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A-1757
Application␣ Information
Proteins
...............................................
Monitoring Disulfide Formation
with P/ACE Capillary Electrophoresis
Robert P. Oda, Jane A. Liebenow, T. C. Spelsberg, and James P. Landers
Mayo Clinic, Rochester, MN 55905
Introduction
Experimental Conditions
The ability to monitor intermolecular disulfide
bond (dimer) formation in biological systems is of
paramount importance for two reasons: 1) in many
peptides and proteins, disulfide bonds are necessary for biological activity; 2) where disulfide
bond formation is undesirable, gentle oxidation of
a peptide can be performed without generation of
the dimer which would form under stronger conditions. In both cases, the ability to rapidly measure
disulfide bond formation is required. Typically,
colorimetric assays are used to determine these
processes. However, while giving qualitative “yes/
no” answers, they are limited with respect to
quantitative information and the type of bond formation (i.e., homo- or hetero-dimer). Capillary
electrophoresis (CE) is a relatively new analytical
technique capable of resolving subtle differences
between proteins and peptide conformations(1,2).
In this Application Information Bulletin, the utility of CE to monitor peptide dimer formation is
demonstrated.
CE instrument:
P/ACE™ System 2050 with Gold™
(version 7.11) software
Polarity:
Normal (cathode at detector end)
Capillary:
Uncoated, 57 cm (50 cm to detector) x 50 µm i.d.
Temperature:
28°C
Run buffer:
20 mM sodium citrate or 50 mM
sodium phosphate, pH 2.5
Applied voltage: 25 kV
Injection:
pressure, 3 s
CE run method
sequence:
3 column volumes rinse with
run buffer, pressure injection, separation, 5 column volumes rinse
with 0.1 M NaOH, 5 column volumes rinse with run buffer
Detection:
UV, 200 nm
Ntc primary
structure:
CFLGIPFAEPPVGSRRFMPPEP
KRPWSGVL
Ctc primary
structure:
TFQTNPDGTIQFRC
Sample
preparation:
See reference 3 for details
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Results and Discussion
faster migration times than their corresponding
monomers. Mixtures of monomers and dimers were
next subjected to strong oxidizing (with H2O2) and
reducing conditions (with dithiotreitol—DTT). The
results of the CE analysis of Ntc peptide are shown
in Figure 1. Reducing a 2:1 mixture of dimer/monomer with 1 mM DTT leaves only 4% of the Ntc
dimer peak (Figure 1, lower left panel). Likewise,
oxidizing a 1:2 dimer/monomer mixture with 0.015%
H2O 2 reduces the monomer peak by 90% (Figure 1,
lower right panel). The conversion between the Ntc
monomeric and dimeric forms is rapid and virtually
complete within 30 min. In the experiment of Figure
1, CE analysis was commenced 2-3 min after adding
the reagent to the dimer/monomer mixture.
Two synthetic peptides were used to evaluate the
utility of CE for the separation of peptide monomers
from their disulfide-linked dimers. One peptide (molecular mass 3369), termed N-terminal cysteine or
Ntc peptide, consisted of a 30-mer which contained
residues 32-60 of mouse mRNA acetylcholine
esterase plus an N-terminal cysteine. The other peptide (molecular mass 1629) was called Ctc peptide,
and consisted of a 14-mer with 13 residues from a
proprietary protein and one C-terminal cysteine residue. The disulfide-linked homo-dimers of these peptides were generated under controlled air oxidizing
conditions(3) and subsequently purified. It was found
that the purified homo-dimers electrophoresed with
Monomer
Dimer
No Treatment
Dimer
Absorbance (200 nm)
Monomer
+ H2O2
+ DTT
1
5
10
15
1
5
10
15
Time (minutes)
Figure 1. Oxidization and reduction of an Ntc monomer/dimer mixture. A 1:2 mixture of monomer/dimer is reduced
in the presence of 1 mM DTT (left panels) while a 2:1 mixture monomer/dimer is oxidized in the presence of 0.015%
H2O2 (right panels). Separation was carried out in 20 mM citrate buffer, pH 2.5. Bar represents 0.002 AU. Arrow
indicates the sulfonic acid derivative.
2
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shown in panel B. The purified Ntc dimer was subjected to the same oxidizing conditions as the monomer; however, in this case no decrease in peak height
is shown (see right hand panels of Figure 2), supporting the argument that the loss in peak height is
caused by the dimerization and not to a degradation
process.
The time-course of peptide dimerization monitored by CE is shown in Figures 2 and 3. The dimerization process is temperature-dependent.
