Disulfide bond, absorption spectrum

Using ultraviolet/visible (UV/Vis) absorption spectroscopy, it is possible to measure the protein concentration using Beer s Law A = e c, where A is the measured absorbance of a solution, e is the absorptivity of the protein, is the pathlength of the cell used to determine the absorbance, and c is the protein concentration. Proteins typically exhibit two strong, broad absorption bands in the UV/Vis part of the spectrum. The first and most intense band is centered at 214 nm and arises from absorption of light by the peptide backbone. The second absorption band is typically found at 280nm. This band arises from absorbance from the aromatic side chains of Trp, Tyr, and Phe. Disulfide bonds may exhibit weak absorption in this range as well. 

In the case of peptides and proteins the spectro-scop) of the amide bonds, the side chains and any prosthetic groups (such as haems) determines the observed UV-visible absorption spectrum. However, as with DNA, intensities and wavelengths can be perturbed by the local environment of the groups. UV spectra of proteins are usually divided into the near and far UV regions. The near-UV in this context means 250-300 nm and is also described as the aromatic region, though transitions of disulfide bonds (cystines) also contribute to the total absorption intensity in this region. The far-UV (< 250 nm) is dominated by transitions of the peptide backbone of the protein, but transitions from some side chains also contribute to the spectrum below 250 nm. 

More complicated molecules, with two or more chemical bonds, have more complicated absorption spectra. However, each molecule has such a characteristic spectrum that the spectrum can be used to detect the presence of that particular molecular substance. Figure 14-17, for example, shows the absorptions shown by liquid carbon tetrachloride, CCfi, and by liquid carbon disulfide, CS2. The bottom spectrum is that displayed by liquid CC14 containing a small amount of C. The absorptions of CS2 are evident in the spectrum of the mixture, so the infrared spectrum can be used to detect the impurity and to measure its concentration. 

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