20

Characterization of Protein-Protein Interactions

via Static and Dynamic Light Scattering
Daniel Some and Sophia Kenrick
Wyatt Technology Corp.
USA
1. Introduction
Light scattering in its various flavors constitutes a label-free, non-destructive probe of
macromolecular interactions in solution, providing a direct indication of the formation or
dissociation of complexes by measuring changes in the average molar mass or molecular
radius as a function of solution composition and time. It is a first-principles technique,
thoroughly grounded in thermodynamics, permitting quantitative analysis of key properties
such as stoichiometry, equilibrium association constants, and reaction rate parameters.
In the past, light scattering experiments on interacting protein solutions have been labor
intensive and tedious, requiring large volumes of sample, and hence impeding widespread
adoption by protein researchers. Recent advances in instrumentation and technique hold the
promise for simplifying and automating measurements, as well as reducing sample
requirements, thus broadening the appeal of these methods to the wider community of
analytical biochemistry, biophysics, and molecular biology research. Pioneering work in
automating and applying these measurements to equilibrium protein-protein interactions
appeared in 2005-2006 (Attri & Minton, 2005a, 2005b; Kameyama & Minton, 2006). This
chapter deals primarily with such automated methods.
2. Theory of light scattering from biomacromolecules in solution
2.1 Static light scattering
Static light scattering (SLS) measurements quantify the excess Rayleigh ratio R, which
describes the fraction of incident light scattered by the macromolecules per unit volume of
solution. Knowledge of R vs. scattering angle () and concentration c may determine molar
mass, size and self-interactions of the sample, while R(t) will describe the kinetics of self-
association or dissociation, via time-dependent changes in the average molar mass or size.
Likewise, characterization of R vs. the composition ([A], [B], ) of a multi-component
system, such as hetero-associating proteins, may determine the stoichiometry and
equilibrium binding affinity of such a system, as well as binding or dissociation kinetics.
The basic theory of static light scattering from macromolecules in solution is available in
myriad publications, including elementary textbooks dealing with physical chemistry (e.g.,
van Holde et al., 1998; Teraoka, 2002) or essential references on polymers (e.g., Young, 1981).
We will cite from these without further reference. More rigorous publications, particularly
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Protein Interactions 402
those dealing with non-ideal, multi-component solutions are found in the scientific
literature (e.g., Blanco et al., 2011, and references therein).
2.1.1 Static light scattering in the ideal limit
Macromolecules in solution are subject to correlations arising from intermolecular
potentials, which in turn affect the magnitude of scattered light. However, if the particles are
few and far between, and the potentials between them sufficiently short-ranged, these
correlations may be ignored, leading to what is known as the ideal limit: essentially, the
particles behave like an ideal ensemble of point particles.
The simplest picture of scattering from proteins in solution invokes the ideal limit, i.e.,
point-like particles with no interactions, much like the more commonly known ideal gas law
for pressure and temperature. In this case, the scattering from particles much smaller than
the wavelength of incident light, with detectors placed in the plane perpendicular to the
incident polarization, can be described by Eq. (1):

2
2 2
0
4
0
4
*
A
n dn
R Mc K Mc
dc N

| |
= =
|
\

(1)
In Eq. (1), N
A
represents Avogadros number, dn/dc is the proteins refractive increment, M
the proteins molar mass, n
0
the solvent refractive index,
0
the wavelength in vacuum, and c
the protein concentration in units of mass/volume. K* incorporates the constants n
0
, N
A
,
0

and dn/dc.
The protein refractive increment dn/dc describes the change in refractive index of a solution
relative to pure buffer, due to a mass/volume protein concentration c; this parameter may
be readily measured by means of a common instrument known as a differential
refractometer and is, fortuitously, nearly invariable for most proteins in standard aqueous
buffers at any given wavelength (dn/dc=0.187 mL/g at =660 nm). High concentrations of
excipient will affect dn/dc; adding for example arginine, which has a refractive index higher
than that of most proteins, can even reduce dn/dc to zero such that no scattering occurs!
For a solution consisting of multiple macromolecular species, e.g., monomer + oligomers or
A+B+AB complex, the total light scattered is the sum of intensities scattered by each species:

* *
i i w
i
R K M c K M c = =

(2)
Here M
i
and c
i
refer to the molar mass and concentration of each species i, M
w
is the weight-
averaged molar mass and c the total protein concentrations. We have assumed that all
species have the same refractive increment and non-ideality may be ignored.
Upon inspection of Eq. (2) it becomes clear that given knowledge of the measurement
conditions (solution refractive index, scattering wavelength), sample parameters (dn/dc),
sample concentration (e.g., by means of a UV absorption or differential refractive index
concentration detector), and excess Rayleigh ratio R, it is possible to determine the weight-
averaged molar mass of macromolecules in the solution. If the solution is monodisperse (as
is often the case in the course of chromatographic fractionation), then the molar mass of the
solvated macromolecule may be determined.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 403
Eq. (2) contains the reason that light scattering is famously sensitive to small quantities of
dust or other particulates: if the mass if the dust particle is a million times that of the
protein, only one-millionth the concentration of dust particles produces the same scattering
intensity as the protein.
Generalization of Eq. (2) to species with different refractive increments is obvious, but for
reasons of simplicity we will assume henceforth equal dn/dc for all proteins. This ideal gas
law for light scattering is generally applicable to characterization of specific protein-
protein binding with equilibrium dissociation constants K
D
1-10 M.
Angular dependence of the scattered intensity comes into play for larger particles such as
protein aggregates whose radii exceed ~/50. In the limit of transparent particles with radii
below ~40 nm, this dependence is described by the Rayleigh-Ganz-Debye (RGD) equation:
( ) ( ) ( ) ( )
2
2
2
2
0
16
* ; 1 sin 2
3
g
R K McP P r

= = + (3)
For this reason SLS is often referred to as multi-angle light scattering (MALS). Here is the
angle between the incident and scattered light rays within the plane perpendicular to the
incident polarization, r
g
is the radius of gyration, and higher order terms in P() have been
ignored. For globular proteins, r
g
will be ~80% the average geometrical radius. When r
g
< 8-
12 nm, P() ~ 1, angular dependence is eliminated, and the molecules are considered
isotropic scatterers. Since the dimensions of most proteins and complexes are below 20 nm,
for the remainder of this chapter we will assume isotropic scattering, i.e., P() = 1.
2.1.2 Analysis of protein complexes via static light scattering coupled to online
chromatographic separation
Analytical size-exclusion chromatography (SEC) is often an unreliable measure of molar
mass, particularly if the standards used to calibrate column elution times do not represent
the sample well in terms of shape or column interactions. Because it does not need to make
any assumptions regarding separation models or column calibration standards, flow-mode
MALS is an invaluable extension of analytical SEC (SEC-MALS) or asymmetric-flow field
flow fractionation (AF4-MALS). The analysis almost invariably occurs at concentrations well
below 1 mg/mL, low enough to fall squarely within the ideal limit. Figure 1 shows a typical
SEC-MALS experimental layout, combining a MALS detector with concentration analysis by
means of UV/Vis absorption or differential refractometry (dRI).

