The conformational transition of horse heart porphyrin c.

This research examines the structural changes in horse heart cytochrome c to porphyrin c using anhydrous HF, and how it reacts to denaturation through guanidine hydrochloride and heat. Notably, the researchers found that porphyrin c refolds in two kinetic phases and is not significantly influenced by the viscosity of the refolding solvent.

Excision of Heme Iron and Conforming to Porphyrin C

• The heme iron of the horse heart cytochrome c was selectively excised using anhydrous HF. The resulting product, porphyrin c, exhibited viscosity, far ultraviolet circular dichroic, and fluorescence properties that are characteristic of native cytochrome c.

Susceptibility to Denaturation

• The research found that porphyrin c was more susceptible to denaturation by guanidine hydrochloride and by heat than its parent molecule, cytochrome c.

Conformational Transitions

• All the conformational parameters of porphyrin c exhibited a common reversible transition at a point centered at 0.95 m guanidine hydrochloride at a temperature of 23 degrees Celsius and a pH of 7.0.

Refolding of Porphyrin C

• Porphyrin c refolded in two kinetic phases with time constants of 20 and 200 ms. These phases were detected by stopped flow absorbance or fluorescence measure.
• About 80% of this change was observed in the faster of the two phases.
• The kinetics of porphyrin c refolding were not significantly altered by increasing the viscosity of the refolding solvent 15-fold by adding sucrose, suggesting that the folding process is not diffusion-limited.

Final Suggestions

• The research suggests that the folding of guanidine denatured cytochrome c is not a diffusion-limited process.
• Also, the requirement for protein axial ligation led to the slow kinetic phase observed during the refolding of cytochrome c. This observation might give insights into the structural dynamics and stability of these protein molecules.