Water requirements in monomer folding and dimerization of triosephosphate isomerase in reverse micelles. Intrinsic fluorescence of conformers related to reactivation.
The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)
Fernández-Velasco
D A
DA
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F. México.
Sepúlveda-Becerra
M
M
Galina
A
A
Darszon
A
A
Tuena de Gómez-Puyou
M
M
Gómez-Puyou
A
A
D000818:Animals; D001704:Biopolymers / Q000737:chemistry; D004789:Enzyme Activation; D008823:Micelles; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D001704:Biopolymers; D008823:Micelles; D014867:Water; D014305:Triose-Phosphate Isomerase