Raman difference spectroscopy in measurements of molecules and molecular groups inside proteins

In order to obtain the Raman spectra of small molecules inside proteins (or other large macromolecules) and/or small molecular groups of a protein, techniques were developed to perform sensitive Raman difference spectroscopy. The macromolecule–small molecule complex Raman spectrum is taken in addition to the Raman spectrum of the macromolecule, and the two are computer subtracted. For studies on proteins, it is necessary to use optical multichannel detectors, as spectrometers employing photomultiplier detectors are too slow optically to collect signals from dilute samples. The apparatus is described and characterized in detail. A subtraction fidelity of 0.1% can be achieved for samples, such as proteins, which contain broad bands in their Raman spectra. Hence it is possible to obtain fairly good Raman spectra of very small molecular groups inside proteins. An example from recent work is presented.