Amino acid spectral characteristics

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

Second-order derivatives of the spectrum of Phe, Tyr, and Trp present characteristic absorption minima at 257, 280, and 290 nm. In addition, each aromatic amino acid has other minima of lower intensity 250 and 264 nm for Phe, 272 nm for Tyr, and 268 and 278 nm for Trp (81). In peptides composed only of Tyr and Phe, the spectral contribution of each aromatic amino acid to the derivative of the peptide spectrum can be distinguished quickly by the Phe (257 nm) and Tyr (278 nm) absorption minima. However, identification of Tyr in the presence of Trp is unclear,... 

The characteristic absorptions in the i.r. spectra of lactones and amino acids are discussed on pp. 302 and 308 respectively the spectra of (DL)-valine and l-tryptophan are given in Figs 3.36 and 3.37. Further descriptive spectral interpretations for a range of substituted carboxylic acids and their derivatives are given in appropriate preparative sections. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

Amino acid side chains, particularly those with aromatic groups, exhibit characteristic frequencies that often are very useful in probing the local environment of the group in the protein (Spiro and Gaber, 1977). From our present point of view, however, we are interested in characterizing spectral features of backbone chain conformation. It is therefore important to know the locations of such bands so that their contribution to the spectrum is not confused with amide and backbone vibrations. We discuss below some features (in the nonstretching region) of such side-chain modes these are summarized in Table XL. 

Assignment to residue type is accomplished readily by systematic spin decoupling, since the spectrum of each amino acid has a characteristic coupling pattern. This is illustrated in Figure 23, where the decoupled residue is identified readily as tyrosine by comparing the decoupling pattern to the spectrum of this amino acid [67]. The only requirement for the success of this procedure is extensive resolution of spectral lines and line widths sufficiently narrow to permit the identification of multiplets. 

Formation of a carbon-carbon bond between indole and E(A-A) results in the rapid accumulation during l/r2 of an intense spectral band centered at 476 nm with a shoulder at approximately 440 nm (Fig. 14, spectrum 3). The spectral characteristics of this intermediate are consistent with the assignment of this species to thet-Trp quinonoid. Accumulation of the t-Trp quinonoidal species is followed by a further increase in absorbance (l/t3) in the 420-nm region of the spectrum (Fig. 14, spectrum 19). If the reaction is carried out in the absence of phosphate ion (an allosteric effector of reactions at the P-site), then the spectral changes during l/r3 are even more pronounced and a distinct spectral band at 420 nm is evident (data not shown). The shape and position of the 420-nm band, in conjunction with the observed sequence of spectral events, is consistent with the assignment of this band to the Schiff base species formed between the cofactor and i-Trp, the product amino acid, E(Aex2) (86, 87). 

Most biological macromolecules are colourless to the human eye and only reveal their spectral characteristics when viewed in the UV range. Absorption spectra are characterized by their shape, the peak wavelength (Amax) and the peak height or molar extinction coefficient (e). Spectra of amino acids, nucleic acids, proteins, DNA and other chromophores are described in the following section. 

A number of characteristic SERS bands originate from the amino acid side chains Trp, Tyr and Phe. The peptide backbone vibrations are not enhanced in this protein (low scattering intensity in the spectral range of 1650-1675 cm amide I). The presence of the strong SERS bands of Trp, Tyr and Phe and the absence of the amide vibrations indicate a preferential interaction of these amino acid side chains with the surface. The strong (S—S) vibration at 509 cm in the NSRS spectrum is also missing in the SERS spectrum. This indicates that the disulfide bonds do not interact directly with the surface. 

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