Peptide bond, absorption spectroscopy

Solid-state spectral studies relating to proteins have not been numerous, but those associated with the peptide-bond absorption are of special interest. In particular, we note Peterson and Simpson s (1957) singlecrystal spectroscopy of myristamide, and the demonstration of dichroism in oriented films of helical polypeptides by Gratzer et al. (1961). The results of these studies are discussed in Section IV, . Since great care and exacting technique is required for solid-state studies, we may do no better than to refer interested readers to the original papers for critical discussions of experimental aspects. 

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. 

Quantitative analysis of proteins can be achieved by UV spectroscopy. The peptide bond has an absorption maximum around k = 205 nm, the aromatic rings on the amino acids Tryptophan and Tyrosine absorb strongly around k = 280 nm. Also commonly used are colorimetric assays, which contain reagents that specifically form coloured complexes with proteins. These quantitative methods usually measure the total protein concentration. Either the protein of interest has to be isolated prior to analysis, or a very specific method has to be found to quantify only the targeted protein. Very sensitive and specific analysis of antibodies and antigens can be achieved with bioassays (section 5.1) or biosensors (section 5.2). 

IR spectroscopy is widely used for structural analysis because many functional groups have characteristic vibrational frequencies. Vibrations of the peptide bond give rise to three major infrared (IR) absorption bands (Table 6.1, Fig. 6.6) ... 

The peptide subunit was easily incorporated into lipid bilayers of liposome, as confirmed by absorption and fluorescence spectroscopy. Formation of H-bonded transmembrane channel structure was confirmed by FT IR measurement, which suggests the formation of a tight H-bond network in phosphatidylcholine liposomes. Liposomes were first prepared to make the inside pH 6.5 and the outside pH 5.5. Then the addition of the peptide to such liposomal suspensions caused a rapid collapse of the pH gradient. The proton transport activity was comparable to that of antibiotics gramicidin A and amphotericin B. 

The wavelengths of IR absorption bands are characteristic of specific types of chemical bonds. In the past infrared had little application in protein analysis due to instrumentation and interpretation limitations. The development of Fourier transform infrared spectroscopy (FUR) makes it possible to characterize proteins using IR techniques (Surewicz et al. 1993). Several IR absorption regions are important for protein analysis. The amide I groups in proteins have a vibration absorption frequency of 1630-1670 cm. Secondary structures of proteins such as alpha(a)-helix and beta(P)-sheet have amide absorptions of 1645-1660 cm-1 and 1665-1680 cm, respectively. Random coil has absorptions in the range of 1660-1670 cm These characterization criteria come from studies of model polypeptides with known secondary structures. Thus, FTIR is useful in conformational analysis of peptides and proteins (Arrondo et al. 1993). 

The effect of N-acetyl substitution in methionine on the nature of transients formed after one-electron oxidation was studied as a function of pH and NAM concentration. The observed absorption bands with X = 290 nm, 360 nm, and 490 nm were respectively assigned to a-(alkylthio)alkyl, hydroxysulfuranyl and dimeric radical cations with intermolecular three-electron bond between sulfur atoms. N-acetylmethionine amide (NAMA) (Chart 7) represents a simple chemical model for the methionine residue incorporated in a peptide. Pulse radiolysis studies coupled to time-resolved UV-Vis spectroscopy and conductivity detection of N-acetyl methionine amide delivered the first experimental evidence that a sulfur radical cation can associate with the oxygen of an amide function vide infra). ... 

All retinal-dependent visual pigments form Schiff bases with lysine side chains of the photoreceptor proteins. How can the same chromophore be "tuned" to absorb across the wavelength range of 360 to 635 nm Modern techniques such as resonance Ra-man and FTIR spectroscopies and study of mutant forms have shown that interaction of the conjugated double bond system of the chromophores with immediately adjacent dipoles of side chain groups and peptide linkages is sufficient to account for the great variability in absorption maxima. 

The metal-peptide stoichiometry of the dimeric Cd peptide was studied by UV-Vis spectroscopy (77) as an absorption band at 238 nm is observed upon addition of Cd(II) to the peptide which is assigned to the ligand-to-metal charge-transfer (LMCT) transition of the newly formed Cd-S bonds. A Job plot demonstrated that the complex consists of 2 peptides and 1 metal ion. These results were supported by spectrophotometric titrations analyzed according to the following equilibrium (1) to yield n = 2 and IQ = 0.65 0.08 pM. 

The conformation of peptides on surfaces has been studied by reflection absorption Fourier transform infrared spectroscopy. The two amide bond signals present in the IR spectra provide information about the secondary structure of the peptide as well as the orientation of a helical peptide with respect to the surface (Koga et al., 2006 Yasutomi et al., 2004,2005). 

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