Fig. 1. SEC-MALS instrumentation.
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Protein Interactions 404
While normally applied to characterize polydisperse ensembles, irreversible oligomers or
other tightly-bound complexes, SEC-MALS may be used to assess reversible protein
interactions, especially self-association (Bajaj et al., 2007). In this approach, the excess
Rayleigh ratio and concentration is measured at multiple points along an eluting peak, and
these data are fit to equations representing mass conservation, mass action and ideal light
scattering identical to those in Section 2.1.3. Since the ratio of monomer to oligomer in a
reversibly associating system is concentration-dependent, the change in weight-average
molar mass across the peak should indicate dissociation of oligomers upon dilution in the
column. The measurement may be repeated at different initial sample concentrations to
enhance the analysis and establish whether the dissociation kinetics are fast or slow.
While this analysis can provide a good semi-quantitative characterization of reversible
association, it is subject to certain systematic errors. The analysis must assume either rapid
or very slow equilibration. As the sample proceeds through the column and detectors, it
dilutes continuously; if the equilibration is neither very fast nor very slow compared to the
elution time, the ratio of complex to monomer will not represent equilibrium conditions.
Also, band-broadening between detectors means that the concentration measured in the UV
or dRI concentration detector is somewhat different than that in the MALS cell, hence
systematic errors in the analysis will arise. An integrated UV-SLS cell can eliminate the latter
source of error (Bajaj et. al., 2004).
The advantages of analyzing reversible complexes via SEC-MALS are low sample quantity
and clean data with little noise due to particulates if the size exclusion column and HPLC
system are very clean. The column will separate any dust or aggregates from the sample.
2.1.3 Quantifying specific, reversible protein-protein binding via composition-gradient
static light scattering
The use of stop-flow injections with well-defined concentrations permits true equilibrium
analysis. In some instances the kinetics of association or dissociation may be analyzed as
well. Composition-gradient light scattering apparatus, described in section 3, is more
generally useful than SEC-MALS for studying protein-protein interactions. This section
presents the principles of this approach.
The analysis of specific, reversible protein-protein interactions in the ideal limit, via light
scattering measurements from a series of compositions, has been presented concisely by
Attri & Minton (Attri & Minton, 2005b). The equations, with a minor change in notation,
include the ideal light scattering law (4), mass action (5) and conservation of mass (6),
shown below assuming up to two constituent monomeric species A and B:

( )
2
,
*
A B i j
i j
R
iM jM A B
K
(
= +

(4)

[ ] [ ]
i j
ij
i j
A B
K
A B
(

=
(5)
[ ] [ ]
, ,
,
i j i j
total total
i j i j
A i A B B j A B
( (
= =


(6)

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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 405
M
A
and M
B
represent the molar masses of constituent monomers A and B, respectively; i and
j represent the stoichiometric numbers of A and B in the A
i
B
j
complex, with A
1
B
0
and A
0
B
1

representing the monomers of A and B; [A
i
B
j
] represents the molar concentration of the A
i
B
j

complex; [A]
total
and [B]
total
represent the total molar concentration of A and B in solution;
and K
i,j
is the equilibrium association constant relating equilibrium molar concentrations of
the A
i
B
j
complex and free monomer. Light scattering and concentration data acquired over a
series of compositionsmultiple concentrations, in the case of self-association of a single
species, or a series of A:B composition ratios in the case of hetero-associationare fit to Eq.
(4) by means of a standard least-squares nonlinear curve fitting procedure. This technique is
known as composition-gradient multi-angle static light scattering, CG-MALS or CG-SLS.
Beyond the usual curve fitting algorithms, there is an added complication of solving first at
each composition and fitted parameter iteration, the nonlinear system of Eqs. (5) (one for
each complex present in equilibrium with monomers) + Eqs. (6). Examples of the system of
equations to be fit are presented in Section 4.4.1.

Fig. 2. Composition gradient for characterization of self- and hetero-association.
A typical composition gradient for the analysis of combined self-association and hetero-
association, shown in Figure 2, includes three segments: I and III are concentration series in
A and B individually to determine any self-association, while II is a crossover gradient
stepping through a series of A:B composition ratios to characterize hetero-association.
Figure 3a simulates LS signals for homodimer and homotrimer association, showing how
the appropriate association model is well-determined by LS.

Fig. 3. Simulated CG-MALS signals. a) self association; b) and c) hetero-association.
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Protein Interactions 406
The crossover gradient, though perhaps not intuitive to those accustomed to sigmoidal
titration curves, is in fact quite efficient for analyzing stoichiometry and binding affinity
even in the presence of a complex interaction that may include simultaneous self- and
hetero-association. Figure 3b and Figure 3c depict a qualitative interpretation of the
behavior of the light scattering signal for such a gradient: the position of the peak along the
composition gradient axis indicates the stoichiometric ratio of the complex, while the height
and shape of the peak indicate both the binding affinity and the true stoichiometry, i.e.,
discrimination between the formation of 1:1 or 2:2 complexes.
2.1.4 Non-ideal static light scattering for characterization of nonspecific protein
interactions
Nonspecific protein interactions arise from various sources such as hard-core molecular
repulsion, net charge, dipoles, hydrophobic patches, van der Waals interactions, hydration
forces, etc. These are generally weak in relation to specific binding and so only become
important at concentrations exceeding about 1 mg/mL. In contrast to site-specific lock-and-
key binding, nonspecific interactions do not exhibit well-defined stoichiometry and do not
generally saturate at some sufficiently high concentration. The dominant interaction may
vary from attractive to repulsive or vice-versa as the concentration increases. The ill-defined,
multi-sourced nature of nonspecific interactions lends itself to thermodynamic analysis in
terms of small deviations from the ideal case described by Eq. (1), hence the designation
thermodynamic non-ideality. These interactions are of interest since they generally
correlate to solubility, viscosity, and propensity for aggregation, and are also key to
understanding crowded biomolecular environments such as the intracellular environment.
Akin to the virial expansion of the osmotic pressure as a power series in concentration, light
scattering of dilute solutions may be analyzed in terms of a virial expansion, which actually
uses the same pre-factors (virial coefficients A
2
, A
3
, or B
22
, B
222
, etc.) as the osmotic pressure
equation though with a slightly different functional form:

2
2 3
2
2 3
* 1
2 3
* 1 2 3
R Mc K c
or A c A c
K R M A Mc A Mc
= = + + +
+ + +

(7)
In many cases a proteins nonspecific interactions may be modeled as those of hard,
impenetrable spheres, albeit with an effective specific volume v
eff
different from that of the
molecules actual specific volume, resulting in a virial expansion containing only one
independent parameter describing the non-ideality (Minton & Edelhoch, 1982):

