title: Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O.
abstract: Guanidine hydrochloride (GdnHCl) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50-100-fold in a 'renaturation buffer'. Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows. Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short-lived intermediate during the folding of the beta 2-subunit of Escherichia coli tryptophan synthase.
authors.affiliation: Département de Biochimie et Génétique Moléculaire, Institut Pasteur, Paris, France.
authors.forename: S
authors.initials: S
authors.lastname: Blond-Elguindi
authors.forename: B
authors.initials: B
authors.lastname: Friguet
authors.forename: M E
authors.initials: ME
authors.lastname: Goldberg
mesh_terms: D003903:Deuterium; D017666:Deuterium Oxide; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D014367:Tryptophan Synthase / Q000378:metabolism*; D014867:Water
chemical_list: D006146:Guanidines; D014867:Water; D003903:Deuterium; D014367:Tryptophan Synthase; D017666:Deuterium Oxide; D019791:Guanidine