Amino acid spectra analysis studies

Because of the long computing time of distances and the difficulty of evaluating the output dendrogram, cluster analysis is ususally performed with a small number of objects, as in the work of Aishima. However, Ooghe et al. > used cluster analysis with 269 objects in the study of French red wines by their amino acid spectrum. 

A protein subject to NMR analysis may have 100-200 amino acid residues, which provide a 1H NMR spectrum of many hundreds of lines. Because the amino acid sequence can be assumed to have been determined previously by non-NMR methods, the first step in the NMR study is to assign each line in the spectrum to a specific moiety (NH, ot-CH, side chain CH3, etc.) of a specific amino acid residue. Without the 2D methods that we have discussed, it would be virtually impossible to make such assignments. For relatively small proteins ( 50—100 residues) it is often possible to use conventional homonuclear 2D methods, such as COSY and HOHAHA, to define some bonding paths and to supplement these results with NOE data for residues that are very close in space as a result of secondary structural elements such as a helices. However, for proteins of moderate size such techniques are insufficient, and special methods had to be developed and now constitute the standard method of making sequential assignments. 

RSSF spectroscopy has also been used to study the effect of active site mutations on the 3-elimination reaction catalyzed by tryptophanase. In many PLP-de-pendent enzymes, the Lys residue that forms the E(Ain) with the cofactor is preceded by a basic residue in the primary amino acid sequence. Phillips et al. (106) have examined the effect of changing Lys 269 to Arg on the formation and accumulation of reaction intermediates. The activity of the mutant enzyme is only 1096 of the native enzyme. Secondly, the mutant enzyme exhibits an altered pH dependence both in the spectrum of the native enzyme and in the catalytic rate profile. RSSF studies of the reaction of/.-alanine, z-Trp, S-methyl-z-cysteine, S-benzyl-z-cysteine (SBC), and oxindolyl-/-alanine show that all these various substrates react with the enzyme to form covalent intermediates. However, the rate and extent of quinonoid accumulation is greatly reduced. Analysis of quinonoid bands formed in the reactions of SBC and oxindolyl-z-alanine with tryptophanase show that mutation effects the equilibrium distribution of intermediates, but does not perturb either the band shape or the A x of the observed quinonoid intermediates. Therefore, the structure of the quinonoid intermediate and the surrounding active site environment are similar to the wild-type enzyme. SWSF characterization of these reactions show that the Keq for E(Aex) formation with each substrate is similar to that found for the wild-type enzyme. Instead, the primary effect of the Lys 269 Arg mutation is at the catalytic step in which the a-proton is removed from E(Aex) to form a quinonoid. These studies show that Lys 269 is not a critical catalytic residue nevertheless it does contribute to the conformational and/or electrostatic environment of the active site that is necessary for the formation and breakdown of quinonoidal species. 

Numerous electronic-spectrum assay procedures for determining concentrations have been devised. For example, the reaction of ninhydrin with most amino acids yields a purple product having a maximum absorption at 570 nm. This provides a very convenient procedure for amino acid analysis. In a biological study we frequently... 

Recent work, whereby the spectrum and concentrations of dissolved free amino acids were monitored by direct analysis of seawater from a fixed station over the period of a day at hourly intervals (K. Mopper, pers. comm., 1979), showed pronounced variations in both composition and concentration. Particularly striking was the sharp increase in basic amino acids (low C N) at the expense of a decrease in acidic-neutral acids (high C N) outside the daylight hours. An explanation can at present only be speculative, but the results suggest that the phenomenon is directly linked with photosynthetic activity. This suggests, therefore, that the approach to study excretion in situ requires the detection of bioactive compounds at their natural levels, which generaUy lie several orders of ms itude below those of bulk parfuneters. 

The following amino acids have been definitely excluded as part of a common structure of the Type 1 center Tryptophan has been eliminated as a ligand for the reasons given in Section IIAl. Arginine is absent in the plastocyanins. Tyrosine has been eliminated by optical absorption studies of azurin and by a recent analysis of the resonance enhanced Raman spectrum of stellacyanin 206). 

Absorption studies of amino acids in the UV region go back to the mid-thirties [15]. An overview on this subject appeared as early as 1952 [16]. Only the aromatic amino acids, phenylalanine, tyrosine and tryptophan, absorb in a conveniently observed region (Fig. 6). The weakest of them is phenylalanine, revealing at least 6 absorption bands it has a molecular absorption coefBcient of fimoi 0.195 X 10 at 257 nm. The UV-spectrum of tyrosine is strongly dependent on pH in the presence of 0.1 N HCl (protonated hydroxyl group) Sj oi = 1-34 x 10 at the maximum, (l x 275 nm) in 0.1 N NaOH (phenolate) = 2.33 x 10 at 293 nm. The absorption of tryptophan, is nearly independent of the pH it shows a maximum at 280 nm with = 5.55 x 10 and a second maximum (shoulder) at 288 nm of = 4.55 x 10. These parameters have often been used in the quantitative analysis of peptides. The sulfur-containing side chains show weak absorption below 250 nm and are of interest only in rare special cases. 

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