( )
2
*
1 8 30
eff eff
R Mc
K
v c v c
=
+ + +
(8)
Each virial coefficient may be expressed in terms of the effective volume. As the lowest
order correction to ideal light scattering, A
2
=4v
eff
/M tends to be of greatest interest for
characterizing nonspecific interactions. Unscreened, long-range charge-charge interactions
are not fit well by the effective hard sphere model.
An interesting and counterintuitive feature of purely (or primarily) repulsive interactions is
that the LS intensity is not monotonic with concentration. Rather it initially rises as expected
from the ideal LS equation, then plateaus, and eventually declines with higher concentration
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 407
(see high-concentration case study, Section 5.5). Many non-associating proteins exhibit light
scattering behavior which is fit well by the effective hard sphere assumption including
scattering that eventually decreases with concentration (Fernndez & Minton, 2008). The
same work described CG-SLS apparatus suitable for high concentrations.
2.1.5 Quantifying repulsive and weakly attractive protein interactions via composition-
gradient static light scattering at high concentration
Attractive nonspecific interactions, though weak, will at high enough concentrations
invariably lead to the formation of transient clusters which can be analyzed in terms of
specific reversible oligomerization, rather than in terms of virial coefficients. The same is not
true of repulsive interactions. Hence it is possible to segregate the repulsive interactions into
the virial coefficients and treat the attractive interactions as specific self-association.
The analysis is further simplified by assuming not only that the monomers and oligomers
behave as effective hard spheres but also that all species have the same effective specific
volume v
eff
. An algorithm has been developed to include the effect of varying
thermodynamic activity on the apparent association constants (Minton, 2007). This approach
has been shown to work well for enzymes (Fernndez & Minton, 2009) as well as antibodies
(Scherer et al., 2010). While not quite as rigorous, we have found that a reduced analysis
based on Eq. (9) serves to reproduce the essential behavior at high protein concentration in
terms of self-associating oligomers subject to an effective hard sphere repulsion:

( )
2
*
1 8 30
i
i
eff eff
iMc
R
K
v c v c
=
+ + +

(9)
2.2 Dynamic light scattering
Rather than measuring the time-averaged intensity of scattered light, dynamic light
scattering (DLS) measures the intensity fluctuations which arise from Brownian motion of
the scattering molecules. The fluctuations are mathematically processed to produce an
autocorrelation spectrum, which is then fit to appropriate functional forms to assess the
translational diffusion constant D
t
. D
t
can be related via the Stokes-Einstein equation to a
characteristic dimension, the hydrodynamic radius r
h
, which is just the radius of a sphere
with the diffusion constant D
t
. The theory of DLS is covered in myriad sources, from
textbooks (e.g., Teraoka, 2002) to peer-reviewed scientific literature.
DLS has certain practical advantages over SLS. In particular, DLS enjoys a relative immunity
to stray light, which permits robust measurements in small volumes with free surfaces, such
as a microwell plate. On the other hand, DLS is not as sensitive as SLS and so requires
higher concentrations, limiting the range of binding affinities it can measure.
2.2.1 Analyzing protein-protein binding via composition-gradient dynamic light
scattering in the ideal limit
The same nonspecific interactions leading to thermodynamic non-ideality in SLS do affect
the diffusion constant and apparent r
h
measured by DLS. In the ideal limit corresponding to
a sufficiently dilute solution, this may be ignored. A solution consisting of multiple species
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Protein Interactions 408
A
i
B
j
will exhibit a diffusion constant which is the z-average of the diffusion constants D
t,ij
of
the individual species, leading to an average hydrodynamic radius as shown in Eq. (10):

2 2
, , ,
1 1
ij i j ij i j
avg
h ij i j i j
h
M A B M A B
r
r
( (
=


(10)
Upon measuring a series of concentrations or compositions like that shown in Figure 2, one
can determine stoichiometry and equilibrium association constants by analyzing the
apparent diffusion constants in terms of Eq. (10) in combination with Eq. (5) and Eq. (6).
This technique is known as composition-gradient dynamic light scattering (CG-DLS).
Unlike the molar mass measured by static light scattering, it is not obvious or
straightforward to construct the hydrodynamic radius or diffusion constant of an
associating complex, given the hydrodynamic radii of its constituent species. This is
especially true for stoichiometries higher than 1:1 where the geometry could vary from
compact to extended, leading to significantly different diffusion constants. Composition-
gradient DLS data has been shown to successfully analyze binding of globular proteins to an
A
i
B
j
complex by assuming a power law relationship (Hanlon et. al, 2010):

( )
1
, , , h ij h A h B
r ir jr

= +
(11)
The best fits for different associating systems resulted in values ranging from ~2.8 for
homodimers and 1:1 complexes, to ~ 2.0 for a 2:1 stoichimetry. In this work, CG-DLS in a
microwell plate reader provided important benefits, including low sample consumption and
the ability to measure the same samples at multiple temperatures in order to obtain
enthalpy and entropy of the interaction via van t Hoff analysis.
2.2.2 Analyzing nonspecific interactions via dynamic light scattering
Nonspecific interactions give rise to thermodynamic non-ideality in DLS as well as MALS.
The first-order correction to the translational diffusion constant incorporates a combination
of parameters: the second osmotic virial coefficient A
2
(B
22
), the specific volume of the
molecule v
sp
, and the first-order correction to the molecules friction coefficient due to
concentration
1
, as presented in Eq. (12) (Teraoka, 2002).

( )
0 2 1
1 ; 2 2
t D D sp
D D k c k A v = + + =
(12)
We can expect v
sp
to remain approximately constant for a given protein in different buffer
systems, while
1
actually includes additional A
2
dependence. A measurement of k
D
for a
series of monoclonal antibodies in different buffers exhibits excellent correlation with A
2

(Lehermayer et al., 2011).
The sample concentrations needed to measure k
D
are comparable to those needed to
measure A
2
, but the volumes can be much smaller. Hence, in order to track trends in
nonspecific interactions with buffer modifications such as pH or ionic strength, k
D

(particularly as measured in a DLS plate reader) can be a low-volume, high-throughput
surrogate for CG-MALS A
2
analysis. Unlike CG-MALS, however, currently the high-
concentration behavior of CG-DLS is insufficiently understood to interpret data in the 10-
200 mg/mL range.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 409
3. Composition-gradient light scattering instrumentation
3.1 Detectors
Light scattering instrumentation for solutions has evolved considerably in the past two
decades, resulting in systems that make characterization of protein interactions via CG-
MALS and CG-DLS both feasible and accessible. Current top-of-the-line commercial MALS
instruments provide a dynamic range covering protein solutions from tens of ng/mL up to
hundreds of mg/mL (HELEOS, Wyatt Technology Corp.), accessing interactions with K
D

