Purification and characterization of a liver-derived beta-N-Acetylhexosaminidase from marine mammal Sotalia fluviatilis.
A beta-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 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees 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 beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.
Gomes Júnior
J E
JE
EMBRAPA Recursos Genéticos e Biotecnologia, PqEB-Final W5 Norte-Cp02372, Brasília, DF, Brazil. jose_edilson@yahoo.com.br
Souza
D S L
DS
Nascimento
R M
RM
Lima
A L M
AL
Melo
J A T
JA
Rocha
T L
TL
Miller
R N G
RN
Franco
O L
OL
Grossi-de-Sa
M F
MF
Abreu
L R D
LR
D000818:Animals; D002241:Carbohydrates; D002850:Chromatography, Gel; D004289:Dolphins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D002241:Carbohydrates; D001619:beta-N-Acetylhexosaminidases