The reaction of S-mercuric-N-dansylcysteine with acetylcholinesterase and butyrylcholinesterase.
S-mercuric-N-dansylcysteine was investigated as a potential probe of protein sulphydryl groups using bovine serum albumin, S-carboxymethyl-bovine serum albumin, lysozyme, and partially reduced lysozyme as test proteins. Criteria used to assess covalent binding through mercury-bridged mercaptide linkages include a finite reaction time (minutes to hours), abolition of the characteristic fluorescence spectrum following addition of a reducing agent, and failure to separate probe and protein after chromatography or electrophoresis. By these criteria, both Torpedo californica acetylcholinesterase and human serum cholinesterase (butyrylcholinesterase) contain four free sulphydryl groups per tetrameric enzyme molecule whereas Electrophorus electricus acetylcholinesterase has none. Labeled acetylcholinesterase and butyrylcholinesterase remain active and responsive to the inactivator Zn2+. Zn2+ promotes an increase in the fluorescence of bound S-mercuric-N-dansylcysteine, whereas activators such as Mg2+ or gallamine promote a decrease, suggesting that the label may be a useful probe of ligand-induced conformational changes. With T. californica acetylcholinesterase, but not with human serum cholinesterase, Zn2+ also promotes access to two additional groups that are reactive towards the sulphydryl reagent.
Tomlinson
G
G
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Kinsch
E M
EM
D000110:Acetylcholinesterase / Q000097:blood*; D000818:Animals; D002091:Butyrylcholinesterase / Q000097:blood*; D055598:Chemical Phenomena; D002621:Chemistry; D002800:Cholinesterase Inhibitors; D002802:Cholinesterases / Q000097:blood*; D002850:Chromatography, Gel; D003545:Cysteine / Q000494:pharmacology / Q000031:analogs & derivatives; D004557:Electric Organ / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004593:Electrophorus; D005453:Fluorescence; D006801:Humans; D066298:In Vitro Techniques; D013045:Species Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis; D014101:Torpedo; D015032:Zinc / Q000494:pharmacology
D002800:Cholinesterase Inhibitors; D013438:Sulfhydryl Compounds; C028374:S-mercuric-N-dansylcysteine; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D015032:Zinc; D003545:Cysteine