from sub-nM to mM. Closed systems employing low-stray-light flow cells are important for
low through moderately high concentrations, but are not suitable for the most concentrated
protein samples that tend to be viscous. Easily-cleaned, microcuvette-based systems are
better suited to the latter measurements.
A large selection of microcuvette-based DLS detectors is commercially available (Zetasizer
series, Malvern Instruments; DynaPro series, Wyatt Technology; etc.). The lowest protein
concentration range that these can analyze is on the order of 10-100 g/mL, accessing an
interaction range from tens of nM to tens of M. Of particular note is the DynaPro DLS plate
reader which can be integrated with standard liquid handling robotics to prepare
automatically low-volume composition gradients.
Some instruments provide simultaneous SLS and DLS detection (HELEOS+QELS (flow cell
or microcuvette), NanoStar (microcuvette), Wyatt Technology Corp; Zetasizer V (flow cell
or microcuvette), Malvern Instruments).
3.2 Automated composition-gradient delivery systems
An automated composition-delivery system for CG-MALS or CG-DLS is similar in many
ways to standard stop-flow apparatus: two or more solutions are mixed by pumping via
syringe pumps through a static mixer, an aliquot is delivered to the detector, and the flow
stopped. The syringe pumps are operated at different flow ratios in order to obtain different
compositions.
The most significant added requirement for light scattering is good in-line filtration of the
solutions in order to eliminate large aggregates and particles generated by airborne dust,
mechanical motion of syringes and valves, or protein films sloughing off surfaces. The pore
size should be on the order of 0.1 m or less. One approach (Attri & Minton, 2005a) is to add
an in-line filter after the mixing point, illustrated in Figure 4. The key disadvantage of this

Fig. 4. Single in-line filter, parallel detectors CG-MALS setup, after Attri & Minton 2005a.
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Protein Interactions 410
single-filter architecture is the changing chemical environment on the filter membrane:
proteins will adhere to and release from the filter unpredictably as the environment
changes. This is of particular concern in a tight-binding hetero-association analysis carried
out at low protein concentrations. An in-line concentration detector is crucial.

Fig. 5. Multiple in-line filters, serial detectors CG-MALS setup (Calypso, Wyatt Technology
Corp.). UV or dRI concentration detector is optional.
Another approach is to flow each solution through a dedicated filter, as shown in Figure 5.
In preparation for the gradient, each solution is pumped through its own filter and
associated lines until saturation is reached so that, in the course of a subsequent composition
gradient, well-defined compositions are produced reliably. This setup potentially eliminates
the need for an in-line concentration detector if the stock protein solution concentrations are
known prior to loading.
CG-MALS systems typically include light scattering and concentration detectors. The setup
of Figure 5 shows a common approach, serially connected detectors, much like that of SEC-
MALS. In order to achieve accurate correspondence between the concentration in the MALS
flow cell and that in the concentration detector, both cells must be fully flushed with each
composition. This can require relatively large sample volume. An alternative approach is to
split the flow between the two detectors, as shown in Figure 4. Careful calibration of the
flow resistance and delay between the two detectors is required to match the concentrations
at the end of each injection. Additional care must be taken to ensure that laboratory
temperature fluctuations, clogged capillaries or viscosity changes do not alter the split ratio
between the detectors. The parallel detector configuration affords a smaller injection volume
per composition.
Automation would not be complete without control and analysis software. Currently the
only commercially available hardware/software package integrating syringe pump control,
MALS data acquisition, and data analysis of equilibrium and kinetic macromolecular
interactions is the Calypso CG-MALS system (Wyatt Technology, Santa Barbara).
4. Practical challenges of composition-gradient light scattering
4.1 Sample and buffer preparation
Due to the sensitivity of light scattering to the presence of just a few large particles, and
especially in the absence of a separation step like SEC or FFF, particle-free solutions are
essential in CG-MALS. Even though the composition-gradient apparatus provides in-line
filtration, samples and solvents must be pre-filtered to the smallest practical pore size into
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 411
very clean glassware or sterile, disposable containers. Solvents and buffers are generally
filtered via bottle-top vacuum filters or large syringe-tip filters with pore sizes of 0.1-0.2 m.
Samples should be diluted to a bit above the appropriate working concentration in filtered
solvent and then filtered with a syringe-tip filter to the smallest allowable pore size, most
commonly 0.02-m (e.g., Anotop filter, Whatman). All filters should be flushed to wash out
any particles prior to introducing sample or final buffers.
4.2 Maintaining clean experimental apparatus
Regular cleaning and maintenance of the LS detectors and sample delivery apparatus are
imperative for reliable, reproducible data. As a general rule, after each experiment, the
instruments should be flushed with a buffer in which the sample is soluble before changing to
storage or cleaning solutions. Common detergents for removing protein and polymer residue
from glass and plastic surfaces include 5% v/v Contrad 70 and 1% w/v Tergazyme. Other
methods useful for cleaning a dirty system include flushing with a high-salt (0.5-1.0 M NaCl)
solution, 20-30% alcohol in water, or 10% nitric acid, as well as manual disassembly and
cleaning. Salt and protein residues may be removed from syringes or valves by sonication.
Cleanliness of the instruments and buffers should be verified by observing noise in the
MALS signals as the solutions flow through the system.
4.3 Designing optimal methods
The key parameters in CG-MALS experiment design are: 1) stock concentrations; 2) number
and spacing of composition steps; 3) injected volume per step; and 4) equilibration time.
4.3.1 Determining optimal concentrations
Since molecular interactions are generally concentration-dependent, it is important to
estimate the right concentration range that will, on the one hand, be high enough to produce
a significant amount of complex, but on the other, be low enough so as not to saturate the
complex leaving no free monomer. A general rule-of-thumb, assuming one has an estimate
of K
D
, is to prepare stock solution concentrations at 5-10x K
D
. A more sophisticated approach
is to perform a series of CG-MALS simulations assuming different concentrations, K
D
values
and even association schemes (e.g. 1:1 or 2:1), selecting composition gradients that best
discriminate between reasonably feasible models.
For self-association, a concentration gradient should include concentrations low enough that
essentially no self-association occurs, as shown at the low-concentration end of Figure 3a
where the no-interaction signal coincides with the associating signal. The gradient should
also include concentrations high enough that at least 20-30% of the LS intensity arises from
oligomers, as shown on the high-concentration end of Figure 3a.
For heteroassociation, the optimal A:B stock concentration ratio is not necessarily the
stoichiometric ratio, but depends on the molar masses of the molecular species. For good
contrast, the total LS signal at 100% A (right side of Figure 3b) and 100% B (left side of
Figure 3b) will be nearly equal, i.e., M
A
c
A
~M
B
c
B
where c
A
and c
B
refer to the stock
concentrations of A and B. This should be balanced against centering the LS peak close to
the center of the crossover gradient. In particular, juggling these competing considerations
can be tricky when the molar masses differ by a factor of 3 or more. If the mass ratio is large
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Protein Interactions 412
it may be better to perform a titration-like gradient where each injection includes a fixed
concentration of the larger molecule, but varies the concentration of the smaller molecule.
Once the concentration ratio has been selected, the overall concentrations of A and B in the
heteroassociation gradient should be chosen to discriminate well between K
D
values within a
reasonably expected range. For example, the conditions of Figure 3b discriminate well
between K
D
values of 1, 10 and 100, but would not be conclusive if the actual K
D
is 0.1 or 1000.
An initial CG-MALS analysis may yield multiple association models that fit the data well.
Simulations of light scattering from new composition gradients can assist in judiciously
designing a follow-on experiment to refine the analysis by eliminating some of the first-
round models. Such simulations are incorporated into the Calypso software.
The concentrations required to measure nonspecific interactions characterized by the second
virial coefficient (A
2
) typically range from 2-20 mg/mL. For proteins, an initial estimate of A
2

may be calculated by assuming a hard sphere of the same molecular weight (M) and
hydrodynamic radius (r). The stock concentration needed to achieve a 15% contribution to
the total scattered intensity from the A
2
term (see Eq. (7)) can be calculated as per Eq. (13):

