S-mercuric-N-dansyl-cysteine labels the free sulfhydryl groups of human serum cholinesterase.
Human serum cholinesterase (BChE) has a putative sulfhydryl group (Cys-66) which is unreactive toward conventional alkylating agents such as iodoacetic acid, raising the possibility that this group is blocked in native BChE. In order to test this further, we examined the reactivity of BChE towards the sulfhydryl-specific reagent S- mercuric-N-dansylcysteine (SMNDC). Stoichiometric binding of 4 mol SMNDC/mol of tetrameric enzyme was observed in fluorescence titration experiments, with retention of catalytic activity. SMNDC remained bound to the protein during SDS-polyacrylamide gel electrophoresis in the absence of reducing agent and the fluorescence pattern observed under U.V. light coincided with the Coomassie Blue stained bands. Addition of excess dithiothreitol to the SMNDC-labeled enzyme resulted in the complete removal of bound SMNDC. Thus, Cys-66 appears to be present in the free sulfhydryl form in BChE, analogous to the corresponding free thiol group (Cys-231) of Torpedo californica acetylcholinesterase. As is the case with the latter species, BChE (labeled or unlabeled) is inactivated by 1.0 x 10(-4)M ZnSO4.
Tomlinson
G
G
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Kinsch
E M
EM
D002091:Butyrylcholinesterase; D002802:Cholinesterases; D003545:Cysteine / Q000031:analogs & derivatives; D006801:Humans; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D015032:Zinc / Q000494:pharmacology
D013439:Sulfhydryl Reagents; C028374:S-mercuric-N-dansylcysteine; D002091:Butyrylcholinesterase; D002802:Cholinesterases; D015032:Zinc; D003545:Cysteine