Page 147 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: GLM/GJK P2: GRB Final Pages
Encyclopedia of Physical Science and Technology En005H-218 June 15, 2001 20:33

Electrophoresis 375

are known (required to give the number of amines substi- peptides, making it possible to study otherwise insoluble
tuted), it is possible to calculate the number of charges mixtures.
on the unknown protein. Inaccuracies are introduced into When a highly charged macroion is produced, the ag-
these calculations because the amount of adsorption of the gregate moves rapidly in the gel matrix under a moderate
charged macroions to the polyacrylamide is dependent on potential gradient. Their relative velocities are related to
the charge, so the position of the polyampholyte after car- size, but since these macroions still carry some residual
bamylation does not remain a simple function of charge. shape from the native molecule, the frictional coefficient
This brief definition of the molecular parameters that is not always equal to the expected sphere. Hence, it is
affect rates of transport in PAGE illustrates the impor- important to use reference standards whose overall origi-
tant assumptions made in converting relative positions to nal shape is similar to the unknown proteins if reasonable
molecular parameters for the procedures discussed in the estimates are to be obtained. (The absolute accuracy is sel-
following sections. dom better than ±10%, although reproducibility is much
higher.) To reduce the contribution from variable shapes
it is usual to perform several experiments in gels formed
C. Estimation of Relative Masses Using
from various concentrations of acrylamide and bisacry-
Polyacrylamide Gel Electrophoresis
lamide. Plotting the logarithm of the relative mobility
Most of the charge of a biological macroion comes from against the concentration of acrylamide gives a straight
dissociation of the intrinsic chemical groups. In the case line whose slope can be related to the molecular size,
of many of the nonparticulate and soluble proteins, these while the intercept on the ordinate (infinite dilution of
groups have isoelectric points in the pH range 4–5, which acrylamide and bisacrylamide) is a measure of the mobil-
means that at neutral pH they are negatively charged. The ity of the SDS–protein in free solution (Ferguson plot).
absolute charge is not independent of mass, because the These plots can be used to determine relative masses of
capacity to carry more amino acids bearing charged side native proteins, because the slope is a measure of the ef-
chains is greater the larger the mass, while the compo- fective ratio of charge to mass at unit charge. Proteins that
sition is determined by genetic factors. This means the contain a significant amount of covalently linked carbohy-
ratio Q/M 1/3 is not constant for all proteins, so that sep- drate can still give anomalous results in this plot because
arations between individual proteins can be obtained ex- the randomly arranged carbohydrate chains change the
perimentally. In the case of nucleic acids the total charge overall shape of the ellipsoid from that given by standards
is generally related to mass for a given type of nucleic using purer proteins. Another factor to be considered is
acid, because here each nucleoside (the effective monomer the dependence on the amount of detergent bound per
of nucleic acids) carries free phosphates that are equally unit weight of peptide. Although this is generally con-
ionized at neutral pH. Thus, the relative positions after stant, there are notable exceptions where the equilibrium
PAGE can be related to size. As a result, masses deter- between free SDS and that bound does not follow the ex-
mined by a single PAGE experiment with native proteins pected relationship. To overcome this problem, high con-
are less readily interpreted in terms of van der Waals radii centrations of SDS (say, 10% solutions) may be required
than those made with nucleic acids (but see the later dis- in some cases, and this has its own limitations.
cussion of the Ferguson plot). It has been found, how- The bands or spots produced by SDS–PAGE are
ever, that when a protein is mixed with certain charged widened by diffusion of the micelles within the pores of
detergents [the most popular being sodium dodecyl sul- the polyacrylamide, but since the electrophoretic mobil-
fate (SDS)] the quantity of detergent associated with a ities of the bands are unidirectional and are greater than
gram of protein is relatively constant. The result of this those produced by diffusion, the leading edge of the band
association is a spheroidal micelle having a charge and is sharper than that expected from a simple diffusional
frictional coefficient proportional to the relative mass of model [Eq. (9)]. The concentration of the micelles at the
the protein [see Eq. (6)]. The addition of SDS dissociates leading edge is an advantage when small amounts of a
multisubunit proteins into their respective components, so macroion are being studied. Some experimental proce-
although adding SDS produces a macroion whose mass dures enhance this sharpening by enlisting the Kohlrausch
can be estimated from PAGE (the intrinsic charge of the regulating function [T ± /C, Eq. (4)]. To produce sharp
protein is swamped by the added charge from the SDS), bands this ratio must be unequal on the two sides of an
the native biologically active units cannot be examined interface, and to achieve this the salt concentrations (and
in the detergent. Despite this deficiency, SDS–PAGE has pH for polyampholytes) must be different across the inter-
become the most popular method of determining relative face. Practically, this is achieved by layering a thin band
masses of protein subunits and has displaced the ultracen- of gel containing different buffers on top of the main gel
trifuge in routine investigations. Another attraction is that and electrophoresing the protein through this band before
the detergent solubilizes otherwise insoluble proteins and entering the main gel. More elaborate arrangements of