Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O

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.