title: Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?
abstract: A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.
authors.affiliation: Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: I
authors.initials: I
authors.lastname: Shin
authors.forename: E
authors.initials: E
authors.lastname: Wachtel
authors.forename: E
authors.initials: E
authors.lastname: Roth
authors.forename: C
authors.initials: C
authors.lastname: Bon
authors.forename: I
authors.initials: I
authors.lastname: Silman
authors.forename: L
authors.initials: L
authors.lastname: Weiner
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000873:Anthracenes; D017838:Bungarus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient / Q000379:methods; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D008823:Micelles; D008832:Microchemistry; D010569:Perylene / Q000031:analogs & derivatives* / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
chemical_list: D000873:Anthracenes; D008823:Micelles; D010743:Phospholipids; D010569:Perylene; C004965:hypericin; D000110:Acetylcholinesterase