Purification and Characterization of a Liver-derived β-N-Acetylhexosaminidase from Marine Mammal Sotalia fluviatilis

• Published in: The Protein Journal Vol. 29; no. 3; pp. 188 - 194
• Main Authors: Gomes Júnior, J. E., Souza, D. S. L., Nascimento, R. M., Lima, A. L. M., Melo, J. A. T., Rocha, T. L., Miller, R. N. G., Franco, O. L., Grossi-de-Sa, M. F., Abreu, L. R. D.
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.04.2010
• Subjects: Animal Anatomy; Animals; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - isolation & purification; beta-N-Acetylhexosaminidases - metabolism; Biochemistry; Bioorganic Chemistry; Carbohydrates; Chemistry; Chemistry and Materials Science; Chromatography, Gel; Dolphins - metabolism; Electrophoresis, Polyacrylamide Gel; Histology; Hydrogen-Ion Concentration; Kinetics; Liver - chemistry; Liver - enzymology; Morphology; Organic Chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Purification; Hepatic extracts; Glycosidases; β-N-Acetylhexosaminidase
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-010-9239-3

A β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis , order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS–PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 × 10 −6 μmol/(min.mg) were found for this enzyme, determined by p-nitrophenyl-β- d -hexosaminide substrate digestion. Optimal pH and temperature for β-N-Acetylhexosaminidase activity were 5.0 and 60 °C, respectively. Enzyme activity was inhibited by sodium selenate (Na 2 SeO 4 ), mercuric chloride (HgCl 2 ) and sodium dodecyl sulfate (C 12 H 25 SO 4 Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the β-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.