgalactosaminidase

Sea squirt alpha-N-acetylgalactosaminidase was purified to homogeneity. Its molecular weight was estimated to be approximately 160,000 by gel filtrati on and 40,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under re ducing condition. The chromatographic and electrophoretic behaviors indicat ed that the enzyme was composed of four subunits. The optimum pH of the enz yme reaction was about 4.0 at 37 degrees C, while the enzyme was stable in the range of pH 5.0 to 6.0 during 4 h preincubation at 37 degrees C. Althou gh the enzyme (0.1 unit) was stable at 0 degrees C for 30 min in the presen ce of 7.5 mM metal ions (Al3+, Ba2+, Ca2+, K+, Mn2+, Pb2+, Sr2+, and Zn2+), almost 40% of the enzyme activity was lost in the presence of Cu2+, Hg2+, monoiodoacetic acid, and EDTA. The enzyme hydrolyzed aryl N-acetyl-alpha-D- galactosaminide as well as GalNAc alpha 1(-->4GalNAc alpha 1-->)(n) 4GalAc- p-aminobenzoic acid ethyl ester (ABEE) (n = 1-4), but GalNAc alpha 1-->4Gal NAc-ABEE only scarcely. Furthermore, an allergenic pentasaccharitol ABEE de rivative, GalNAc alpha 1-->2Fuc alpha 1-->3(GalNAc beta 1-->4) GlcNAc beta 1-->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 st ructural analyses of the carbohydrate epitopes of the sea squirt allergen m olecules.