170 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
of either low molecular weight molecules or small
peptides. Under a range of high pressures up to 5000
psi (which approximates to 3.45 107 Pa), the reso-
lution is greatly enhanced with a faster flow rate and
a shorter run time. The solvent used for elution,
referred to as the mobile phase, should be an HPLC
grade that contains low contaminants; the insoluble
media is usually referred to as the stationary phase.
Two types of mobile phase are used during elution;
one is an isocratic elution whose composition is not
changed, and the other is a gradient elution whose
concentration is gradually increased for better reso-
lution. The three HPLC methods most commonly
used in the separation steps of enzymatic assays
are reverse phase, ion-exchange, and size-exclusion
chromatography.
The basis of reverse phase HPLC is the use of a
nonpolar stationary phase composed of silica cova-
lently bonded to alkyl silane, and a polar mobile
phase used to maximize hydrophobic interactions
with the stationary phase. Molecules are eluted in a
solvent of low polarity (e.g., methanol, acetonitrile,
or acetone mixed with water at different ratios) that
is able to efficiently compete with molecules for the
hydrophobic stationary phase.
The ion-exchange HPLC contains a stationary
phase covalently bonded to a charged functional
group; it binds the molecules through electrostatic
interactions, which can be disrupted by the increas-
ing ionic strength of the mobile phase. By modify-
ing the composition of the mobile phase, differential
elution, separating multiple molecules, is achieved.
In the size-exclusion HPLC, also known as gel fil-
tration, the stationary phase is composed of porous
beads with a particular molecular weight range of
fractionation. However, this method is not recom-
mended where molecular weight differences be-
tween substrates and products are minor, because of
overlapping of the elution profiles (Oliver 1989).
Selection of the HPLC detector depends on the
types of signals measured, and most commonly the
UV/visible light detectors are extensively used.
Electrophoretic Methods
Agarose gel electrophoresis and polyacrylamide gel
electrophoresis (PAGE) are widely used methods for
separation of macromolecules; they depend, respec-
tively, on the percentage of agarose and acrylamide in
the gel matrix. The most commonly used method is
the sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis (SDS-PAGE) method; under denaturing
conditions, the anionic detergent SDS is coated on
peptides or proteins giving them equivalently the
same anionic charge densities. Resolving of the sam-
ples will thus be based on molecular weight under an
electric field over a period of time. After elec-
trophoresis, peptides or proteins bands can be visual-
ized by staining the gel with Coomassie Brilliant
Blue or other staining reagents, and radiolabeled
materials can be detected by autoradiography. Ap-
plications of electrophoresis assays are not only for
detection of molecular weight and radioactivity dif-
ferences, but also for detection of charge differences.
For instance, the enzyme-catalyzed phosphorylation
reactions result in phosphoryl transfer from sub-
strates to products, and net charge differences be-
tween two molecules form the basis for separation by
electrophoresis. If radioisotope^32 P-labeled phos-
phate is incorporated into the molecules, the reactions
can be detected by autoradiography, by monitoring
the radiolabel transfer after gel electrophoresis, or by
immunological blotting with antibodies that specifi-
cally recognize peptides or proteins containing phos-
phate-modified amino acid residues.
Native gel electrophoresis is also useful in the
above applications, where not only the molecular
weight but also the charge density and overall mole-
cule shape affect the migration of molecules in gels.
Though SDS-PAGE causes denaturing to peptides
and proteins, renaturation in gels is possible and can
be applied to several types of in situ enzymatic
activity studies such as activity staining and zymog-
raphy (Hames and Rickwood 1990). Both methods
assay enzyme activity after electrophoresis, but
zymography is especially intended for proteolytic
enzyme activity staining in which gels are cast
with high concentrations of proteolytic enzyme sub-
strates, for example, casein, gelatin, bovine serum
albumin, collagen, and others. Samples containing
proteolytic enzymes can be subjected to gel elec-
trophoresis, but the renaturation step has to be per-
formed if a denaturing condition is used; then the
reaction is performed under conditions suitable for
assaying proteolytic enzymes. The gel is then sub-
jected to staining and destaining, but the entire gel
background will not be destained because the gel
is polymerized with protein substrates, except in
clear zones where the significant proteolysis has
occurred; the amount of staining observed will be