Amino acids Protein/peptide analysis

Modern methods of amino-acid and peptide analysis, have enabled the complete amino-acid sequence of a number of proteins to be worked out. The grosser structure can be determined by X-ray diffraction procedures. Proteins have molecular weights ranging from about 6 000 000 to 5 000 (although the dividing line between a protein and a peptide is ill defined). Edible proteins can be produced from petroleum and nutrients under fermentation. 

The most complicated speciation analysis is in plants and biological samples. Besides the compounds and ions already mentioned metals in living systems participate in a lot more complicated species. They may be bound to amino acids, proteins, peptides and the separation methods may influence their original distribution. For the determination of organo-arsenic, selenium, lead and tin hydride generation combined with different preconcentration steps may be used (Dedina and Tsalev, 1995). 

Sequential analysis of amino acids in purified peptides and proteins is best initiated by analysis of the terminal amino acids. A peptide has one amino acid with a free a-amino group (NH2-terminus) and one amino acid with a free a-carboxyl group (COOH-terminus). Many chemical methods have been developed to selectively tag and identify these terminal amino acids. 

In this chapter selected examples from our group are discussed to show how metal coordination to ligand-modified amino acids or peptides can be used for induction or fixing of defined conformations in amino acid residues or di- and tripeptides. In this context Ramachandrarfs method for conformational analysis of peptide or protein structures will be introduced. 

Enzymes such as protease in conjunction with pancreatin and amylase have been extensively used to liberate Se species from proteins for analysis [43, 57, 128, 133-136]. Relatively long times ( 24 h) are required to fully hydrolyze proteins using enzymes. However, not all Se is released as simple amino acids. Some peptides, and small molecular weight proteins remain. Thus, ultrafiltration (< 1 kDa) before analysis will be needed to separate amino acids from other material with higher molecular weight. In the presence of cysteine, selenomethionine and selenocysteine are stable to enzyme attack (Fig. 20.2). However, although large amounts of Se are released from marine tissues (30-60 percent), little (less than 10-20 percent) is characterizable by HPLC-ICP-MS. 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

The identification of N-terminal amino acids in peptides and proteins is of considerable practical importance because it constitutes an essential step in the process of sequential analysis of peptide structures. 

D9. de Verdier, C. H., and Agren, G., Paper chromatographic analysis of amino acids and peptides in tissue extracts and enzyme hydrolyzed proteins. Acta Chem. Scand. 2, 783-796 (1948). 

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