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Electrophoretic study of aged soman inhibited butyrylcholinesterase ec

Biochimie (Paris) 66(3): 235-250

Electrophoretic study of aged soman inhibited butyrylcholinesterase ec

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 noninhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the crosslink products were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peak areas of the crosslinked species and the parameters of crosslinkability were calculated from densitometric data, vs. 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-procainamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities vs. 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-procainamide 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 .fwdarw. B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition (.hivin.A = 0.5) was slightly greater for the aged enzyme than for the native one. The dealkylation of soman-cholinesterase conjugate (aging) does not seem to induce structural changes detectable in the crosslinkability of lysyl residues at the subunit interfaces and on the surface of the tetrameric enzyme. The affinity of the anionic site and ligand-induced isomerization process were altered in soman-inhibited and aged enzymes. The occurrence of a weak conformational change of the active center and/or the formation of noncovalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction was suggested.

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Accession: 005349023

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