Use of procainamide gels in the purification of human and horse serum cholinesterases

Abstract

Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase.

Purification and Anticholinesterase Sensitivity of Cholinesterase Extracted from Liver Tissue of Puntius Javanicus

“…The supernatant was collected and stored at -25°C. Procainamide-sephacrayl 6B synthetization: Procainamide-based affinity matrix was synthesised based on the method of Ralston et al (1983) with slight modification from Son et al (2002). Sephacryl 6B (50 mL settled gel) was washed with 10 volumes of deionised water using a sintered glass tunnel as recommended by the manufacturer, the Sigma Chemical Company (Poole, UK), sucked dry to a wet cake, then suspended in a volume of 0.6 M NaOH containing 50mM sodium borohydrate (Sigma, St. Louis, USA) and stirred.…”

Acetylcholinesterase active centre and gorge conformations analysed by combinatorial mutations and enantiomeric phosphonates

“…Several litres of media were subjected to affinity chromatography to purify the mutant enzymes, typically in amounts of 2-10 mg. Procainamide affinity resin utilized CNBr-activated Sepharose CL-4B resin with a hexanoic alkyl chain [15]. Harvested ultraculture medium containing the expressed enzyme was centrifuged (2000 g for 15 min at 4…”

Design of acetylcholinesterases for biosensor applications

“…Two approaches are usually applied for the purification of the enzymes: ion exchange chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998) and affinity chromatography (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Villatte et al, 1998;Villatte et al, 2000) based on the chemical modification of the support with reversible AChE inhibitors (Massoulie and Bon, 1976;Ralston et al, 1983) or on the addition of a His-tag to the enzyme (Estrada-Mondaca and Fournier, 1998;Heim et al, 1998;Hussein et al, 1999;Hussein et al, 2000).…”