The folding pathway of human epidermal growth factor (EGF) has been charact
erized by structural and kinetic analysis of the acid-trapped folding inter
mediates. Oxidative folding of the fully reduced EGF proceeds through 1-dis
ulfide intermediates and accumulates rapidly as a single stable 2-disulfide
intermediate (designated as EGF-II), which represents up to more than 85%
of the total protein along the folding pathway. Among the five 1-disulfide
intermediates that have been structurally characterized, only one is native
, and nearly all of them are bridges by neighboring cysteines, Extensive ac
cumulation of EGF-II indicates that it accounts for the major kinetic trap
of EGF folding. EGF-II contains two of the three native disulfide bonds of
EGF, Cys(14)-Cys(31) and Cys(33)-Cys(42). However, formation of the third n
ative disulfide (Cys(6)-Cys(20)) for EGF-II is slow and does not occur dire
ctly. Kinetic analysis reveals that an important route for EGF-II to reach
the native structure is via rearrangement pathway through 3-disulfide scram
bled isomers. The pathway of EGF-II to attain the native structure differs
from that of three major 2-disulfide intermediates of bovine pancreatic try
psin inhibitor (BPTI). The dissimilarities of folding mechanism(s) between
EGF, BPTI, and hirudin are discussed in this paper.