DISSOCIATION CONSTANTS FOR REVERSIBLE HOMODIMERS OF RECOMBINANT MONOMERIC HUMAN ACETYLCHOLINESTERASE

Abstract

Size exclusion chromatography profiles of highly purified monomeric recombinant human acetylcholinesterase (hAChE; EC 3.1.1.7) at micro molar concentration range reveal elution volumes smaller than expected for a monomeric globular protein comparable in size to the bovine serum albumin. Dilution of hAChE to the nano molar concentration range results in the elution peak shifts towards larger volumes indicating formation of hAChE monomers. This observation is consistent with small angle X‐ray scattering (SAXS) experiments on hAChE that indicate formation of reversible homodimers of the micro molar monomeric recombinant enzyme in solution (Blumenthal et al., EB2016 abstract).FPLC chromatography of a series of concentrations of recombinant monomeric hAChE in the range of 0.00001 – 1 mg/ml (150 pM‐15uM) was performed to assess concentration dependent elution shifts and determine dimer dissociation constants in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 4 ºC. Peaks of enzyme activity in FPLC elution fractions were detected using Ellman spectrophotometric activity assay. Wild type hAChE constructs with and without N‐terminal FLAG tag sequence as well as Tyr337Ala/Phe338Ala double mutant hAChE not containing FLAG tag were assayed. Size of the hAChE active center gorge in the choline binding domain was increased in this double mutant. Corresponding dimer dissociation constants were determined to be in the range of 1 – 2 uM for both wild type and mutant hAChE, consistent with hAChE oligomerisation interface residing outside of the active center gorge opening as indicated by crystallographic dimers of hAChE.Support or Funding InformationThis work was supported by the Counter ACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.