Amino acid optical absorption

Complexes between chiral polymers having ionizable groups, and achiral small molecules become, under certain conditions, optically active for the absorption regions of the achiral small molecules. Dyes such as acridine orange and methyl orange have been used as achiral species, since they are in rapport with biopolymers through ionic coupling. This phenomenon has been applied to the detection of the helix chirality in poly-a-amino acids, polynucleotides, or polysaccharides when instrumental limitations prevent direct detection of the helices. 

Similar curves are obtained with other synthetic polypeptides, and in most cases they are reasonably independent of the nature of the amino acid side chains. In synthetic polypeptides and proteins the observed Cotton effects do not arise from isolated chromophores but are composite curves resulting from several transitions assigned to the amide bonds in the 200-m/x region. The a-helical curve, for example, results from three optically active absorption bands. One around 222 m/ arises from an n — 7T transition of nonbonding electrons, and the other two at 208 and 191 m/ji are attributed to w — tt transitions parallel and perpendicular to the axis of the helix. These transitions of the a-helix and the resulting Cotton effects characteristic of the a-helix are at present of great interest in interpreting ORD curves of membranes. 

IR spectroscopy is not a very sensitive analytical tool and is, therefore, not well suited to the detection of small amounts of material. If, however, intermediates have intense and well-resolved IR absorptions, the progress of their chemical transformation can be followed by IR spectroscopy [83,88,91-93], Near-infrared spectroscopy, in combination with an acousto-optic tunable filter, can be sufficiently sensitive to enable the on-bead identification of polystyrene-bound di- and tripeptides, even if the peptides have very similar structures (e.g., Leu-Ala-Gly-PS and Val-Ala-Gly-PS) or differ only in their amino acid sequence (e.g., Leu-Val-Gly-PS and Val-Leu-Gly-PS) [94]. Special resins displaying an IR and Raman barcode have been developed, which may facilitate the deconvolution of combinatorial compound libraries prepared by the mix-and-split method [48]. 

Among the properties of amino adds that are most pertinent to the biomedical scientist are their optical rotations, already discussed, which are listed for each amino acid in Table 4.1. Note the dramatic differences between optical rotations in the zwitterionic (water) and fully protonated (HC1) forms. Further, all amino acids absorb ultraviolet light in the range 190-220 nm. The C=0 bond in carboxyl residues is largely responsible. Moreover, aromatic amino acids, especially tryptophan, absorb in the 260-285 nm range. Protein concentrations in solutions are often determined via absorption at 210 or 280 nm. 

Oxazolones (73), the saturated azlactones, have been studied intensively (B-57MI41801, B-57MI41802, 65AHC(4)75,69MI41800,77AHC(21)175). They show carbonyl and C=N absorptions in the 1820 and 1660 cm-1 regions, respectively. Azlactones derived from chiral a-amino acids, e.g. compound (74), can be obtained in optically active forms which racemize easily. The derived salts (75 R2 = H, Me or Ph) likewise exhibit optical activity they show intense carbonyl bands at 1890-1880 and C=N+ absorptions at ca. 1650 cm-. ... 

Heat and alkaline treatments have been known since the early part of the century to raoemize amino acid residues in proteins (1,2,). Dakin and Dudley (3) also studied digestibility of casein in vitro and in vivo after hydroxide treatment. Heating casein with 0.5 N NaOH at 37° for about 30 days completely prevented enzymatic hydrolysis and intestinal absorption when the treated casein was fed to a dog. The kinetics of base-catalyzed racemization of proteins was investigated by Levene and Bass (4-6). In these early studies, the extent of racemization was measured by changes in optical rotation. 

Another important factor for the polymer conformation is the solvent effect. As the usual solvents for the copolymers in question are DMSO and DMF, which have absorption in the UV region, CD spectral measurements are impossible. However, the optical rotation measurements and analyses using the Moffitt-Yang equation give the Moffitt parameter bo for the copolymers (Table 21). Hie parameter is known to be related to the helix content of poly-(a-amino acids). The b0 value of polycarboben-... 

The redox states of the flavin cofactor in a purified flavoenzyme can be conveniently studied by optical spectroscopy (see also Elavoprotein Protocols article). Oxidized (yellow) flavin has characteristic absorption maxima around 375 and 450 nm (Fig. lb and Ic). The anionic (red) and neutral (blue) semiquinone show typical absorption maxima around 370 nm and 580 nm, respectively (Fig. lb and Ic). During two-electron reduction to the (anionic) hydroquinone state, the flavin turns pale, and the absorption at 450 nm almost completely disappears (Fig. lb and Ic). The optical properties of the flavin can be influenced through the binding of ligands (substrates, coenzymes, inhibitors) or the interaction with certain amino acid residues. In many cases, these interactions result in so-called charge-transfer complexes that give the protein a peculiar color. 

The potential advantages of selective nitration of tyrosyl residues in native proteins are numerous. The reaction is performed under mild conditions, giving rise to a 3-nitrotyrosyl derivative (pK 7), which in the acid form absorbs intensely at 350 nm. Hence, the nitrotyrosine content may be readily determined spectrophotometrically, as well as by amino acid analysis ( 2.2.3). The absorption spectrum of 3-nitro-tyrosine is highly sensitive to solvent polarity and exhibits significant optical activity in the long wavelength absorption band. Consequently, nitrotyrosyl residues can be utilized as indicators of conformational change, or of interactions of proteins with other macromolecules or small molecules (e.g. Kirschner and Schachman 1973). Any perturbation in the pK of nitrotyrosyl residues is readily determined spectrophotometrically. 

The first reported circular dichroism (CD) study of chiral PPy s was by Delabouglise and Gamier105 on the polymers 4 (R = CH2OH, CHMc2, and Ph), in which a series of amino acids are covalently bound at the 3-position of each pyrrole ring. They exhibit a CD band at ca. 470 nm associated with the absorption band at 460 nm. This optical activity is believed to be induced by the presence of the chiral amino acid substituents, leading to the adoption of a one-handed helical structure by the PPy chains. 

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