3
2
2
2
0.15 16
;
3 2
sphere
A
stock
sphere
N r
c A
M A M

= =
(13)
4.3.2 Composition steps
An adequate number of compositions must be evaluated for proper fitting of CG-
MALS/DLS data to an appropriate interaction model. For nonspecific interactions or simple
homodimerization, at least five non-zero concentrations are recommended. Likewise, at
least 5 composition steps are required for 1:1 binding. More complicated interactions
forming larger numbers of species in equilibrium typically require 8-10 different
compositions or more.
Sometimes the composition steps, rather than being distributed evenly across a gradient, can
be focused in a specific region in order to make best use of the available sample, as shown in
Figure 6.

Fig. 6. Simulated interaction data for four possible interaction models (left) and
corresponding CG-MALS method (right) focusing compositions around region of interest.
Dashed vertical lines indicate plateau compositions.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 413
4.3.3 Step volumes and equilibration time
The volume of sample introduced to the detectors at each composition must suffice to flush
out completely the previous contents of the cell. At low sample concentration, or for
particularly sticky samples, adsorption onto surfaces (especially the in-line filter of Figure
4) may necessitate increased step volumes. The required injection size may also vary with
flow rate as well as detector configuration, and so should be determined experimentally for
any set of conditions. The proper step volume may be assessed by running an ascending
gradient followed by a descending gradient at a series of injection volumes: as the volume
increases, the signals will match more closely.
After each injection, flow is stopped and the sample given time to reach equilibrium. Often
the time scale for the reaction is faster than the dead time (the time between mixing and
reaching the flow cell), but when this is not the case, ample time should be allotted after
each injection for equilibration.
Where sample volume is scarce or where high concentration or viscosity prevents
performing stop-flow experiments, CG-MALS analyses may be performed using a
microcuvette. Stock solutions for each composition are prepared in advance. The light
scattering intensity from each sample is measured using a calibrated cuvette and analyzed
as for a flow system. Microcuvettes must be carefully cleaned and dried between samples.
4.4 Data analysis
CG-MALS data analysis protocols include two distinct segments: pre-processing and model
fitting. The former comprises basic steps common to many measurements: baseline
subtraction, application of proportionality (calibration or conversion) factors, smoothing,
and selecting the data points for analysis. Specific to multi-angle light scattering are
despiking and detector selection, since the main source of noise is foreign particles that
primarily affect lower scattering angles and always generate positive signals. For
equilibrium analysis, data should be selected after equilibration at each step, and usually a
range of data points from each composition step are averaged to provide a single value from
each detector.
4.4.1 Equilibrium models: Fitting and interpretation
The essential parameters in a CG-MALS model are monomer molar mass M
A
and M
B
;
association stoichiometries ij; association constants K
A,ij
; and incompetent (inactive) fractions
f
incomp,A
, f
incomp,B
. The latter refer to protein molecules in the stock solution that are
incompetent to participate in the interaction due to mutation, misfolding, aggregation, etc.
In the course of fitting the data, a set of stoichiometries ij must be selected. Parameters such
as monomer molar mass and incompetent fractions may be constrained to known values or
floated to be optimized in the fit. Additionally, constraints may be imposed on the
association constants, e.g., models of equivalent binding sites or isodesmic association
confer specific relationships between the K
A,ij
, as described in Table 1.
Standard iterative non-linear curve fitting algorithms, such as Levenberg-Marquardt, are
implemented. For each composition, the total concentration of each monomer species is
known either from precise dilution or by measurement with a UV or dRI detector. At each
iteration of the free parameters, the equations for mass action and mass conservation (Eqs.
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Protein Interactions 414
(5) and (6)) are solved; then the light scattering is computed (Eq. (4)) and compared to the
measured value, the difference thereof serving as the minimization function. The result of
fitting the data to a particular model will provide association constants plus any other free
parameters, as well as a measure of goodness of fit, such as
2
.
A broad range of useful equilibrium association models may be implemented, including any
combination of self-interactions (formation of oligomers) and hetero-associations
(stoichiometries of 1:1, 2:2, 1:n, etc.). Several common association models for proteins are
presented in Table 1. Examples of these and more complex association schemes are
discussed in Section 5.
Although useful, appropriate fitting of CG-MALS data does not require a priori
knowledge of the interaction stoichiometry or system constraints. For a well-designed
experiment, the best fit of the data should naturally converge on a single solution. This is
illustrated in Figure 7 where incorrect models are applied to LS data for 1:1 and 1:2
interactions. In Figure 7A, which depicts a 1:2 interaction with equivalent binding sites as
for antibody-antigen binding, a first guess of 1:1 interaction creates a fitted curve that
does not peak at the correct stoichiometric ratio and clearly does not fit the data.
Similarly, applying combined 1:1 and 1:2 stoichiometries, unconstrained for equivalent
and independent binding sites, to a system with only one binding site results in the fitting
algorithm eliminating the contribution from the 1:2 species (LS contribution from AB
2
= 0
for all compositions, Figure 7B).
In many instances, the expected interaction scheme fits the data well, resulting in low
2
and
random residuals. Otherwise different stoichiometric models should be tested until the
measured LS behavior is well matched. If the experiment design was far from optimal for
the true system behavior or the interaction is particularly complex, more than one model
may fit the data equally well. Several strategies may be brought to bear on selecting the
most appropriate scheme, including Occams razor (i.e., the simplest model that fits the
data) and information from other techniques such as crystallographic structure or NMR
analysis of binding sites. Simulation tools are useful in designing follow-up experiments to
discriminate between multiple possibilities.

