Biomedical Engineering Reference
In-Depth Information
A novel pump-damp-probe method (PDPM), which allows the characterization of
solvation dynamics of a fluorescence probe not only in excited but also in the ground
states has been recently developed (Changenet-Barret, 2000 and references therein). In
PDPM , a pump produces a nonequilibrium population of the probe excited, which, after
media relaxation, is simulated back to the ground states. The solvent relaxation of the
nonequlibrium ground state is probed by monitoring with absorption technique. The
inramolecular protein dynamics in a solvent-inaccessible region of calmodulin labeled
with coumarin 343 peptide was examined by PDPM. In the pump-dump-probe
experiments, part of a series of laser output pulses was frequency-doubled and softer
beams were used as the probe. The delay of the probe with respect to the pump was fixed
at 500 ps.
Single molecule and two photon fluorescence spectroscopy
Recent developments in fluorescence spectroscopy and microscopy have made it
possible to detect and image single molecules (Denk et al., 1990; Xue and Yeung, 1995;
Craig et al., 1996; Edman et al., 1996; Xie et al., 1998; Heinze et al., 2000; and
references therein). These techniques allow the conducting of spectroscopic
measurements for studying chemical and biological species and their interaction with the
environment. Single molecular measurement (SMM) offers time resolution to monitor
dynamic processes such as translation, orientation and enzymatic reactions on a time
scale from milli-seconds to ten- seconds. Confocal fluorescence methodologies are based
on the detection of laser-induced fluorescence of single molecules in a very small focal
volume of approximately 1 fl. At present, molecules with an extinction coefficient larger
than and a fluorescent quantum efficiency greater than 0.1 can be studied with SMM.
The techniques which have evolved to the level of single molecule sensitivity at room
temperature are as follows: flow cytometry, confocal fluorescence correlation
spectroscopy, and micro-droplet technique.
The single-molecule fluorescence technique was used for the study of differences in
the chemical reactivity of individual molecules of enzymes. The kinetics of producing
fluorescent NADH from lactate and catalyzed by lactate dehydrogenase has been
monitored by this technique (Xue and Yeung, 1995). The enzyme molecules are
presented at very low concentration in a narrow capillary and
each discrete molecule produces a discrete zone of the fluorescent NADH. The activity
of individual enzyme molecules was found to be variable up to a factor of four. The
kinetics of synthesis of a fluorescent product, 2'-(2-benzothiazol)-6'-hydrobenzthiazol,
produced by single alkaline phosphotase molecule has been investigated (Craig et al.,
1996). Single enzyme moleculesshow a range of activity from 1 to 10. The experimental
values of activation energy of the enzymatic reaction vary more than a factor of 2. Some
of the above mentioned results may be explained by a partial adsorption of enzyme
molecules on the capillary walls' surface which exhibit different activity as compared
with molecules in bulk. This problem can be solved by comparing parameters of the
enzyme kinetics in the single-molecules regime and in “regular” conditions with high
concentrations of enzymes. The single-molecule fluorescence technique has been useful
in the study of conformational transition of biopolymers (Edman et al. 1996). It has
been shown that single DNA molecules labeled with a fluorescence probe exhibit
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