Substrate: S-Butyrylthiocholine chloride (BTC) (Sigma Catalog # B-3128), 20 mM stock in DMSO
5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130), 10 mM stock in DMSO
96 well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rmBCHE to 0.01 µg/mL in Assay Buffer.
Dilute BTC to 10 mM in deionized water.
Combine equal amounts of diluted BTC and DTNB. Dilute substrate mixture to a final concentration 200 µM BTC and DTNB in deionized water.
Load into plate 50 µL of 0.01 µg/mL rmBCHE 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 (nmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 109 nmol/M
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:
rmBCHE: 0.0005 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 (~3 mg/L) in human plasma, no endogenous physiological substrate has been described for this enzyme. It can degrade a large number of ester-containing compounds in addition to acylcholines. Thus, it is likely to play significant pharmacological and toxicological roles. It is thought to be involved in the pathological process 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 acetylcholine availability (3). Its involvement in a cholinergic anti-inflammatory pathway connect BCHE and ACHE with a possible marker of low-grade systemic inflammation observed in Type-2 diabetes, obesity, hypertension, coronary heart disease, and AD (4). BCHE can exist in monomeric and multimeric forms (1). The expressed recombinant mouse BCHE contains multiple forms that consist of soluble monomers, dimers, and tetramers.
Darvesh, S. et al. (2003) Nat. Rev. Neuroscience 4:131.
Diamant, S. et al. (2006) Proc. Natl. Acad. Sci. USA 103:8628.
Campbell, V. A. and Gowran, A. (2007) Br. J. Pharm. 152:655.
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