Fig. 7. Proper fitting of CG-MALS data requires the correct association model. A) Fitting of
1:2 interaction by 1:1 or 1:2 stoichiometry. B) Best fit of interaction between chymotrypsin
(A) and bovine pancreatic trypsin inhibitor (B) from crossover gradient in Section 5.2.1
requires only 1:1 interaction.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 415
Interaction Model Unknowns Governing Equations
Dimerization
(e.g., Section 5.1.1)
[ ]
[ ]
2
2 20
A
A
K = [ ] [ ]
( )
2
2
20
* 4
A
R K M A K A = +
[ ] [ ] [ ]
2
20
2
total
A A K A = +
1:1 hetero-association
(e.g., Section 5.2.1)
[ ]
[ ][ ]
11
AB
A B
K = [ ] [ ] ( ) [ ][ ]
2
2 2
11
*
A B A B
R K M A M B M M K A B = + + +
[ ] [ ] [ ][ ]
11
total
A A K A B = +
[ ] [ ] [ ][ ]
11
total
B B K A B = +
1:2 hetero-association,
equivalent binding
sites
(e.g., Section 5.2.2)
[ ]
[ ][ ]
11
AB
A B
K =
[ ]
[ ][ ]
( )
2 11
2
2
12
2
AB K
A B
K = =
[ ] [ ] ( ) [ ][ ]
( ) [ ][ ]
2
2 2
11
2 2
2 1
11
4
*
2
A B A B
A B
R K M A M B M M K A B
M M K A B
= + + +
+ +

[ ] [ ] [ ][ ] [ ][ ]
2
2 1
11 11
4 total
A A K A B K A B = + +
[ ] [ ] [ ][ ] [ ][ ]
2
2 1
11 11
2 total
B B K A B K A B = + +
Isodesmic self-
association to
n-mers
(e.g., Section 5.1.2; see
Kameyama & Minton
2006)
[ ]
[ ]
2
2 20
A
A
K =
[ ]
[ ]
1
0 20
n
n
A
n
n
A
K K

= =
[ ]
2 2 1
20
1
*
n
i
i
A
i
R K M i K A

=
=


[ ] [ ]
1
20
1
n
i
i
total
i
A iK A

=
=


Table 1. Common equilibrium association models that can be quantified by CG-MALS.
4.4.2 Kinetics models: Fitting and interpretation
Reaction kinetics for reversible and irreversible associations can be observed and
quantified by light scattering to provide a direct measure of association, dissociation, or
aggregation via the evolution of M
w
. Quantifying characteristic rate constants from CG-
MALS data requires knowledge of the final stoichiometry and, in the case of reversible
associations, the appropriate equilibrium association constants. For example, LS data for
covalent inhibition of an enzyme by an inhibitor may be fit at varying inhibitor
concentrations to yield a second-order rate constant for the interaction, k
a
. In the case of
irreversible dissociation, the apparent first-order kinetics can be described by an
exponential function, and the apparent dissociation rate constant, k, can be related to
applicable biomolecular constants:

( ) [ ] [ ]
2 2
0
*
kt
R K A A e

=

(14)
More complex analyses, such as the association of two proteins into an equilibrium complex,
involve solving the rate equations that govern the system of interest. The equilibrium
association constant K
A
and final stoichiometry must be determined in addition to the time-
dependent change in light scattering. For the simplest heteroassociation A B AB + , Eq.
(15) relates the CG-MALS data to the second order association rate constant k
a
=K
A
k
d
:
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Protein Interactions 416

[ ] [ ]
[ ] [ ] ( ) [ ] [ ] ( )
[ ] 1
2 ;
*
A B a
total total
A
d AB d AB AB dR
M M k A AB B AB
K dt dt dt K


= =
`

)
(15)
5. CG-MALS examples
5.1 Self-association
5.1.1 Dimerization of chymotrypsin
Dimerization has been observed by CG-SLS for the enzyme -chymotrypsin with pH-
dependent affinity (Kameyama & Minton, 2006; Fernndez & Minton, 2009). Figure 8
presents dependence of the reaction on ionic strength (Hanlon & Some, 2007), closely
matching results obtained via sedimentation equilibrium (Aune et al., 1971).


(A) (B)
Fig. 8. Self-association of chymotrypsin forming dimers vs. ionic strength. (A) LS and UV
280

concentration data over a series of concentration gradients (B) K
A
vs. [NaCl].
5.1.2 Isodesmic self-association
Some proteins tend to self assemble into chains, fibrils, or other large oligomers, such as
amyloid- plaques in Alzheimers disease and -synuclein aggregates in the Lewy bodies of
Parkinsons disease. A model of isodesmic self-association, i.e., the assumption that each
protein monomer binds to the growing chain with equal affinity, can often be used to
describe such an interaction, especially in the early nucleation phase of the assembly.
Insulin changes its self-association state as a function of pH and the presence of zinc ions
(Attri et al., 2010a, 2010b, and references therein). At physiological conditions in the
presence of Zn
2+
, insulin exists as a hexamer that further associates isodesmically to higher
order oligomersdimers of hexamers (12-mers), trimers of hexamers (18-mers), etc. (Attri et
al., 2010b). This interaction was studied using both static and dynamic light scattering.
Based on the reported equilibrium and diffusion constants, M
w
, D
t
, and the molar
composition of insulin oligomers could be reproduced (Figure 9).
In contrast, in the absence of Zn
2+
, insulin monomers exist in isodesmic equilibrium with
dimers, trimers, and higher order complexes with pH-dependent affinity (Figure 10). Rather
than constraining the maximum oligomerization state as in Table 1, both studies considered
the possibility of infinitely large oligomers.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 417

Fig. 9. Infinite self-association of insulin hexamers at neutral pH in the presence of Zn
2+
. A)
LS signal and r
h
avg
vs. protein concentration, calculated per K
A
and D
t
in Attri et al., 2010b.
B) Calculated molar distribution of species.


Fig. 10. Molar distribution of insulin self-association products and light scattering signal in
the absence of Zn
2+
at pH 3 (left), 7.2 (middle), and 8 (right), calculated per Attri et al., 2010a.
5.2 Hetero-association
5.2.1 Reversible enzyme-inhibitor binding with 1:1 stoichiometry
Following Kameyama & Minton 2006, we characterized a standard 1:1 reversible association
between -chymotrypsin (CT) and bovine pancreatic trypsin inhibitor (BPTI). A CG-MALS
experiment consisting of self-association gradients for each binding partner CT and BPTI
and a crossover hetero-association gradient was performed as per Figure 2. The self-
association gradients yield the molecular weight for each monomer and confirm the lack of
self-association for CT and BPTI at neutral pH. Fitting the LS data in Figure 11A as a
function of composition (Figure 7B) results in an equilibrium dissociation K
D
= 119 nM (K
A
=
8.5x10
6
M
-1
), consistent with measurements by other techniques (referenced in Kameyama &
Minton, 2006). The LS contribution from each species is then transformed to a concentration,
giving the species distribution shown in Figure 11B. As expected for a 1:1 interaction, the
plateau with the highest amount of complex formation occurs at a molar ratio of CT:BPTI
~1:1 (~11 M each CT and BPTI). Since the experiment was performed at concentrations
>10x K
D
, nearly all available free monomer is consumed in the (CT)(BPTI) complex. This is
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Protein Interactions 418
evident in Figure 11B where the mole fraction of CT is ~0 for all compositions [CT]<[BPTI],
and the mole fraction of BPTI is ~0 for all compositions [BPTI]<[CT].

