Micro-Spectrophotometer

Off-line and on-line micro-spectrophotometry is proving to be an important tool for assessing the catalytic properties of enzymes in the crystal (Bourgeois et al., 2002Pearson et al., 2007). It can be applied to metallo-enzymes, photosensitive proteins or proteins with co-factors, substrates or products that absorbs in UV-VIS. Micro-spectrophotometry can be used to confirm the presence or absence of a substrate or a co-factor, as well as the redox state of a protein. It can also aid the design of efficient triggering of a reaction in the crystals or trapping of intermediate states. On-line micro-spectrophotometry is also particularly useful for assessing radiation damage, including the unwanted redox processes triggered by intense x-ray beams, and for determining the best data collection strategy for avoiding the extensive radiation damage (Berglund et al., 2002Pearson et al., 2007)

BioCARS micro-spectrophotometer is available to users for crystal absorption measurements both on-line, in the 14-BM-C station (pictured), and a stand-alone instrument for off-line measurements. We use modified 4DX XSPECTRA micro-spectrophotometer system. For testing of photo-sensitive samples, external illumination with laser or LED light sources at various wavelengths can be accommodated.

REFERENCES

  • Berglund, G.I., Carlsson, G.H., Smith, A.T., Szoke, H., Henriksen, A., and Hajdu, J.
    The catalytic pathway of horseradish peroxidase at high resolution.
    Nature 417, 463-468 (2002)
    abstract
  • Bourgeois, D., Vernede, X., Adam, V., Fioravanti, E., and Ursby, T.
    A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals.
    Journal of Applied Crystallography 35, 319-326 (2002)
    abstract
  • Pearson, A.R., Pahl, R., Kovaleva, E.G., Davidson, V.L., and Wilmot, C.M.
    Tracking X-ray-derived redox changes in crystals of a methylamine dehydrogenase/amicyanin complex using single-crystal UV/Vis microspectrophotometry.
    Journal of Synchrotron Radiation 14, 92-98 (2007)
    abstract