Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes


KAYA H. B. , OZCAN B., ŞİŞECİOĞLU M. , ÖZDEMİR H.

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, cilt.170, ss.198-209, 2013 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 170 Konu: 1
  • Basım Tarihi: 2013
  • Doi Numarası: 10.1007/s12010-013-0177-3
  • Dergi Adı: APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
  • Sayfa Sayıları: ss.198-209

Özet

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and L-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5%, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5'-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K-m, V-max, optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25-35 degrees C. The Michaelis Menten constant (K-m) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V-max was 0.090 mu mol/mL/min. Maximum binding was achieved at 2 degrees C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.