Synthetisation of an affinity matrix (Procainamide Sepharose Cl-6b) for brain cholinesterase purification and separation source from Monopterus albus

Abstract

Affinity chromatography for acetylcholinesterase; AChE namely Procainamide Sepharose CL-6B was synthesised through the coupling method between soluble procainamide hydrochloride immobilised by a cross-linked agarose size exclusion, sepharose CL-6B. 1, 4-butanediol diglycidyl ether plays a role in building up a productive and rigid connecting of a biospecific ligand (Procainamide hydrochloride) to an insoluble matrix (Sephacryl CL-6B). Local freshwater eel brain was extracted and centrifuge at high speed. The supernatant was collected, and seven different volumes were separately loaded on to the column for isocratic purification where 12 fractions were collected at the end of elution stage. This study shows 1000 μL of extractant; considered as the maximum volume to load onto the column. Lastly, a stepwise elution was performed with five different concentrations of NaCl, and each of 1 mL fraction was collected then assay for determination of enzyme activity and protein content. The data shows AChE was successfully purified with percentage recovery of 38 % after 21 purification fold. Kinetic study strengthens the data where the efficient coefficient ratio of ATCi was much higher compared to PTCi and BTCi at 3.03, 2.67 and 1.52 Vmax⋅Km −1, respectively, prove that the collected fraction predominantly contained with AChE, which is a targeted enzyme to be used as a sensitive biosensor to detect the presence of carbamate and organophosphate contamination in the environment.