Screening of acetylcholinesterase inhibitors in snake venom by electrospray mass spectrometry

Abstract

An electrospray ionization/mass spectrometry (ESI/MS)-based assay for the determination of acetylcholinesterase (AChE)-inhibiting activity in snake venom was developed. It allows the direct monitoring of the natural AChE substrate acetylcholine (AC) and the respective product choline. The assay scheme was employed in the screening for neurotoxic activity in fractions of the venom of Bothrops moojeni. AChE inhibition was assessed in two fractions. As a positive control, the established AChE inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) was used, a dose-response curve for this compound was generated and the IC 50 value for the inhibitor was determined to be 1.60 ± 0.09 × 10 -9 mol L -1 . The dose-response curve was used as "calibration function" for the venom inhibition activity, resulting in BW284c51-equivalent concentrations of 1.76 × 10 -9 mol L -1 and 1.07 × 10 -9 mol L -1 for the two fractions containing activity. The ESI/MS-based assay scheme was validated using the established Ellman reaction. The data obtained using both methods were found to be in good agreement. The ESI/MS-based assay scheme is therefore an attractive alternative to the standard colorimetric assay.

Development of a fast liquid chromatography/mass spectrometry screening method for angiotensin‐converting enzyme (ACE) inhibitors in complex natural mixtures like snake venom

“…The aim of our study was the development of a fast and robust HPLC/ESI‐MS‐based screening method for ACE inhibitors in crude natural mixtures allowing application of the natural substrate AI. As ACE is only active in the presence of non‐volatile buffers comprising salts and NaCl in the millimolar concentration range,25, 29, 45 a direct FIA analysis as recently published for the screening for acetylcholine‐esterase inhibitors46 was not possible due to ion‐suppression effects and the risk of clogging of the ESI interface. For the removal of buffer salts, ACE, proteins and peptides from the analyzed crude reaction‐mixture samples, TFC was applied as online SPE in a backflush mode.…”

“…Snake venom is a well‐known source of biologically active peptides and proteins. Recently, we have shown the presence of acetylcholine‐esterase inhibitors46 and proteases48 in the venom of Bothrops moojeni , which may be responsible for the toxic effects of the venom. Among the active peptides of the pitviper venom, the ACE‐inhibiting peptides are important toxic components.…”

Synthesis and characterization of a novel potato starch derivative with cationic acetylcholine groups

“…ACh has been considered as an important excitatory neurotransmitter [15], and its physiological and pharmacological effects have been well documented [16][17][18]. Furthermore, ACh also can be absorbed and decomposed in biological body, changed into acetic and choline under acetylcholinesterase (AChe), both of which are useful for biological body [19]. Therefore, it is suggested that the product we synthesized has biology activity and can be used in the areas of food, cosmetics and medicine.…”

Inhibitory Effects of Sodium Arsenite and Acacia Honey on Acetylcholinesterase in Rats

“…Following the transmission of an impulse across the synapse by the release of ACh, AChE is released into the synaptic cleft [ 27 ]. This enzyme hydrolyzes ACh to choline and acetate, and transmission of the nerve impulse is terminated [ 28 ]. In this study, we report the effects of sodium arsenite and Acacia honey on acetylcholinesterase activity as well as the relationship with electrolyte levels.…”