Fig. 11. CG-MALS quantifies binding of CT and BPTI.
Under acidic conditions, the affinity of CT for BPTI decreases, and CT can form reversible
dimers, as in Section 5.1.1. At pH 4.4, the K
D
for the association of CT and BPTI is of the
same order as for the dimerization of CTK
D
= 10 M and 50 M respectively (Kameyama
& Minton, 2006). Based on these results, we can simulate the expected LS signals for
simultaneous self and hetero-associations (Figure 12). Discrimination between 1:1 binding
only, and self + heteroassociation, is readily evident where [CT]>[BPTI] (Figure 12A).
Despite the additional self-association, the fraction of hetero-association product still peaks
at a molar ratio of CT:BPTI ~1:1 (Figure 12B).

Fig. 12. CT-BPTI interaction at pH 4.4. A) Predicted LS for simultaneous CT self-association
and CT-BPTI hetero-association (blue) compared to CT-BPTI hetero-association alone with
K
D
= 10 M (red) or no interaction (green). B) Molar distribution of species for simultaneous
self- and hetero-association model, based on Kameyama & Minton, 2006.
5.2.2 Antibody-antigen binding with 1:2 stoichiometry, two equivalent binding sites
The power of CG-MALS lies in its ability to identify multiple stoichiometries in solution. For
example, a single multivalent receptor A may bind multiple protein ligands B, leading to the
simultaneous presence of AB, AB
2
, AB
3
, etc. The increasing prevalence of therapeutic
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 419
antibodies brings this type of multivalent binding to the forefront of biotechnology.
Moreover, CG-MALS is able to characterize this type of interaction with affinities as low as
K
D
~0.1 nM, typical of antibody-antigen interactions. Our antibody-antigen binding data
(Figure 13) indicates the presence of four species in solution: free antibody (Ab), free antigen
(Ag), the 1:1 complex (Ab)(Ag), and the 1:2 complex (Ab)(Ag)
2
. The CG-MALS K
D
value of
10 nM agrees well with the literature value.

Fig. 13. Light scattering (A) and composition (B) distributions for crossover gradient
between an antibody and monovalent antigen, K
D
~10 nM.
Conversely, Some et al. (2008a) found that CG-MALS data for a dimeric Fc receptor (FcR)
binding to the Fc of a recombinant human Ab (rhumAb), shown in Figure 14, is only fit well
by a model assuming two equivalent binding sites on each FcR dimer (B) for rhumAb (A),
producing equilibrium between monomers (A and B), AB, and A
2
B (Figure 14C). CG-MALS
data do not support other binding models, including 1 A : 1 B association alone (Figure 14A)
and 1 A : 2 B with equivalent binding sites (Figure 14B). The calculated single-site affinity of
50 nM agrees closely with surface plasmon resonance (SPR) analysis.

Fig. 14. Best fits (red lines) of measured CG-MALS data (blue circles) to different association
models, IgG (rhumAb) : dimeric receptor (FcR). Stoichiometry: (A) 1:1; (B) 1:2; (C) 2:1.
Only the {2 mAb per receptor} model fits the data.
5.2.3 Association of multivalent protein complexes
Combinations of multivalent binding partners can lead to the formation of metacomplexes
in solution that are not identified by other techniques. As a homo-tetramer, streptavidin
(SA) is composed of four identical binding sites capable of binding either of two Fab
domains of an anti-streptavidin IgG. As we have observed, the combination of multivalent
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Protein Interactions 420
proteins enables higher-order stoichiometries to present themselves in solution, including
multiple IgG molecules binding a single SA molecule and self-assemblies of IgG-SA
complexes (Figure 15). Indeed, the LS signal measured for such a system by CG-MALS is
nearly twice the value expected for a simple 1:2 association (Figure 15). Careful analysis of
the data indicates that the solution is best described as 1:1 (IgG)(SA) complexes that self-
associate (Figure 16). The infinite self-association (ISA) model employed here assumes that
each base unit(IgG)(SA) complexassembles with other base units with the same affinity;
however, this may differ from the binding-site affinity (K
D
) for a single IgG-SA interaction.
The binding-site K
D
for one SA molecule binding one IgG was determined as 22 nM, while
these 1:1 base units assemble with an average affinity K
D
= 50 nM.

Fig. 15. Light scattering and concentration data for association of SA and anti-SA IgG.
Theoretical LS plateaus are indicated for the case of no IgG-SA interaction and a 1:2
equivalent binding site model (Section 5.2.2). Additional stoichiometries that contribute to
the measured LS signal, including infinitely self-associating 1:1 complexes, are shown.

Fig. 16. A) Best fit of LS data for SA + anti-SA IgG includes infinite self assembly (ISA) of 1:1
metacomplexes B) Concentration distribution for hetero-association plateaus (#5-15).
5.3 Dissociation kinetics induced by a small molecule inhibitor
Although other techniques, such as SPR and FRET-based methods, are capable of
quantifying association and dissociation kinetics, many require modification of the protein
of interest, i.e., immobilization in the case of SPR and labeling with fluorescent tags for
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 421
FRET. In contrast, CG-MALS enables real-time observation of reaction kinetics in solution
without protein modification. For example, chymotrypsin self-association at low pH is
inhibited by the small molecule 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). When
introduced to a chymotrypsin solution, AEBSF covalently binds the monomer active site and
prevents dimerization. Varying concentrations of AEBSF were mixed with a constant stock
solution of chymotrypsin, and the resulting dissociation kinetics quantified with a model of
an irreversible dissociation (Some & Hanlon, 2010). For each composition, the solution was
allowed to react for >1 hr while observing the decrease in weight-average molar mass of the
solution. The characteristic reaction time (1/k in Eq. (14)) varies inversely with the AEBSF
concentration, consistent with the rate models defined for the system (Figure 17), indicating
a rate constant of 0.064 M
-1
s
-1
.

Fig. 17. Decrease in LS signal (left) and change in characteristic reaction time (right)
corresponding to irreversible dissociation of chymotrypsin dimers in the presence of AEBSF.
5.4 Nonspecific interactions of non-self-associating proteins
5.4.1 Nonspecific self-interactions
As discussed in Section 2.1.4, all macromolecules at high concentrations exhibit some degree
of nonspecific interactions, quantified by the second virial coefficient, A
2
. This property is of
particular interest in the development of pharmaceutical formulations where A
2
is one
metric for the stability of a formulation and the propensity of biomolecular therapeutics to
aggregate in solution. Formulations that may appear stable at moderate concentrations (~10
mg/mL or less) may indeed form self-association products at relevant formulation
concentrations of 100 mg/mL or more (see Section 5.5). For a well-formulated protein,
however, repulsive interactions should dominate for all concentrations of interest. BSA, for
example, exhibits nonspecific repulsion even at 100 mg/mL in PBS, as shown in Fig. 18.
Long-range interactions are well-screened in this buffer, resulting in an A
2
=1.0x10
-4

mol*mL/g, consistent with a hard-sphere of radius 3.5 nm and M
w
= 67 kDa.
5.4.2 Nonspecific attraction quantified by the cross-virial coefficient
Carrier proteins, drug delivery vehicles, and other polymers attract their biomolecular
targets via nonspecific interactions (e.g., Dong et al., 2011) which cannot be described by an
equilibrium association constant. A virial expansion may be employed to quantify
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Protein Interactions 422
nonspecific attraction or repulsion between molecules of the same species or different
species. In the example below, the net negative charge of BSA, in PBS with 50 mM NaCl at
pH 7, yields repulsion between BSA molecules. Lysozyme exhibits a slight positive charge
with a net self-attraction as evidenced by the negative A
2
. The charge-mediated attraction
between BSA and lysozyme molecules is evident in Figure 19 as the increase in LS when
BSA and lysozyme are mixed together. The data are best fit by a model of nonspecific
attraction, quantified by the cross-virial coefficient A
11
. The results can be normalized to a
unitless value as per Sahin et al., 2010:
( ) 2 2 2 2
meas exc exc
a A A A =
.




