In proteins, there are three types of chromophores relevant for UV/vis spectroscopy:
• peptide bonds (amide bond)
• certain amino-acid side chains (mainly tryptophan and tyrosine)
• certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem).
The electronic transitions of the peptide bond occur in the far UV. The intense peak at 190 nm, and the weaker one at 210–220 nm are relevant transitions. A number of amino acids (Asp, Glu, Asn, Gln, Arg and His) have weak electronic transitions at around 210 nm. Usually, these cannot be observed in proteins because they are masked by the more intense peptide bond absorption. The most useful range for proteins is above 230 nm, where there are absorptions from aromatic side chains. While a very weak absorption maximum of phenylalanine occurs at 257 nm, tyro sine and tryptophan dominate the typical protein spectrum with their absorption maxima at 274 nm and 280 nm, respectively (Figure 1). In praxi , the presence of these two aromatic side chains gives rise to a band at ∼278 nm. Cystine (Cys 2 ) possesses a weak absorption maximum of similar strength as phenylalanine at 250 nm. This band can play a role in rare cases in protein optical activity or protein fluorescence.
Fig1. The presence of larger aggregates in biological samples gives rise to Rayleigh scatter visible by a considerable slope in the region from 500 to 350 nm. The dashed line shows the correction to be applied to spectra with Rayleigh scatter, which increases with λ −4 . Practically, linear extrapolation of the region from 500 to 350 nm is often performed to correct for the scatter. The corrected absorbance is indicated by the double arrow.
Proteins that contain prosthetic groups (e.g. haem, flavin, carotenoid) and some metal–protein complexes, may have strong absorption bands in the UV/vis range. These bands are usually sensitive to local environment and can be used for physical studies of enzyme action. Carotenoids, for instance, are a large class of red, yellow and orange plant pigments composed of long carbon chains with many conjugated double bonds. They contain three maxima in the visible region of the electromagnetic spectrum (∼420 nm, 450 nm, 480 nm).
Porphyrins are the prosthetic groups of haemoglobin, myoglobin, catalase and cytochromes. Electron delocalisation extends throughout the cyclic tetrapyrrole ring of porphyrins and gives rise to an intense transition at ∼400 nm called the Soret band . The spectrum of haemoglobin is very sensitive to changes in the iron bound ligand. These changes can be used for structure–function studies of haem proteins.
Molecules such as FAD (flavin adenine dinucleotide), NADH and NAD + are import ant coenzymes of proteins involved in electron-transfer reactions (redox reactions). They can be conveniently assayed by using their UV/vis absorption: 438 nm (FAD), 340 nm (NADH) and 260 nm (NAD + ).
In genetic material, the absorption of UV light occurs between 260 nm and 275 nm. The absorption spectra of the purine (adenine, guanine) and pyrimidine (cytosine, thymine, uracil) bases in nucleic acids or in polymers are sensitive to pH and greatly influenced by electronic interactions between bases.
