During their activity only certain parts of the proteins undergo molecular changes and their identification from the infrared spectrum is impeded by the strong background absorbance of the entire protein. This obstacle is overcome by forming the IR difference between two stable reaction states, an ingenious procedure which renders IR spectroscopy selective. The difference spectrum exhibits bands characteristic only for the molecular groups that are modified during protein activity.
The most common infrared cell is the transmission cuvette. The protein solution is dried on a infrared transparent window, inside of a circular groove and approximately 30 µl buffer are overlaid. A counter window squeezes the construct, forming a reservoir, with the thickness between 3 and 10 µm.
A significant advance in the understanding of membrane protein structure and functionality is achieved using the attenuated total reflection (ATR) cell. Here, the protein film is formed at the surface of a highly refractive crystal material (Diamond, Ge, Si, ZnSe or ZnS). In the crystal, the infrared beam undergoes total reflection and the sample is probed by an evanescent field. With the ATR technique, the strong absorption of water is circumvented.
Step-scan FT-IR spectroscopy benefits from all advantages of the continuous-scan FT-IR spectroscopy and imposes no mechanical restrictions for the achievement of high time resolutions (ns-µs).During a step-scan experiment, the movable mirror of the interferometer moves step-wise from one sampling position to the next. At each position the mirror is held fixed and the kinetic process is initiated, for example by a laser pulse. After covering the entire set of sampling positions, the kinetic data are rearranged into interferograms and Fourier transformed. Finally, absorption difference spectra are calculated at different times after triggering the reaction.
Raman spectroscopy is based on a scattering mechanism for exciting molecular vibrations. Under resonant conditions, the sensitivity of Raman spectroscopy can be drastically enhanced while probing only chromophore scattering.