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., 2002; Pearson 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., 2002; Pearson 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.
- 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