Handbook of Size Exclusion Chromatography and Related ...

most proteins to be maximally stable at a certain pH value or range of values, and
to display diminishing stability as the pH is varied in either direction from this
optimal value (or range). As has been pointed out previously (67,68), considerable
evidence exists that some proteins (those that can be described as deformable, or
“soft”, in that they have relatively low structural stability) are bound to surfaces in
a two-step process (69). First, the native protein forms a fairly weak interaction
with the surface (this interaction may be either hydrophobic or electrostatic,
depending on the nature of the surface and of the exterior of the protein). A
subsequent conformational change in the loosely bound protein allows a
substantial increase in the extent of contact between the protein and the surface,
and therefore in the number of binding interactions (68–71). If the second step
(the conformational change in the bound protein molecule) proceeds to a sufficient
extent, this may result in an overall tight binding of such a soft protein to the
packing material, even under conditions such that the equilibrium in the first step
(the original association of the protein with the packing material) is in favor of the
protein remaining in the mobile phase. In contrast to this behavior, a more
structurally stable, relatively “hard”, or nondeformable protein, even though it has
the same surface chemistry as the soft protein, will exhibit only the first, weak step
of binding, and will remain principally in the mobile phase.
The relevance of the foregoing to the question of adsorptive interactions in
SEC is that as the pH of the mobile phase is moved away from the pH of maximum
protein stability, the protein will be progressively softened, becoming much less
resistant to structural changes induced upon contact with the packing material. It is
important to note that this softening of the structure can proceed to a significant
extent, long before the pH change reaches the point of causing denaturation of the
protein in solution.
The result of all of these concurrent and often opposing effects is that an
experimenter who wishes to use protein SEC data to support specific, quantitative
conclusions concerning protein sizes and shapes will be required to test
multidimensional arrays of sets of conditions, rather than a one-dimensional array
in which only the variable of specific interest is changed.
5 USE OF MALS DETECTION FOR ABSOLUTE MOLAR
MASS DETERMINATION IN SEC
As discussed in the previous section, the molecular weight of protein measured by
column calibration in SEC may be erroneous due to the nonsize effect and the
assumption that the conformation of the protein sample is the same as that of the
standard proteins used for column calibration. Because of its ability to measure
absolute molecular weight of protein eluted from the SEC column and easy
interface with any SEC system, on-line multi-angle light scattering (MALS)
© 2004 by Marcel Dekker, Inc.