Protein Protocols Protein Protocols

574 Irvine
As well as being a standard chromatographic mode for desalting and purifying proteins,
size-exclusion chromatography can be used for estimation of molecular weights.
This is true only for ideal size-exclusion chromatography, in which the support does
not interact with solute molecules (see Note 1). Because the physical basis for discrimination
in size-exclusion chromatography is size, one would expect that there
would be some parameter related to the dimensions and shape of the solute molecule
that would determine its K d value (see Note 2). All molecules with the same value for
this parameter should have identical K d values on a size-exclusion chromatography
column. Dimensional parameters that have been suggested for such a “universal calibration”
include two terms related to the hydrodynamic properties of proteins, namely
Stokes radius (R S) and viscosity radius (R h). Current evidence appears to indicate that
R h is more closely correlated to size-exclusion chromatography behavior than is R S (5).
However, even this parameter is not suitable for a universal calibration that includes
globular proteins, random coils, and rods (6). Hence calibration curves prepared with
globular proteins as standards cannot be used for the assignment of molecular weights
to proteins with different shapes, such as the rodlike protein myosin. Several mathematical
models that relate size-exclusion chromatography to the geometries of
protein solutes and support pores have been proposed (see Note 3). However, in
practical terms, for polymers of the same shape, plots of log molecular weight against
K d have been found to give straight lines within the range 0.1 < K d < 0.9 (see Note 4).
It has been found that the most reliable measurements of molecular weight by HPSEC
are obtained under denaturing conditions, when all proteins have the same random
coil structure. Disulfide bonds must be reduced, usually with dithiothreitol, in a buffer
that destroys secondary and tertiary structure. Buffers containing guanidine hydrochloride
(7,8) or sodium dodecyl sulfate (SDS) have been used for this purpose. However,
the use of denaturants has many drawbacks, which are described in Note 5. In
any case, polyacrylamide gel electrophoresis in SDS-containing buffers is widely
used for determining the molecular weights of protein subunits. This technique can
also accommodate multiple samples in the same run, although each run takes longer
than for HPSEC.
The method described in this chapter is for the estimation of molecular weights of
native proteins. The abilities to measure bioactivity, and to recover native protein in high
yield, make this an important method, even though a number of very basic, acidic, or
hydrophobic proteins will undergo non-ideal size exclusion under these conditions.
2. Materials
2.1. Apparatus
An HPLC system for isocratic elution is required. This comprises a pump, an injector,
a size-exclusion column and a guard column (see Note 6), a UV-detector, and a
data recorder. There are many size-exclusion columns based on surface-modified silica
on the market. The results described here were obtained using a Zorbax Bioseries
GF-250 column (0.94 × 25 cm), of particle size 4 µm, sold by Agilent Technologies.
This column can withstand very high back pressures (up to 380 Bar or 5500 psi) and
can be run at flow rates up to 2 mL/min with little loss of resolution (10). It has a
molecular weight exclusion limit for globular proteins of several hundred thousand.