Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin.
The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.
D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D013879:Thioredoxins / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D006146:Guanidines; D013879:Thioredoxins; D019791:Guanidine