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Biochimie

[Electrophoretic study of aged butyrylcholinesterase after inhibition by soman].


PMID 6331528

Abstract

The following states of purified tetrameric form (C4) of human plasma butyrylcholinesterase were studied by electrophoretic techniques: native, inhibited by soman and by methane sulfonyl fluoride and soman-aged. In order to detect a significant conformational change of the aged cholinesterase as compared to the non-inhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the cross-link products were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The peak areas of the cross-linked species and the parameters of cross-linkability were calculated from densitometric data, versus the maximal effective reagent length. The effect of occupancy of the esteratic site by substituted phosphonyl group and by methyl-sulfonyl residue on the binding activity of the anionic site was studied by affinity electrophoresis at varying temperatures with immobilized-procaïnamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities versus ligand concentration. Corresponding thermodynamic quantities were calculated from Van't Hoff plots and basic thermodynamic equations. The reactivity of aged-cholinesterase with diimidates was similar to that of the native enzyme. Affinity for immobilized-procaïnamide was slightly lowered in aged and inhibited enzymes as compared to the native and sulfonylated enzymes. As for the ligand-induced isomerization of anionic site (A----B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition (A = 0,5) was slightly greater for the aged enzyme than for the native one. From these results, the following conclusions can be drawn: the dealkylation of soman-cholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces and on the surface of the tetrameric enzyme. On the other hand, the affinity of the anionic site and ligand-induced isomerization process are altered in soman-inhibited and aged enzymes. These data suggest the occurrence of a weak conformational change of the active center and/or the formation of non-covalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction.