Purification and immunological characterization of pigeon serum butyrylcholinesterase. Implications on environmental monitoring and toxicological testing of birds.
Butyrylcholinesterase (EC 3.1.1.8) (BChE) was purified from pigeon serum to electrophoretic homogeneity by a four-step procedure involving blue sepharose CL-6B chromatography, ion exchange chromatography, procainamide affinity chromatography and gel filtration. An overall 2789-fold purification was achieved, with a final specific activity of 61.35 mumol/min/mg. The purified enzyme separated into two peaks when filtered through a column of Sephacryl S-300, a smaller peak containing the tetrameric form of BChE (C4) and a larger peak containing the monomeric form of BChE (C1). Native polyacrylamide gel electrophoresis (PAGE) of both peaks revealed single protein bands which coincided with esterase activity, with approximate M(r) values of 84,000 and 340,000, respectively. The C1 monomer represented 85-90% of the activity found in the pigeon serum. It is not clear whether this polymorphism of BChE in vertebrates contributes to the wider inter-individual variations observed in xenobiotics elimination kinetics and in the response to the pharmacological and toxic effects of pesticides. PAGE of the monomeric form of the enzyme in the presence of sodium dodecyl sulphate showed only one protein band with a M(r) of 84,000, while that of the tetrameric form revealed two bands, a major protein band (84,000) and a minor band (170,000), representing the monomer and the dimer of the dissociated tetrameric BChE enzyme under reducing conditions. Highly specific polyclonal antibodies were raised in rabbits against the purified enzyme. These antibodies cross-reacted with other avian BChEs, a criterion which make them useful for the immunopurification of other BChEs from different species as well as for biomonitoring and toxicological studies on the role of esterases as an indicator of avian exposure to organophosphorous pesticides.
Khattab
A D
AD
Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, U.K.
Walker
C H
CH
Mackness
M I
MI
Saphier
P W
PW
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002091:Butyrylcholinesterase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D002853:Chromatography, Liquid; D010856:Columbidae; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay; D007306:Insecticides / Q000378:metabolism / Q000633:toxicity; D011817:Rabbits; D013045:Species Specificity
D000906:Antibodies; D007306:Insecticides; D002091:Butyrylcholinesterase