Measurement of Electrostatic Interactions of Proteins Using Multi-Channel Surface Plasmon Resonance

Measurement of Electrostatic Interactions of
Proteins Using Multi-Channel
Surface Plasmon Resonance

Conan J. Fee, Rayleen Fredericks-Short, Jaganmohan Billakanti, and Vinod Babu Damodaran
Department of Chemical & Process Engineering and the Biomolecular Interaction Centre,
University of Canterbury, Christchurch, New Zealand
Introduction and Objective
Cytochrome C 2B4Z
The charge-pH behaviour of proteins is of interest for
30.00
ion exchange processes. In this poster, we describe a
Native
new technique that utilises a multi-channel surface
20.00
e
1 PEG
2 PEG
plasmon resonance (SPR) instrument to measure
a
r
g 10.00
3 PEG
electrostatic interactions of cationic proteins in solu-
t
Ch
0.00
4 PEG
Ne
0
2
4
6
8
10
12
14
tion under a range of conditions.
-10.00
5 PEG
6 PEG
-20.00
Multi-Channel SPR
pH
Figure 1 shows the matrix arrangement of the ProteOn
Figure 3. Prediction of net charge-pH behaviour of na-
XPR36 SPR system (Bio-Rad Laboratories, Hercules,
tive and PEGylated Cytochrome C, calculated from PDB
CA), with 6 ligand channels and 6 analyte channels
sequence [1] using PropKa [2].
flowing at right angles. Cationic and anionic channels
can be prepared and proteins flowed across each si-
multaneously to measure interactions.
Relating SPR Interaction to Charge
From protein molecular weight and predictions of net
charge, Z, (Figure 3) at a given pH, the initial responses of
the cation exchange SPR to the proteins (Figure 4) were
related to the charge concentration in solution (Figure 5).
Cytochrome C

e
140
p
lo
120
S
m
100
r
a
r
g
]
o
s
80
Figure 1. Multi-Channel SPR. Individual channels can
s
n
e
[
RU/
60
be used for cation exchange, anion exchange and con-
l S
ia
40
it
In
trol surfaces.
20
R
P
S
0

0
0.2
0.4
0.6
0.8
1
Concentration [μM]
Chip Surface Preparation
Figure 4. Initial slope of SPR response for various Cyto-
A 1000 Da MW α-amino, ω-carboxyl-PEG was im-
chrome C concentrations on the cation exchange SPR chip.
mobilised on the surface of a GLC SPR chip (Bio-
Rad Laboratories) by amine coupling at pH 8.5 for 24
Cation Exchange SPR Response
hours. After blocking for 24 hours with ethanola-
Initial slopes of the SPR response were related to the charge
mine, taurine was amine coupled to the tethered car-
concentration in solution for Cytochrome C, Lysozyme and
boxy-PEG for 24 hours, floowed by further blocking.
Bovine Lactoferrin at three pH values (Figure 5). A
Taurine contains a sulfonic acid group that forms a
straight-line relationship exists, suggesting that the method
strong cation exchanger on the chip surface. Figure 2
may be useful for determining the net charge of unknown or
shows the SPR response to BSA (no interaction) and
modified (e.g. PEGylated) proteins.
Cytochrome C at pH 8.5 with (a) a native chip sur-

face and (b) the taurine surface. The native chip sur-
140
face contains carboxyl groups, so shows cation ex-
Lactoferrin pH 8.5
change activity.
Lactoferrin pH 7.5
120
Lactoferrin pH 6.5
]
/s
Cytochrome C pH 8.5
BSA
U
Cytochrome C pH 7.5
R
(a)
Cyt C 10,000 ng/mL
[ 100
Cytochrome C pH 6.5
Cyt C 5,000 ng/mL
e
Cyt C 2,500 ng/mL
s
Lysozyme pH 8.5
Cyt C 1,250 ng/mL
n
o
Running Buffer
p
Lysozyme pH 7.5
s
e
80
Lysozyme pH 6.5
R
R2 = 0.9525
R
P
l S
60
ia
it
f
In
o
e
40
p
lo
S
20
BSA
Cyt C 10,000 ng/mL
0
Cyt C 5,000 ng/mL
Cyt C 2,500 ng/mL
(b)
Cyt C 1,250 ng/mL
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Running Buffer
Charge Concentration [Z/mL]
Figure 5. Cation exchange interaction as a function of the
sum of net charge and concentration.
References:
[1] PDB ID:2B4Z Mirkin, N., Jaconcic, J., Stojanoff, V., Moreno, A. (2008) High resolution X-ray crystallographic structure of bovine
Figure 2. Cation exchange SPR interactions (a) native
heart cytochrome c and its application to the design of an electron transfer biosensor. Proteins 70: 83-92 .
[2] Hui Li, Andrew D. Robertson, and Jan H. Jensen (2005). Very Fast Empirical Prediction and Interpretation of Protein pKa Values. Proteins,
chip (b) taurine-modified chip.
61, 704-721.

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