Unfolding and refolding of sol-gel encapsulated carbonmonoxymyoglobin: An orchestrated spectroscopic study of intermediates and kinetics?

The pH-induced unfolding and refolding of the carbon monoxide-bound derivat ive of horse skeletal myoglobin (COMb) encapsulated in porous sol-gels is p robed using several optical techniques in conjunction with different unfold ing/refolding protocols. UV resonance Raman (UVRR) spectroscopy and fluores cence are used to monitor the unfolding of the globin and exposure of the A helix to solvent. Absorption spectra, visible resonance Raman (VRR) spectr a, and geminate recombination are used to probe the heme and the heme envir onment. Encapsulation slows the kinetics of acid-induced unfolding and dram atically slows the kinetics of refolding. The spectra and the kinetics impl y that this approach allows for the detailed study of the burst phase of un folding. Using different encapsulation protocols and sequences of solvent r eplacements, it is possible to trap and probe not only low-pH forms observe d in solution-phase studies but also novel partially unfolded species that are likely to be important unfolding and folding intermediates. The role of water as a chaotropic agent is indicated by the spectral changes that occu r in the introduction and subsequent removal of glycerol from the solution bathing the unfolded and partially unfolded, sol-gel encapsulated COMb. The results directly support the view that unfolding or increasing the exposur e to solvent of at least some segment of the A helix is the initial step in the unfolding pathway. In addition, the results indicate that the refoldin g of the A helix is likely to be the last process in the refolding pathway.