192
(
N
Control24 h
m
3 h
l h
5 min
J. Y. CHANG
I
N
Control +
24 II
1 h
5 min
Fig. 13.1. HPLC profiles of acid-trapped folding intermediates of Hirudin core domain residues
1-49. Folding was performed in the Tris-HCI buffer (0.1 M, pH S.4) in the absence (control minus)
and presence (control plus) of b-mercaptoethanol (0.25 mM). The samples were analyzed by HPLC
using the following conditions. Solvent A was water containing 0.1 % trifluoroacetic acid. Solvent B
was acetonitrile/water (9:1, by volume) containing 0.1 % trifluoroacetic acid. The gradient was 14 % to
32 % solvent B linear in 50 mins. Column was Vydac C-lS for peptides and proteins, 4.6 mm, 10 mm.
Column temperature was 23oC. R (orange color) and N (blue color) indicate the elution positions of
the fully reduced and the native species. Fractions containing 1- and 2-disulfide species are colored
with green. Those containing 3-disulfide species are colored with red
disulfide (scrambled) species, which overlap and advance sequentially along the
folding pathway. Those which become trapped in the case "control minus" folding are 3-disulfide, scrambled species, unable to convert to the native species
because of the depletion and absence of free thiol as catalyst. ~-mercaptoethanol
is not the only reagent that is useful for "control plus" folding experiment, other
thiol reagents, such as cysteine and reduced glutathione are equally effective. If
one compares the patterns of folding intermediates performed in the absence and
presence of ~-mecaptoethanol, it becomes very clear that the only difference is
the relative level of accumulation of scrambled 3-disulfide species and the recovery of the native species. We have found this phenomenon in all four proteins
that have been analyzed in our lab.
(
N
Control24 h
m
3 h
l h
5 min
J. Y. CHANG
I
N
Control +
24 II
1 h
5 min
Fig. 13.1. HPLC profiles of acid-trapped folding intermediates of Hirudin core domain residues
1-49. Folding was performed in the Tris-HCI buffer (0.1 M, pH S.4) in the absence (control minus)
and presence (control plus) of b-mercaptoethanol (0.25 mM). The samples were analyzed by HPLC
using the following conditions. Solvent A was water containing 0.1 % trifluoroacetic acid. Solvent B
was acetonitrile/water (9:1, by volume) containing 0.1 % trifluoroacetic acid. The gradient was 14 % to
32 % solvent B linear in 50 mins. Column was Vydac C-lS for peptides and proteins, 4.6 mm, 10 mm.
Column temperature was 23oC. R (orange color) and N (blue color) indicate the elution positions of
the fully reduced and the native species. Fractions containing 1- and 2-disulfide species are colored
with green. Those containing 3-disulfide species are colored with red
disulfide (scrambled) species, which overlap and advance sequentially along the
folding pathway. Those which become trapped in the case "control minus" folding are 3-disulfide, scrambled species, unable to convert to the native species
because of the depletion and absence of free thiol as catalyst. ~-mercaptoethanol
is not the only reagent that is useful for "control plus" folding experiment, other
thiol reagents, such as cysteine and reduced glutathione are equally effective. If
one compares the patterns of folding intermediates performed in the absence and
presence of ~-mecaptoethanol, it becomes very clear that the only difference is
the relative level of accumulation of scrambled 3-disulfide species and the recovery of the native species. We have found this phenomenon in all four proteins
that have been analyzed in our lab.