Peptides ultraviolet spectrum

The changes in the ultraviolet spectrum of creatine phosphokinase (Fig. 15) on treatment with NBS point to the presence of thirteen to fourteen tryptophan residues per molecule compared with eleven to twelve tryptophan residues suggested by chemical analysis (Friedberg, 1956). Although the oxidation of the tryptophan residues in the molecule seems to be complete, the amount of cleavage of peptide bonds next to tryptophan is very small. 

Sorm and Keil (535) recently studied the structure of phalloidin in a preparation whose homogeneity had been checked by partition chromatography on starch and by ionophoresis. The ultraviolet spectrum was identical to that determined by Wieland (628). The total hydrolysis of this phalloidin yielded only the following amino acids, identified by chromatography alanine, cysteine, allohydroxyproline, and hydroxy-tryptophan. An investigation of the various peptides obtained on partial hydrolysis led Sorm and Keil to propose for phalloidin, considered to be a... 

The aromatic rings in the protein absorb ultraviolet light at an absorbance maximum of 280 nm, whereas the peptide bonds absorb at around 205 nm. The unique absorbance property of proteins could be used to estimate the level of proteins. These methods are fairly accurate with the ranges from 20 p,g to 3 mg for absorbance at 280 nm, as compared with 1 to 100 p,g for 205 nm. The assay is non-destructive as the protein in most cases is not consumed and can be recovered. Secondary, tertiary and quaternary structures all affect absorbance therefore, factors such as pH, ionic strength, etc can alter the absorbance spectrum. This assay depends on the presence of a mino acids which absorb UV light (mainly tryptophan, but to a lesser extent also tyrosine). Small peptides that do not contain such a mino acids cannot be measured easily by UV. 

SBA, prepared as described herein, possessed an ultraviolet (UV) absorption spectrum and an extinction coefficient comparable to those previously reported (5). Polyacrylamide gel electrophoresis in the presence of SDS resolved SBA into two closely spaced peptide bands comparable to those reported by Lotan et al. (6). ... 

The major limitation of both UV and MS detectors is that neither can provide quantitative or even semiquantitative information without reference standards. Ultraviolet response depends on the presence of a chromophore in a molecule and evidently might vary from one molecular species to another in a library. Although successful application of electrospray mass spectrometry for quantitative analysis of peptides has been reported [35], one should always keep in mind that signal intensity in a mass spectrum depends on the ability of a molecule to ionize. The ability to produce ions, especially with soft ionization techniques, might be very different for different molecules within one library, and the difference might be even bigger from one library to another. 

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. 

Figure 5 Far ultraviolet region of the CD spectra of (a) YINLIYRLRY-NH2 (compound 58, solid line) and YINLITRQRY-NH2 (compound 11, dashed line) in methanol and (b) INPIYRLRY-NH2 (compound 66) as a function of solvent. Solid lines correspond to 10 mM peptide in 10 mM sodium phosphate buffer, pH 7.5, and dashed lines correspond to 10 mM peptide in methanol. The CD spectrum in aqueous buffer correspond to a mixture of predominantly unfolded conformers. Type C spectra are observed in methanol, corresponding to the presence of predominantly helical conformers. (Reproduced from J. Med. Chem. 1995 38, 1150-1157.)...

VIII. The Ultraviolet Absorption Spectrum of the Peptide Bond and of the Polypeptide Fabric. 352... 

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