BLBS102-c08 BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come
8 Enzyme Activities 177chromogenic method(one of the spectrofluorometric methods).
The reaction is incubated for a fixed period of time. Then, be-
cause the development of color requires the inhibition of enzyme
activity, the reaction is stopped and the concentration of colored
product of the substrate is measured. This is thediscontinuous
orend point assay. Assays involving product separation that
cannot be designed to continuously measure the signal change,
such as electrophoresis, high performance liquid chromatogra-
phy (HPLC), and sample quenching at time intervals, are also
discontinuous assays. The advantage of discontinuous assays is
that they are less time consuming for monitoring a large num-
ber of samples for enzyme activity. However, additional control
experiments are needed to ensure that the initiation rate is linear
for the measuring period of time. Otherwise, the radiolabeled
substrate of an enzyme assay is a highly sensitive 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.Detection MethodsSpectrophotometric MethodsBoth the spectrophotometric method and the spectrofluoromet-
ric method discussed below use measurements at specific wave-
lengths of light energy (in the wavelength regions of 200–400 nm
(UV, ultraviolet) 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:A=−logT=εcl,whereAis the absorbance at certain wavelength,Tis the trans-
mittance, representing the intensity of transmitted light,εis the
extinction coefficient, andcis 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,thatis,vi=A/εlt.
Choices of appropriate materials for spectroscopic cells (cu-
vettes) depend on the wavelength used; quartz cuvettes must be
used at wavelengths less than 350 nm because glass and dis-
posable plastic cuvettes absorb too much light in the UV light
range. However, the latter glass and disposible plastic 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 spec-
tra may be found between the reactant and product molecules.
Thus, a different spectrum between two molecules can be calcu-lated to determine the most sensitive analytical wavelengths for
monitoring 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 ex-
cited state; this short-lived transition decays through various
high-energy, vibrational substrates at the excited electronic state
by heat dissipation, and then relaxes to the ground state with a
photon emission, the fluorescence. The emitted fluorescence is
less energetic (longer wavelength) 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 instrument is
designed to excite the sample and detect the emitted light at dif-
ferent wavelengths. The ratio of quanta fluoresced over quanta
absorbed offers a value of quantum yield (Q), which measures
the efficiency of the reaction leading to light emission. The light
emission signals vary with concentration of fluorescent mate-
rial by the Beer-Lambert law, where the extinction coefficient
is replaced by the quantum yield, and gives:If= 2. 31 I 0 εclQ,
whereIfandI 0 are the intensities of light emission and of in-
cident 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 fluorescent material concentration, which has
to be determined independently, due to the not strictly linear
relationship betweenIfandc(Lakowicz 1983).
Spectroflurometric methods provide highly sensitive capac-
ities for detection of low concentration changes in a reaction.
However, quenching (diminishing) 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 wavelength. The acceptor
molecule will either decay to its ground state if quenching occurs
or fluoresce at a characteristic wavelength if energy is trans-
ferred (Lakowicz 1983). Both situations can be overcome by
using an appropriate peptide sequence, up to 10 or more amino
acid residues, to separate the fluorescence 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 spectrofluorometric
method. For instance, the construction of a calibration curve
as the experiment is assayed is essential, and the storage con-
ditions for the fluorescent molecules are important because of
photodecomposition. In addition, the quantum yield is related to
the temperature, and the fluorescence signal will increase with
decreasing temperature, so a temperature-constant condition is
required (Bashford and Harris 1987, Gul et al. 1998).Radiometric MethodsThe principle of the radiometric methods in enzymatic catal-
ysis studies is the quantification of radioisotopes incorporated
into the substrate and retained in the product. Hence, success-
ful radiometric methods rely on the efficient separation of the