title: Purification and characterization of beta-N-acetylhexosaminidase from bovine tick Boophilus microplus (Ixodide) larvae.
abstract: beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
authors.forename: F A
authors.initials: FA
authors.lastname: Del Pino
authors.forename: A
authors.initials: A
authors.lastname: Brandelli
authors.forename: C
authors.initials: C
authors.lastname: Termignoni
authors.forename: J C
authors.initials: JC
authors.lastname: Gonzales
authors.forename: J A
authors.initials: JA
authors.lastname: Henriques
authors.forename: H
authors.initials: H
authors.lastname: Dewes
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002417:Cattle; D002845:Chromatography; D019075:Electrophoresis, Capillary; D004795:Enzyme Stability; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D013379:Substrate Specificity; D013987:Ticks / Q000196:embryology / Q000201:enzymology* / Q000469:parasitology; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D006027:Glycosides; D008670:Metals; D001619:beta-N-Acetylhexosaminidases