Amino Acid Residues and Peptide Bonds

TABLE 7.2 Spectral parameters for selected amino acids at pH 7.0. 

The contribution of Phe in the range is minimal if Tyr and Trp are present in a protein. Trp has the most intense absorbency in this range, which is the basis for the measurement of protein concentrations by UV spectrometry. The number of moles of Tyr and 

Trp per mole of protein, Mty, and M p can be determined from the absorption of the protein solution (6M guanidine hydrochloride in 0.02 M phosphate buffer, pH 6.5) measured at 288 and 280 nm  

Variations in pH affect the spectra of Tyr and Trp, especially the deprotonated Tyr whose shift can be monitored at 295 nm. Another chromophore is the disulfide group of cystine, which has a very week absorption at 250 nm. It has been common practice to determine protein concentrations spectrometrically at 280 nm by assuming an absorbency of 1.0 for a 1 mg/mL protein solution. 

Some of the prosthetic groups displaying electronic absorption in the region of 400-600nm such as flavins ( 455 1.27 x lO ), pyridoxal phosphate ( 415 = 0.65 x lO ) and hemes ( 550 = 2.77 x lO ). Their absorption bands are usually sensitive to the local environment and can be used to monitor structural and environmental changes. 

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In the analysis of the energetics of protein denaturation, knowledge of the structure of the unfolded state is as important as the knowledge of the native one. The thermodynamical approach to protein denaturation usually assumes that the result of denaturation is the complete exposure to the solvent of all the amino acid residues and peptide bonds, which means that the unfolded state is one single population of highly expanded, fully solvated and disordered conformations, whatever the protein, the denaturation process, or the solvent conditions considered. 

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