The Protein Journal

, Volume 28, Issue 6, pp 300–304 | Cite as

Fast and Slow Tracks in Lysozyme Folding Elucidated by the Technique of Disulfide Scrambling



GdmCl (6 M) unfolded lysozyme was previously shown to refold via kinetically partitioned pathways (Kiefhaber in Proc Natl Acad Sci 92:9029–9033, 1995). About 80% of the unfolded lysozyme molecules refold on a slow pathway with well-populated intermediates. The remaining 20% of denatured lysozyme refold on a fast track without detectable intermediate. This kinetic heterogeneity has been proposed to originate from the collapsed state of lysozyme folding. Using the method of disulfide scrambling, we demonstrate in this report that these two populations of unfolded lysozyme can be isolated and analyzed separately. GdmCl (6 M) denatured lysozyme actually comprises two major populations of unfolded isomers, namely X-LYZ-a and X-LYZ-b with molar ratio of about 80:20. X-LYZ-a and X-LYZ-b exist in equilibrium in the unfolded state. Their disulfide structures and CD properties indicate that X-LYZ-a is more extensively unfolded than X-LYZ-b. Refolding experiments using the method of disulfide scrambling also show that folding kinetics of X-LYZ-a is about 8–10 times slower than that of X-LYZ-b and folding intermediates of X-LYZ-a is far more heterogeneous than that of X-LYZ-b. The results highlight the implication of the conformational heterogeneity of 6 M GdmCl denatured proteins for the interpretation of the initial stage of protein folding mechanism.


Method of disulfide scrambling Isomers of unfolded protein Denaturation and unfolding Scrambled lysozyme Conformational isomers Lysozyme 





Scrambled lysozyme




High pressure liquid chromatography



The author acknowledges the support of IsoVax Therapeutic Inc. and the endowment from the Robert Welch foundation.


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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Research Center for Protein ChemistryBrown Foundation Institute of Molecular MedicineHoustonUSA
  2. 2.Department of Biochemistry and Molecular BiologyThe University of TexasHoustonUSA

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