Amino acids infrared analysis

Infrared Analysis of the Structure of Amino Acids, Polypeptides, and Proteins... 

Molecular weight of heavy and light chains Peptide mapping Amino acid analysis Intrinsic fluorescence spectroscopy Thermal denaturation monitored by fluorescence Fourier transfrome infrared spectroscopy Binding (e.g., ELISA, BiaCore, etc) Potency (e.g., cell based, ELISA)... 

Infrared analysis of the structure of amino acids, polypeptides and proteins. Advanc. Protein. Chem. 7, 291—318 (1952). 

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

Neural networks have been applied to infrared spectrum interpreting systems in many variations and applications. Anand introduced a neural network approach to analyze the presence of amino acids in protein molecules with a reliability of nearly 90% [37]. Robb used a linear neural network model for interpreting infrared spectra in routine analysis purposes with a similar performance [38]. Ehrentreich et al. used a counterpropagation (CPG) network based on a strategy of Novic and Zupan to model the correlation of structures and infrared spectra [39]. Penchev and colleagues compared three types of spectral features derived from infrared peak tables for their ability to be used in automatic classification of infrared spectra [40]. 

An interesting fact, first noticed by Wright (1937, 1939) is that the infrared spectrum of the DL-form of an amino acid is usually markedly different from the spectrum of either the d- or the L-form of the same acid when each is examined in the solid state. Wright attributes this to compound formation between the d- and L-forms. Darmon et al. (1948) have confirmed this observation, which is extremely important if infrared methods are to be used for the analysis of mixtures of amino acids, e.g., the estimation of leucine iso-leucine ratios in protein hydrolysates. In this connection, Gore and Petersen (1949) have reported differences between the spectra of L-threonine and D-threonine when examined in the solid state. They point out that this might arise from a polarization effect in the spectrometer. 

The actual presence of large amounts of cysteic acid in bleached hair had at one time been in doubt [55, 56]. It had been theorized that the cysteic acid found in bleached hair hydrolysates was formed by decomposition of intermediate oxidation products of cystine during hydrolysis prior to the analytical procedure [55]. However, differential infrared spectroscopy [4] and electron spectroscopy for chemical analysis by Robbins and Bahl [5] on intact unhydrolyzed hair have conclusively demonstrated the existence of relatively large quantities of cysteic acid residues in chemically bleached hair. Evidence for other sulfur acids (e.g., sulfinic or sulfenic acids) in bleached hair has not been provided. It is unlikely that these amino acids exist in high concentrations in hair because these species are relatively unstable. 

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