Isolation, purification and characterization of human butyrylcholinesterase [HuBuChe]: A potential in vivo treatment for organophosphorous nerve poisoning

Acetylcholine is a very important chemical neurotransmitter in the central nervous system (CNS) as well as in the parasympathetic nervous system in many organisms including humans. There are two major forms of cholinesterase (ChEs) containing in the nerve system of mammals, acetylcholinesterase (ACNE) and butyrylcholinesterase (BuChE) which can hydrolyze acetylcholine to choline and acetate. BuChE is considered as the protection of AChE against toxic xenobiotics.; Human serum butyrylcholinesterase (HuBuChE) is the most suitable BuChE for human use. In order to have a rapid process to isolate and purify HuBuChE from human plasma, several chromatographies were applied. During the separation we found that HuBuChE didn't exactly follow the theory of ion exchange chromatography. The general procedure of ion exchange chromatography is increasing the pH from 4 to 8 and increasing the sodium chloride concentration to elute the enzyme. But HuBuChE wasn't retained in the ion exchange column. We used ultrafiltration to desalt and concentrate the collected fractions. It reduced the experimental hour into half, which is suited our goal to develop a rapid process for HuBuChE purification. In the purification procedures we obtained approximately 35 mg from 500 mL Cohn Fraction IV-4.; In this project we also investigated two major categories: enzyme kinetics and inhibition studies. The investigations were confirmed that AChE can only hydrolyze acetylthiocholine, but BuChE can work on both acetylthiocholine and butyrylthioncholine. Water hydrolysis took place on both substrates, ATC and BTC.; The results of NaCl and procainamide inhibition studies supported the modified procedures of purification. Procainamide inhibited HuBuChE but NaCl didn't, so in affinity chromatography we chose sodium chloride buffer solution to elute the enzyme instead of using procainamide solution. DIFP is organophosphate analogous, and we used it as a substitute of higher toxic compound for inhibition study. DIFP inhibited more than 50% of enzyme activity in a very low concentration, 25 mM.