Refolding of surface epitopes on horse cytochrome c has been measured by monoclonal antibody binding. Two antibodies were used to probe re-formation of nativelike surface structure: one antibody (2B5) binds to native cytochrome c near a type II turn (residue 44) while the other (5F8) binds to a different epitope on the opposite face of the protein near the amino terminus of an α-helical segment (residue 60). The results show that within the first ∼ 100 ms of refolding all of the unfolded protein collapses to nativelike folding intermediates that contain both antibody binding sites. All three absorbance/fluorescence-detected kinetic phases in the folding of cytochrome c (k1 ∼ 5 s−1, k2 ∼ 0.4 s−1, k2 ∼ 0.03 s−1) are slower than the rates of re-formation of the antibody binding sites (kobs > 10.0 s−1), suggesting that the formation of antibody binding sites precedes the refolding reactions observed in kinetically resolved optically-detected refolding phases. Kinetically unresolved folding processes account for 79% and 19% of the total fluorescence change and absorbance change, respectively, observed in equilibrium unfolding. Thus, kinetically unresolved folding reactions appear to be responsible for re-formation of the M Ab binding sites within partially folded intermediate species. These species are nonnative (incompletely folded) in that their optical properties are in between those of the unfolded and the fully folded protein. As a test of whether antibody binding to folding intermediate(s) perturbs further folding, the rate of the absorbance-detected slow refolding phase has been measured for folding intermediate(s) of cytochrome c complexed with antibodies. Neither antibody had a significant effect on the rate of the absorbance-detected slow folding reaction, suggesting that changes in protein conformation associated with the absorbance-detected slow folding phase are not affected by possible antibody-induced changes in structure or stabilization of the epitopes. Since epitopes of protein antigens cover an extensive surface area (700–800 Å2), the results show the major aspects of the nativelike surface topography are formed early (<100 ms) in folding intermediates.
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