The Folding Pathway of Disulfide Containing Proteins
191
Folding in the presence of redox agents
The procedures of unfolding and refolding are as those described in the control
folding experiments. Selected concentrations of redox agents were introduced
immediately after reduced and denatured proteins were collected from the PD-lO
column. Folding intermediates were similarly trapped by acidification or carboxymethylation as those described above.
Carboxymethylation of acid trapped intermediates isolated by HPLC
Acid trapped intermediates were separated and isolated by HPLC. The samples
were dried in a speedvac and immediately treated with 1M of iodoacetic acid in
0.1 ml of Tris-HCI buffer (0.5 M, pH 6.5) containing 40 % (by volume) of dimethylformamide (Chang, 1993). The reaction was allowed for 20 mins and the carboxymethylated intermediates were removed from the excess reagent and salt by a
NAP-5 column (Pharmacia).
Amino acid analysis, amino acid sequencing and MALDI mass spectrometry
Amino analysis was performed with the dabsyl chloride precolumn derivatization method which permits direct evaluation of the disulfide (cystine) content
(Chang & Knecht, 1991). Amino acid sequencing was done with a HewlettPackard G-lOOOA sequencer. The MALD! mass spectrometer was a home-built
time of flight (TOF) instrument with a nitrogen laser of 337 nm wavelength and
3ns pulse width (Boernsen et aI., 1990). The apparatus has been described in
detail elsewhere. The calibration was performed either externally or internally, by
using standard proteins (Hypertensin, M.W. 1031.19; Synacthen, 2934.50 and
Calcitonin, 3418.91).
4
Results and Discussion
4.1
The Folding Intermediates of Hirudin and TAP are Highly Heterogeneous
Reduced and denatured hirudin and TAP were first allowed to refold in the TrisHCI buffer in the absence and presence of thiol catalyst (0.25 mM ~mercaptoethanol). These two folding experiments were designated as "control
minus" (without ~-mercaptoethanol) and "control plus" (with ~-mercaptoethanol). Acid trapped intermediates were analyzed by HPLC and the results are
shown in Fig. l3.1 (Hirudin) and Fig. 13.2 (TAP). The HPLC profiles demonstrate
the heterogeneity of the intermediates, their progression along the folding pathway and the formation of native structure. In the two cases of "control plus"
experiments, the recovery of the native structure is nearly quantitative. However,
in the two cases of "control minus" experiments, about 50 % of the protein
becomes trapped as non-native species, unable to convert to the native species
even during prolonged folding (e.g up to 72 hours).
Each of these time-course trapped intermediates was extensively characterized
for the disulfide content (by dabsyl chloride method) and the composition of 1-,
2- and 3-disulfide species (by MALD! mass spectrometry). The data reveals that
there are three groups of intermediates, namely I-disulfide, 2-disulfide and 3-
191
Folding in the presence of redox agents
The procedures of unfolding and refolding are as those described in the control
folding experiments. Selected concentrations of redox agents were introduced
immediately after reduced and denatured proteins were collected from the PD-lO
column. Folding intermediates were similarly trapped by acidification or carboxymethylation as those described above.
Carboxymethylation of acid trapped intermediates isolated by HPLC
Acid trapped intermediates were separated and isolated by HPLC. The samples
were dried in a speedvac and immediately treated with 1M of iodoacetic acid in
0.1 ml of Tris-HCI buffer (0.5 M, pH 6.5) containing 40 % (by volume) of dimethylformamide (Chang, 1993). The reaction was allowed for 20 mins and the carboxymethylated intermediates were removed from the excess reagent and salt by a
NAP-5 column (Pharmacia).
Amino acid analysis, amino acid sequencing and MALDI mass spectrometry
Amino analysis was performed with the dabsyl chloride precolumn derivatization method which permits direct evaluation of the disulfide (cystine) content
(Chang & Knecht, 1991). Amino acid sequencing was done with a HewlettPackard G-lOOOA sequencer. The MALD! mass spectrometer was a home-built
time of flight (TOF) instrument with a nitrogen laser of 337 nm wavelength and
3ns pulse width (Boernsen et aI., 1990). The apparatus has been described in
detail elsewhere. The calibration was performed either externally or internally, by
using standard proteins (Hypertensin, M.W. 1031.19; Synacthen, 2934.50 and
Calcitonin, 3418.91).
4
Results and Discussion
4.1
The Folding Intermediates of Hirudin and TAP are Highly Heterogeneous
Reduced and denatured hirudin and TAP were first allowed to refold in the TrisHCI buffer in the absence and presence of thiol catalyst (0.25 mM ~mercaptoethanol). These two folding experiments were designated as "control
minus" (without ~-mercaptoethanol) and "control plus" (with ~-mercaptoethanol). Acid trapped intermediates were analyzed by HPLC and the results are
shown in Fig. l3.1 (Hirudin) and Fig. 13.2 (TAP). The HPLC profiles demonstrate
the heterogeneity of the intermediates, their progression along the folding pathway and the formation of native structure. In the two cases of "control plus"
experiments, the recovery of the native structure is nearly quantitative. However,
in the two cases of "control minus" experiments, about 50 % of the protein
becomes trapped as non-native species, unable to convert to the native species
even during prolonged folding (e.g up to 72 hours).
Each of these time-course trapped intermediates was extensively characterized
for the disulfide content (by dabsyl chloride method) and the composition of 1-,
2- and 3-disulfide species (by MALD! mass spectrometry). The data reveals that
there are three groups of intermediates, namely I-disulfide, 2-disulfide and 3-