(A) (B)

Fig. 18. BSA behaves as an effective hard sphere with A
2
= 1.0x10
-4
mol*mL/g for all
concentrations studied. (A) CG-MALS data (B) fit to effective hard sphere model.


BSA self-virial coefficient
A
2
(mol*mL/g)
a
2
+1.1x10
-4
+0.2
Lysozyme self-virial coefficient
A
2
(mol*mL/g)
a
2
-3.6x10
-4
-2.1
Cross-viral coefficient
A
11
(mol*mL/g)
a
11
-4.0x10
-4
-2.7


Fig. 19. Determination of self-and cross-virial coefficients for nonspecific interactions in
BSA-lysozyme solution. Normalized virial coefficients are also presented.
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Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering 423
5.5 Interactions of monoclonal antibodies formulated at high concentration
Recently, CG-MALS was applied to investigate interactions between IgG1 monoclonal
antibodies at concentrations up to ~300 mg/mL (Scherer et al., 2010). Although the two
mAbs studied here were identical except for the CDR sequence, their self-association
properties were remarkably different. MAb2 forms dimers with K
A
~10
3
M
-1
(K
D
~1 mM),
whereas mAb1 associates into dimers with K
A
~10
3
-10
4
M
-1
(K
D
~0.1-1 mM) and appears to
further associate into higher order oligomers of stoichiometry 4-6. The dependence of
association properties on ionic strength also differs dramatically between mAb1 and mAb2:
while the affinity of the mAb2 homodimer increases with [NaCl], that of mAb1 homodimers
is essentially constant. Most significantly, the higher oligomer order of mAb1 decreases
from 6 to 4 as [NaCl] increased from 40 to 600 mM.
Based on these results, we reproduce in Figure 20 the relative LS signal for mAbs1 and 2 and
the fraction of each oligomer present in solution. Each calculation includes the appropriate
correction for non-specific repulsion using v
eff
= 1.8 cm/g (r
eff
= 4.6 nm) for mAb1 and v
eff
=
1.4 cm/g (r
eff
= 4.3 nm) for mAb2 (Scherer et al., 2010). Although the antibody molecules
continue to self-associate into higher molecular weight species, the LS signal is not
monotonically increasing, as would be expected from ideal scattering (Eq. (1)); instead, the
LS intensity reaches a maximum at ~100 mg/mL (Figure 20A). Only by accounting for both
nonspecific repulsion and specific oligomerization can the light scattering data be fully
described for these high-concentration solutions.



Fig. 20. A) LS signals for mAbs 1 and 2 in buffer containing 75 mM NaCl, calculated to
represent results of Scherer et al., 2010. B) and C) Corresponding distribution of oligomers.
6. Conclusion
The power of light scattering, CG-MALS and CG-DLS, for investigating protein interactions
lies in their great versatility. These techniques quantify a wide range of protein-protein
phenomena in solution and without labeling. Both equilibrium and kinetics may be
addressed directly since light scattering provides, from first principles, the molar mass and
size of complexes, rather than an indirect indicator such as fluorescence. Hence light
scattering is particularly well suited to analyzing higher-order complexes, multiple
stoichiometries, and simultaneous self- and hetero-association. The fundamental
thermodynamic nature of static light scattering provides a critical window into interactions
at high concentration. The development of automation and advanced instrumentation
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Protein Interactions 424
suggests that common use of CG-MALS and CG-DLS is feasible, and hence these are
important additions to the protein scientists toolbox.
7. Acknowledgement
The authors would like to thank Allen P. Minton for many helpful discussions and
collaboration in the development of automated CG-MALS; Shawn Cao (Amgen), Joey
Pollastrini (Amgen), and Jihong Yang (Genentech) for contributing antibody samples; and
the entire team at Wyatt Technology Corp. We are also indebted to the many early adopters
of the Calypso CG-MALS system for their support and sharing samples.
8. References
Attri, A.K., Fernndez, C., & Minton, A.P. (2010a). pH-dependent self-association of zinc-
free insulin characterized by concentration-gradient static light scattering.
Biophysical Chemistry. Vol. 148, No. 1-3, (May 2010), pp. 28-33, ISSN 0301-4622
Attri, A.K., Fernndez, C., & Minton, A.P. (2010b). Self-association of Zn-insulin at neutral
pH: investigation by concentration-gradient static and dynamic light scattering.
Biophyisical Chemistry. Vol. 148, No. 1-3, (May 2010), pp. 23-27, ISSN 0304-4622
Attri, A.K. & Minton, A.P. (2005a). New Methods for Measuring Macromolecular
Interactions in Solution via Static Light Scattering: Basic Methodology and
Application to Nonassociating and Self-Associating Proteins. Anal. Biochem.
Vol.337, No.1, (February 2005), pp. 103-110, ISSN 0003-2967
Attri, A.K. & Minton, A.P. (2005b). Composition Gradient Static Light Scattering (CG-SLS):
A New Technique for Rapid Detection and Quantitative Characterization of
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Protein Interactions
Edited by Dr. Jianfeng Cai
ISBN 978-953-51-0244-1
Hard cover, 464 pages
Publisher InTech
Published online 16, March, 2012
Published in print edition March, 2012
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Protein interactions, which include interactions between proteins and other biomolecules, are essential to all
aspects of biological processes, such as cell growth, differentiation, and apoptosis. Therefore, investigation
and modulation of protein interactions are of significance as it not only reveals the mechanism governing
cellular activity, but also leads to potential agents for the treatment of various diseases. The objective of this
book is to highlight some of the latest approaches in the study of protein interactions, including modulation of
protein interactions, development of analytical techniques, etc. Collectively they demonstrate the importance
and the possibility for the further investigation and modulation of protein interactions as technology is evolving.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Daniel Some and Sophia Kenrick (2012). Characterization of Protein-Protein Interactions via Static and
Dynamic Light Scattering, Protein Interactions, Dr. Jianfeng Cai (Ed.), ISBN: 978-953-51-0244-1, InTech,
Available from: http://www.intechopen.com/books/protein-interactions/characterization-of-protein-protein-
interactions-via-static-and-dynamic-light-scattering