7 Enzyme Activities 169fluorescent material by the Beer-Lambert law, where
the extinction coefficient is replaced by the quantum
yield, and gives: If2.31 I 0 clQ, where Ifand I 0 are
the intensities of light emission and of incident light
that excites the sample, respectively. However con-
version of light emission values into concentration
units requires the preparation of a standard curve of
light emission signals as a function of the fluores-
cent material concentration, which has to be deter-
mined independently, due to the not strictly linear
relationship between Ifand c(Lakowicz 1983).
Spectroflurometric methods provide highly sensi-
tive capacities for detection of low concentration
changes in a reaction. However, quenching (dimin-
ishing) or resonance energy transfer (RET) of the
measured intensity of the donor molecule will be
observed if the acceptor molecule absorbs light and
the donor molecule emits light at the same wave-
length. The acceptor molecule will either decay to
its ground state if quenching occurs or fluoresce at a
characteristic wavelength if energy is transferred
(Lakowicz 1983). Both situations can be overcome
by using an appropriate peptide sequence, up to 10
or more amino acid residues, to separate the fluores-
cence donor molecule from the acceptor molecule if
peptide substrates are used. Thus both effects can be
relieved by cleaving the intermolecular bonding
when protease activity is being assayed.
Care should be taken in performing the spectro-
fluorometric method. For instance, the construction
of a calibration curve as the experiment is assayed is
essential, and the storage conditions for the fluores-
cent molecules are important because of photo-
decomposition. In addition, the quantum yield is
related to the temperature, and the fluorescence sig-
nal will increase with decreasing temperature, so a
temperature-constant condition is required (Bash-
ford and Harris 1987, Gul et al. 1998).
Radiometric Methods
The principle of the radiometric methods in enzy-
matic catalysis studies is the quantification of
radioisotopes incorporated into the substrate and
retained in the product. Hence, successful radiomet-
ric methods rely on the efficient separation of the
radiolabled 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 of particles; however,
(^125) I decays through emission of particles, 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 disintegra-
tions 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 radioactiv-
ity per mass or per molarity of the sample (e.g.,
Ci/mg or dpm/mol).
Methods of separation of radiolabeled product
and residual radiolabeled substrate include chroma-
tography, electrophoresis, centrifugation, and sol-
vent extraction (Gul et al. 1998). For the detection of
radioactivity, one commonly used instrument is a
scintillation counter that measures light emitted
when solutions of p-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
radioactivity on surfaces in close contact either with
X-ray film or with plates of computerized phosphor
imaging devices. Whenever operating the isotopes-
containing 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 re-
actions and is usually used in conjunction with other
detection methods. The most commonly used
chromatographic methods include paper chromatog-
raphy, column chromatography, thin-layer chroma-
tography (TLC), and high performance liquid chro-
matography (HPLC). Paper chromatography is a
simple and economic method for the readily separa-
tion of large numbers of samples. By contrast, col-
umn chromatography is more expensive and has
poor reproducibility. The TLC method has the ad-
vantage of faster separation of mixed samples, and
like paper chromatography, it is disposable and can
be quantified and scanned; it is not easily replaced
by HPLC, especially for measuring small, radiola-
beled molecules (Oldham 1992).
The HPLC method featured with low compress-
ibility resins is a versatile method for the separation