Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -70 °C as supplied.
3 months, -70 °C under sterile conditions after opening.
Assay Buffer: 100 mM Sodium Phosphate, pH 7.5
Recombinant Human Butyrylcholinesterase/BCHE (rhBCHE) (Catalog # 6137-CE)
Substrate: Butyrylthiocholine chloride (BTC) (Sigma Catalog # B3128), 20 mM stock in DMSO
5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D8130), 10 mM stock in DMSO
96 well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rhBCHE to 0.02 µg/mL in Assay Buffer.
Dilute BTC and DTNB to 200 µM final concentrations in deionized water.
Load into plate 50 µL of 0.02 µg/mL rhBCHE and start the reaction by adding 50 µL of the BTC/DTNB mixture to the wells. As a Substrate Blank, load 50 µL of Assay Buffer and 50 µL of the BTC/DTNB mixture.
Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Using the extinction coefficient 13260 M-1cm-1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Per Well:
rhBCHE: 0.001 µg
DTNB and BTC: 100 µM
Butyrylcholinesterase (BCHE) is a major acetylcholine hydrolyzing enzyme in the circulation (1). Although it is present in significant amounts in human plasma, no endogenous physiological substrate has been described for this enzyme. It can degrade a large number of ester-containing compounds besides acylcholines, including neurotoxic organophosphate esters. Thus, it plays significant pharmacological and toxicological roles. It is thought to be involved in the pathological progression of Alzheimer’s disease (AD) by depleting acetylcholine. In contrast to ACHE, it attenuates amyloid fibril formation in vitro (2). BCHE inhibitors have been used to delay symptoms of AD patients by virtue of their ability to enhance ACH availability (3). Its involvement in the cholinergic anti-inflammatory pathway connects BCHE and ACHE as possible markers of low-grade systemic inflammation observed in Type-2 diabetes, obesity, hypertension, coronary heart disease, and AD (4). BCHE can exist as monomers, dimers, or tetramers (1).
Darvesh, S. et al. (2003) Nat. Rev. Neurosci. 4:131.
Diamant, S. et al. (2006) Proc. Nat. Acad. Sci. 103:8628.
Campbell, V. A. and Gowran, A. (2007) Br. J. Pharm. 152:655.
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