168 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
catalyzed reaction itself does not produce a measur-
able signal, a suitable reagent that has no effect on
enzyme activity can be added to the reaction mixture
to react with one of the products to form a measura-
ble signal.
Nevertheless, sometimes no easily readable dif-
ferences between the spectrum of absorbance of the
substrate and that of the product can be measured. In
such cases, it may be possible to measure the ap-
pearance of the colored product, by the chromo-
genic method(one of the spectrofluorometric meth-
ods). The reaction is incubated for a fixed period of
time. Then, because the development of color re-
quires the inhibition of enzyme activity, the reaction
is stopped and the concentration of colored product
of the substrate is measured. This is the discontinu-
ous orend point assay. Assays involving product
separation that cannot be designed to continuously
measure the signal change, such as electrophoresis,
high performance liquid chromatography (HPLC),
and sample quenching at time intervals, are also dis-
continuous assays. The advantage of discontinuous
assays is that they are less time consuming for mon-
itoring a large number of samples for enzyme activ-
ity. However, additional control experiments are
needed to assure that the initiation rate is linear for
the measuring period of time. Otherwise, the radio-
labeled substrate of an enzyme assay is a highly sen-
sitive method that allows the detection of radioactive
product. Separation of the substrate and the product
by a variety of extraction methods may be required
to accurately assay the enzyme activity.
DETECTIONMETHODS
Spectrophotometric Methods
Both the spectrophotometric method and the spec-
trofluorometric method discussed below use meas-
urements at specific wavelengths of light energy [in
the wavelength regions of 200–400 nm (UV, ultravi-
olet) and 400–800 nm (visible)] to determine how
much light has been absorbed by a target molecule
(resulting in changes in electronic configuration).
The measured value from the spectrophotometric
method at a specific wavelength can be related to the
molecule concentration in the solution using a cell
with a fixed path length (lcm) that obeys the Beer-
Lambert law:
AlogTcl,
where Ais the absorbance at certain wavelength, Tis
the transmittance, representing the intensity of trans-
mitted light, is the extinction coefficient, and cis
the molar concentration of the sample. Using this
law, the measured change in absorbance, A,can be
converted to the change in molecule concentration,
c, and the change in rate, vi, can be calculated as a
function of time, t, that is, viA/lt.
Choices of appropriate materials for spectroscop-
ic cells (cuvettes) depend on the wavelength used;
quartz cuvettes must be used at wavelengths less
than 350 nm because glass and disposable plastic
cuvettes absorb too much light in the UV light
range. However, the latter glass and disposible plas-
tic cuvettes can be used in the wavelength range
of 350–800 nm. Selection of a wavelength for the
measurement depends on finding the wavelength
that produces the greatest difference in absorbance
between the reactant and product molecules in the
reaction. Though the wavelength of measurement
usually refers to the maximal wavelength of the
reactant or product molecule, the most meaningful
analytical wavelength may not be the same as the
maximum wavelength because significant overlap of
spectra may be found between the reactant and
product molecules. Thus, a different spectrum be-
tween two molecules can be calculated to determine
the most sensitive analytical wavelengths for moni-
toring the increase in product and the decrease in
substrate.Spectrofluorometric MethodsWhen a molecule absorbs light at an appropriate
wavelength, an electronic transition occurs from the
ground state to the excited state; this short-lived
transition decays through various high-energy, vi-
brational substrates at the excited electronic state by
heat dissipation, and then relaxes to the ground state
with a photon emission, the fluorescence. The emit-
ted fluorescence is less energetic (longer wave-
length) than the initial energy that is required to
excite the molecules; this is referred to as the Stokes
shift. Taking advantage of this, the fluorescence in-
strument is designed to excite the sample and detect
the emitted light at different wavelengths. The ratio
of quanta fluoresced over quanta absorbed offers a
value of quantum yield (Q),which measures the effi-
ciency of the reaction leading to light emission. The
light emission signals vary with concentration of