OTHER INSTRUMENTAL METHODS 143
inx and y (horizontal) directions and 4.3 μ m in the z (vertical) direction; and
(3) better control systems with increased speed of the feedback loop that
controls the height of the AFM tip to maintain minimal imaging force and
high image accuracy.
3.7.2 Fast and Time - Resolved Methods
3.7.2.1 Stopped-Flow Kinetic Methods. Enzyme kinetics happen on very
fast time scales; for instance, it is known that the rate of reaction for copper –
zinc superoxide dismutase (CuZnSOD), ∼ 1 × 10^9 M − 1 s − 1 , approaches the diffu-
sion - controlled rate. Chemists use various methods to study fast reactions. One
of the most frequently used rapid kinetic techniques is that of stopped - fl ow in
which the reactants (enzyme and substrate) are rapidly mixed. The lower
practical limit for mixing to take place is about 0.2 ms. The stopped - fl ow prin-
ciple of operation allows small volumes of solutions to be driven from high -
performance syringes to a high - effi ciency mixer just before passing into a
measurement fl ow cell. As the solutions fl ow through, a steady - state equilib-
rium is established and the resultant solution is only a few milliseconds old as
it passes through the measurement cell. The mixed solution then passes into
a stopping syringe, which allows the fl ow to be instantaneously stopped. Some
of the resultant solution will be trapped in the fl ow cell and as the reaction
proceeds, the kinetics can be followed using the appropriate measurement
technique. The most common method of following the kinetics is by absor-
bance or fl uorescence spectrometry, and in these cases the measurement cell
is an appropriate spectrometer fl ow cell. Many commercially available absor-
bance and fl uorescence spectrometers may be modifi ed to accept stopped - fl ow
accessories.
In order to use the stopped - fl ow technique, the reaction under study must
have a convenient absorbance or fl uorescence that can be measured spectro-
photometrically. Another method, called rapid quench or quench - fl ow, oper-
ates for enzymatic systems having no component (reactant or product) that
can be spectrally monitored in real time. The quench - fl ow is a very fi nely tuned,
computer - controlled machine that is designed to mix enzyme and reactants
very rapidly to start the enzymatic reaction, and then quench it after a defi ned
time. The time course of the reaction can then be analyzed by electrophoretic
methods. The reaction time currently ranges from about 5 ms to several
seconds.
M. Fabian and co - workers have studied the protein ’ s role in internal elec-
tron transfer to the catalytic center of cytochrome c oxidase using stopped -
fl ow kinetics.^48 Mitochondrial cytochrome c oxidase, CcO, an enzyme that
catalyzes the oxidation of ferrocytochrome c by dioxygen, is discussed more
fully in Section 7.8. In the overall process, O 2 is reduced to water, requiring
the addition of four electrons and four protons to the enzyme ’ s catalytic
center. Electrons enter CcO from the cytosolic side, while protons enter from
the matrix side of the inner mitochondrial membrane. This redox reaction,