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178 Part 2: Biotechnology and Enzymologyradiolabled product from the residual radiolabeled substrate and
on the sensitivity and specificity of the radioactivity detection
method. Most commonly used radioisotopes, for example,^14 C,(^32) P, (^35) S, and (^3) H, decay through emission ofβparticles; how-
ever,^125 I decays through emission ofγparticles, whose loss is
related to loss of radioactivity and the rate of decay (the half-life)
of the isotope. Radioactivity expressed in Curies (Ci) decays at a
rate of 2.22× 1012 disintegrations per minute (dpm); expressed
in Becquerels (Bq), it decays at a rate of 1 disintegrations per
second (dps). The experimental units of radioactivity are counts
per minute (cpm) measured by the instrument; quantification of
the specific activity of the sample is given in units of radioac-
tivity per mass or per molarity of the sample (e.g.,μCi/mg or
dpm/μmol).
Methods of separation of radiolabeled product and residual
radiolabeled substrate include chromatography, electrophoresis,
centrifugation, and solvent extraction (Gul et al. 1998). For the
detection of radioactivity, one commonly used instrument is a
scintillation counter that measures light emitted when solutions
ofp-terphenyl or stilbene in xylene or toluene are mixed with
radioactive material designed around a photomultiplier tube.
Another method is the autoradiography that allows detecting ra-
dioactivity on surfaces in close contact either with X-ray film or
with plates of computerized phosphor imaging devices. When-
ever operating the isotopescontaining experiments, care should
always be taken for assuring safety.
Chromatographic Methods
Chromatography is applied in the separation of the reactant
molecules and products in enzymatic reactions and is usually
used in conjunction with other detection methods. The most
commonly used chromatographic methods include paper chro-
matography, column chromatography, thin-layer chromatogra-
phy (TLC), and HPLC. Paper chromatography is a simple and
economic method for the readily separation of large numbers
of samples. By contrast, column chromatography is more ex-
pensive and has poor reproducibility. The TLC method has the
advantage of faster separation of mixed samples, and like pa-
per chromatography, it is disposable and can be quantified and
scanned; it is not easily replaced by HPLC, especially for mea-
suring small, radiolabeled molecules (Oldham 1992).
The HPLC method featured with low compressibility resins
is a versatile method for the separation of either low molecular
weight molecules or small peptides. Under a range of high pres-
sures up to 5000 psi (which approximates to 3.45× 107 Pa),
the resolution 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 concentra-
tion is gradually increased for better resolution. The three HPLC
methods most commonly used in the separation steps of enzy-
matic 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 covalently 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 co-
valently bonded to a charged functional group; it binds the
molecules through electrostatic interactions, which can be dis-
rupted by the increasing ionic strength of the mobile phase.
By modifying the composition of the mobile phase, differential
elution, separating multiple molecules, is achieved.
In the size-exclusion HPLC, also known as gel filtration, the
stationary phase is composed of porous beads with a particular
molecular weight range of fractionation. However, this method is
not recommended where molecular weight differences between
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 elec-
trophoresis (PAGE) are widely used methods for separation of
macromolecules; they depend, respectively, on the percentage
of agarose and acrylamide in the gel matrix. The most com-
monly used method is the sodium dodecyl sulfate (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 samples will
thus be based on molecular weight under an electric field over a
period of time. After electrophoresis, peptides or proteins bands
can be visualized by staining the gel with Coomassie Brilliant
Blue or other staining reagents, and radiolabeled materials can
be detected by autoradiography. Applications of electrophoresis
assays are not only for detection of molecular weight and
radioactivity differences, but also for detection of charge
differences. For instance, the enzyme-catalyzed phosphoryla-
tion reactions result in phosphoryl transfer from substrates to
products, and net charge differences between two molecules
form the basis for separation by electrophoresis. If radioisotope
(^32) P-labeled phosphate is incorporated into the molecules, the
reactions can be detected by autoradiography, by monitoring
the radiolabel transfer after gel electrophoresis, or by immuno-
logical blotting with antibodies that specifically recognize
peptides or proteins containing phosphate-modified amino acid
residues.
Native gel electrophoresis is also useful in the above ap-
plications, where not only the molecular weight but also the
charge density and overall molecule 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 zymography (Hames and Rickwood