A reaction involving a protein can be induced by several mechanisms. This reaction induces molecular changes, often involving several intermediates. If it is possible to form the infrared difference spectra between well defined states of the protein, the difference spectra will only reflect those groups which are involved in the reaction.

 Since the absorbance changes of single groups have to be detected against the absorbance background of hundreds of other absorbing groups, the requirements for the sensitivity and the temporal stability of the experimental setup are very high. We are presently able to resolve absorbance changes of the order 10-5, as shown in the picture to the right.

In the upper panel, IR absorption spectra of the visual pigment rhodopsin before and after light activation are shown. In the absorption spectra, the changes due to the photoreaction seem to be negligible compared to the background absorption of up to 0.7. In the difference spectrum below, however, we can see a distinct pattern of absorption difference bands, which reflects in this example the absorption changes due to the transition from the dark state of rhodopsin to its active state, Meta II, or to its inactive precursor, Meta I, respectively. In this panel, the arrow corresponds to 10^-3 absorbance units (this means that the largest observed changes in the difference spectra are roughly only 1% of the absolute absorption). In this representation, bands caused by the initial state (here the dark state) point downwards, while those of the products (here photoproducts) point upwards.

The complicated band pattern in the difference spectrum is composed of bands arising from changes of the vibrational modes of the chromophore (due to the changed geometry after isomerization), to changes of the protein backbone (the so-called amide I and II bands), and to changes of specific amino acid side chains (e.g. protonation changes). In this example, it is evident that the conformational changes of the protein backbone (amide I and amide II) are much larger for the transition to the active state Meta II compared to those of the transition to Meta I.

Our Bruker IFS 28 FTIR Spectrometer equipped with a cryostat for low temperature experiments down to 90 Kelvin (- 183 °C). This spectrometer is used for static experiments, where both initial and photoproduct state are stable, or for slow processes (50 milliseconds to several minutes). In addition, the IFS 28 has an FT-Raman accessory, allowing to monitor chromophores and their changes in chromoproteins. Since the Raman scattering is excited with a Nd:YAG laser (wavelength 1064 nm), no fluorescence or photoreaction is usually evoked by the probing beam.