Unfolding and refolding of Sol - Gel encapsulated carbonmonoxymyoglobin: An orchestrated spectroscopic study of intermediates and kinetics

U. Samuni, M. S. Navati, L. J. Juszczak, D. Dantsker, M. Yang, J. M. Friedman

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

The pH-induced unfolding and refolding of the carbon monoxide-bound derivative of horse skeletal myoglobin (COMb) encapsulated in porous sol-gels is probed using several optical techniques in conjunction with different unfolding/refolding protocols. UV resonance Raman (UVRR) spectroscopy and fluorescence are used to monitor the unfolding of the globin and exposure of the A helix to solvent. Absorption spectra, visible resonance Raman (VRR) spectra, and geminate recombination are used to probe the heme and the heme environment. Encapsulation slows the kinetics of acid-induced unfolding and dramatically slows the kinetics of refolding. The spectra and the kinetics imply that this approach allows for the detailed study of the burst phase of unfolding. Using different encapsulation protocols and sequences of solvent replacements, it is possible to trap and probe not only low-pH forms observed 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 occur 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 exposure 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 refolding of the A helix is likely to be the last process in the refolding pathway.

Original languageEnglish (US)
Pages (from-to)10802-10813
Number of pages12
JournalJournal of Physical Chemistry B
Volume104
Issue number46
StatePublished - Nov 23 2000

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

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