Figure 2 shows that approximately 32% of the
Ntc monomer (peak at 8.2 min) was converted to the
dimer (peak at 7.2 min) after 2 h of incubation at
27oC (Panel C). Approximately the same conversion
rate was observed after 24 h incubation at 4oC, as
A
D
Monomer
Dimer
Absorbance (200 nm)
0 hours
B
E
24 hours, 4°C
C
F
2 hours, 27°C
1
5
10
15
1
5
10
15
Time (min)
Figure 2. Oxidization of Ntc peptide. Purified Ntc monomer and dimer were dissolved in water (2 mg/mL), analyzed
immediately (0 h) and after incubation at 27°C for 2 h and 4°C for 24 h. Bar represents 0.005 AU.
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Figure 3 shows the time-course of CE analysis
with Ctc peptide. The dimerization process of Ctc
peptide is markedly slower than that observed with
Ntc peptide. After 8 h under mild oxidizing conditions, only negligible conversion to the dimer (indicated with an arrow in Figure 3) was observed. As
was the case with the Ntc peptide, the Ctc dimer has
a faster electrophoretic mobility than its monomer.
The two forms are baseline resolved by CE. To induce substantial oxidation, 0.015% H2O2 was added.
It can be seen that another 8 h of incubation caused a
59% conversion of the monomer to the dimer. It appears that the rate of dimerization is influenced by
the peptide structure (size, amino acid composition
and sequence) and/or the location of the cysteine
residues (N- vs. C-terminal).
8 h 07 min
e (2
00 n
m)
2 h 29 min
orb
anc
immediate
Abs
H2O2
8 h 02 min
3 h 28 min
immediate
1
5
10
15
Time at 27°C
Migration Time (min)
Figure 3. CE time-course analysis of the Ctc dimerization process. Ctc peptide (1 mg/mL in water) was incubated at
27°C and analyzed at 0 min (immediately), 3 h 28 min, and 8 h 2 min. Hydrogen peroxide was added and analysis
carried out at 0 min (immediately), 2 h 29 min, and 8 h 7 min. Separation was carried out in 20 mM citrate buffer,
pH 2.5. Bar represents 0.005 AU.
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Evidence for hetero-dimer formation is presented in Figure 4. A mixture of the purified Ntc and
Ctc monomers was incubated at 27°C and analyzed at
0 min (panel A), 2 h 46 min (panel B), 5 h 31 min
(panel C) and 11 h (panel D). Substantial Ntc:Ntc
homo-dimer formation can be observed in panel B,
A
whereas Ctc:Ctc dimerization is relatively slow. The
peak at 8.6 min, appearing between the Ntc monomer
and dimer, is the Ntc:Ctc hetero-dimer. Only after 11
h (panel D) has a definable amount of the Ctc homodimer been formed. This result would be expected in
view of the slow dimerization rate of the Ctc peptide.
C
Ntc
Ctc
Ntc:Ctc
Absorbance (200 nm)
Ctc:Ctc
B
D
Ntc:Ntc
Ctc:Ctc
Ctc:Ctc
1
5
10
15
1
5
10
15
Time (minutes)
Figure 4. Co-oxidization of the Ctc and Ntc peptides. A mixture of the purified Ntc and Ctc monomers was
incubated at 27°C and analyzed at 0 min (Panel A), 2 h 46 min (Panel B), 5 h 31 min (Panel C) and 11 h 1 min
(Panel D). Separation was carried out in 20 mM citrate buffer, pH 2.5. Bar represents 0.005 AU.
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Conclusion
References
CE appears to be an excellent tool to monitor the
dimerization process of proteins and peptides. As
this study with two model peptides has demonstrated, CE can potentially resolve the monomeric
and dimeric forms of a species, as well as discriminate between homo- and hetero-dimers. With
CE, time-course analysis of the oxidation of a
peptide can be easily automated yielding quantitative information on dimerization kinetics.
1. Schwartz, H. E., Palmieri, R. H., Brown, R.
Separation of Proteins and Peptides by Capillary Electrophoresis. Capillary Electrophoresis:
Theory and Practice, pp. 201-253. Edited by
P. Camillieri. CRC Press, Boca Raton, 1993.
2. Palmieri, R. H., Nolan, J. Protein Capillary
Electrophoresis: Theoretical and Experimental
Considerations for Method Development. Capillary Electrophoresis: A Practical Approach,
pp. 313-362. Edited by J. Landers. CRC Press,
Boca Raton, 1993.
3. Landers, J. P., Oda, R. P., Liebenow, J. A.,
Spelsberg, T. C. J. Chromatogr. (in press)
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