title: Characterization of butyrylcholinesterase in bovine serum.
abstract: Human butyrylcholinesterase (HuBChE) protects from nerve agent toxicity. Our goal was to determine whether bovine serum could be used as a source of BChE. Bovine BChE (BoBChE) was immunopurified from 100 mL fetal bovine serum (FBS) or 380 mL adult bovine serum by binding to immobilized monoclonal mAb2. Bound proteins were digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The results proved that FBS and adult bovine serum contain BoBChE. The concentration of BoBChE was estimated to be 0.04 μg/mL in FBS, and 0.03 μg/mL in adult bovine serum, values lower than the 4 μg/mL BChE in human serum. Nondenaturing gel electrophoresis showed that monoclonal mAb2 bound BoBChE but not bovine acetylcholinesterase (BoAChE) and confirmed that FBS contains BoBChE and BoAChE. Recombinant bovine BChE (rBoBChE) expressed in serum-free culture medium spontaneously reactivated from inhibition by chlorpyrifos oxon at a rate of 0.0023 min-1(t1/2= 301 min-1) and aged at a rate of 0.0138 min-1(t1/2= 50 min-1). Both BoBChE and HuBChE have 574 amino acids per subunit and 90% sequence identity. However, the apparent size of serum BoBChE and rBoBChE tetramers was much greater than the 340,000 Da of HuBChE tetramers. Whereas HuBChE tetramers include short polyproline rich peptides derived from lamellipodin, no polyproline peptides have been identified in BoBChE. We hypothesize that BoBChE tetramers use a large polyproline-rich protein to organize subunits into a tetramer and that the low concentration of BoBChE in serum is explained by limited quantities of an unidentified polyproline-rich protein.
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: adafferner@unmc.edu.
authors.forename: Alicia J
authors.initials: AJ
authors.lastname: Dafferner
authors.affiliation: Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, 4, Kosygina St., Moscow 119334, Russia. Electronic address: sofya.lushchekina@gmail.com.
authors.forename: Sofya
authors.initials: S
authors.lastname: Lushchekina
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: pmasson@unmc.edu.
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
authors.affiliation: Syd Labs, Inc., Natick, MA 01760, USA. Electronic address: gpxiao@sydlabs.com.
authors.forename: Gaoping
authors.initials: G
authors.lastname: Xiao
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: lmschopf@unmc.edu.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Schopfer
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: olockrid@unmc.edu.
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002091:Butyrylcholinesterase / Q000097:blood* / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D002091:Butyrylcholinesterase
keywords: Bovine butyrylcholinesterase; Immunopurification; Mass spectrometry; Molecular dynamics; Serum