Biomedical Engineering Reference
In-Depth Information
chemical bond, it is directly related to its bond length (Deng and Callender, 1999).
Therefore, vibrational spectroscopy is well suited to the studies of chemical bond
distortion during enzymatic catalysis.
The conventional methods of investigating nuclear vibrational properties of proteins,
namely spontaneous infrared, Raman resonance spectroscopy, have serious limitations,
because many vibrational modes contribute to the spectrum of a protein at any given
frequency. To overcome these limitations, new experimental approaches have been
developed during the last decade. Among such approaches are differential and time
resolved IR and Raman spectroscopy, coherent anti-Stockes Raman scattering (CARS),
Fourier transform infrared spectroscopy (FTIR), multidimentional IR and RR
spectroscopy, two-dimentional infrared echo and Raman echo (Hamaguchi and
Gustafson, 1994; Deng and Callender 1999; Asplund et al., 2000; Uchida et al., 2000;
Mukamel, 2000; Fourkas, 2001; and references herein).
In Raman differential spectroscopy, a conventional Raman spectrometer was adapted
to measure small differences in the Raman spectra (Deng and Callender 1999). The
spectrometer system permits detection with an accuracy of 0.1%. Laser light is focused
on a specially fabricated split cell from underneath. The Raman-scattering light at is
frequency shifted from the incoming laser light by the frequency of a vibrational
mode. Scattered light is collected from one side of the split cell. The cell is translated
and the scattered light is collected from the cell's second side. The two spectra are then
subtracted in a computer to form the difference spectrum.
Raman differential spectroscopy was applied to the investigation of enzyme-substrate
complexes. The protein phosphoglucomutase (PGM) catalyzes the interconversion of
glucose 1-phosphate to glucose 6-phosphate. It was shown that the difference in the
spectra of enzyme complexes with the substrate samples, the phosphate group of which
was enriched with and correspondingly, belong to the P-O symmetric stretch
with a frequency of The differential Raman spectra of complexes of lactate
dehydrogenase with cofactors NAD and NADH and substrates lactate and pyruvate were
detected. These techniques in combination with site-directed mutagenase and isotope
editing of pyruvate's carboxamid group and the C-4-H fragment of the
NAD, allowed the establishment of the correct geometry of the reactive complex.
Time-resolved anti-Stokes Raman spectroscopy is used for monitoring vibrational
relaxation dynamics in solution and provides information about specific modes in
molecules under investigation (Nakabayashi et al., 1997; Uchida et al. 2000). The
experimental setup of a picosecond time-resolved Raman spectrometer is schematically
shown in Fig. 1.2. A pump pulse excites a molecule, and the anti-Stokes Raman
spectrum of vibrationally excited state of the molecule is obtained by a probe pulse
following the pump pulse after the delay time. The method was used for the
investigation of Fe-ligand interactions, an active site of carbonmonoxy CooA
hemoprotein (Uchida et al., 2000). This protein acts as a transcriptional activator for the
expression of CO oxidation system in bacteria. To identify the axial ligand of CO-bound
CoooA, the protein samples, with and without imidazol ligand, were photodissociated by
a picosecond laser pulse, and vibrations of the transiently formed five-coordinate species
were monitored by the subsequent picosecond probe pulse. It is shown that His77 is the
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