Time-Resolved Infrared Spectroscopy Applied to Photobiological Systems

The main interest in the study of photobiological systems is the role of the chromophore. Its structure and molecular changes during the photoreaction can be investigated by uv-vis and Resonance Raman Spectroscopy. However, there is a need to learn more about the role of the protein in the functioning of these systems. Especially, the interaction and interplay between specific amino acid residues and the chromophore are of great interest. In principle, infrared spectroscopy can be employed for such problems. But, since it is generally considered to be a relatively insensitive method, it appears, at first sight, questionable that it would enable one to detect molecular changes of a few functional groups against the large background absorption caused by the total system, which is a protein or even a biological membrane. With the development of new infrared detectors, however, which are fast and at least two orders of magnitude more sensitive than the usual thermopile detector, such an undertaking now appears feasible. In addition to acquiring the spectral information, it is also important to obtain information on the kinetics of the molecular changes. It appears interesting, for example, to ascertain whether or not the time-course of protein changes parallels that of chromophore changes. Therefore, the method of time-resolved infrared spectroscopy has become a natural extension of time-resolved uv-vis and Resonance Raman Spectroscopy for the investigation of photobiological systems. Since the time-resolved difference spectra reflect only those groups which undergo molecular transformations, their interpretation is facilitated. Nevertheless, additional techniques, such as chemical modifications and isotopic labelling influencing the difference spectra, may be required. Such methods would help to distinguish between molecular changes caused by the protein as apposed to the chromophore,and provide detailed information on the molecular events.