title: Quantitative monitoring by high-performance liquid chromatography of the dissociation of human serum cholinesterase by limited proteolysis.
abstract: Proteolytic action on human serum cholinesterase, a tetrameric enzyme, results in a partial disintegration which can be recorded only qualitatively by time-consuming electrophoretic techniques. In this study, a rapid high-performance liquid chromatographic method was used for the separation and determination of the active dissociation products. Separation of the cholinesterase subunits was accomplished by high-performance gel permeation chromatography on a combination of DIOL columns (Zorbax GF 450/GF 250) in 0.2 M phosphate buffer (pH 7.0). Detection and quantification of enzyme activity in the fractionated eluate were carried out using a Flexigem analyser (substrate, butyrylthiocholine). On limited tryptic digestion of partially purified human ChE, up to three peaks of enzyme activity could be identified. Their elution volumes corresponded to apparent molecular masses of 480,000, 270,000 and 120,000, indicating, in addition to the tetrameric holoenzyme, a dimeric and a monomeric form. Quantification of the relative amounts of individual enzyme activity peaks revealed that in the course of degradation, the dimer appeared first, followed by the monomer. This suggests that the first step in the sequence of dissociation is cleavage of the tetramer into a pair of dimers, then further into the monomeric subunit. During the incubation with trypsin, a significant change in the pattern of the different peaks had already occurred when the total enzyme activity was only slightly reduced.
authors: Abteilung Klinische Chemie, Robert-Bosch-Krankenhaus, Stuttgart, F.R.G.
authors: K P
authors: KP
authors: Kohse
authors: H
authors: H
authors: Wisser
mesh_terms: D002802:Cholinesterases / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008991:Monitoring, Physiologic; D014357:Trypsin / Q000494:pharmacology
chemical_list: D002802:Cholinesterases; D014357:Trypsin