PURIFICATION AND DETERMINATION OF THE AMINO ACID SEQUENCE OF EQUINE SERUM BUTYRYLCHOLINESTERASE

Abstract

Butyrylcholinesterase (Eq-BChE) from equine (horse) serum has been used extensively to demonstrate the efficacy of cholinesterase as a single pretreatment drug (bioscavenger) for organophosphate toxicity in rodents and nonhuman primates. It has the longest mean retention time in animals among all ChE preparations examined. The authors describe the complete amino acid sequence of Eq-BChE. Like other mammalian BChEs, it is a glycosylated protein composed of four identical subunits. The apparent molecular weight of the subunit containing 574 amino acids and the estimated carbohydrate content of 26% is 84,551 Da. It has eight carbohydrate-modified sites at positions 57, 106, 241, 256, 341, 455, 481, and 486 as N-linked Asn residues. The active site charged triad aminoacids are Ser198, Glu325, and His438. The N-terminal aminoacids are GluGluAspIle, consistent with this being the mature form of the enzyme. In addition, a monomeric form of this enzyme that corresponds to 4% of the total BChE activity was isolated and purified. Comparison of Eq-BChE with other species shows that there is a high degree of primary sequence identity. There is a 90.1% identity with the human serum BChE sequence, although the equine enzyme has one less N-glycosylation site and Cys residue. If conserved amino acid substitutions are included then the proteins share 93.4% homology.