Amino acid separation from protein hydrolyzate

Phenylalanine was first obtained by Schultze and Barbieri in 1879 (779, 780) from etiolated lupine sprouts. Its structure was shown (781) by comparison with synthetic material prepared independently by Erlenmeyer and co-workers in 1882 (228). Synthetic nn-phenylalanine was first optically resolved in 1900 (267). The difficulty of isolating phenylalanine from protein hydrolyzates prevented recognition of its widespread occurrence in proteins until after Emil Fischer had developed his method for separation of the amino acids by fractional distillation of their esters. 

The amino acid composition of a protein can be determined by methods that are referred to as amino acid analysis. The protein of interest is hydrolyzed in concentrated acid (usually HCIO4 or HCl) to generate the free amino acids which are separated from one another, nsnally by means of ion exchange chromatography. 

Fischer used Curtius method to separate mixtures of amino acids from protein hydrolyzates by fractionally... 

Mondino and Bongiovanni 262) observed that cystine, tryptophan and to a lesser extent threonine and serine were degraded when a mixture of all the common amino acids found in proteins was hydrolyzed in 6N HCl by an open-flask technique and analyzed on an automatic amino acid analyzer. More recently, Gruen 157) carefully studied the separate effect of each of the other commonly occuring amino acids on the recovery of tryptophan, using the standard procedure for amino acid analysis, whereby tryptophan is eluted from the short column along with the basic amino acids as a well resolved peak appearing before lysine 135, 369). 

Discarded fish bones and cutoffs may contain considerable amounts of muscle proteins. These muscle proteins are nutritionally valuable and easily digestible with well-balanced amino acid composition (Venugopal et al., 1996). Therefore, fish proteins derived from seafood processing by-products can be hydrolyzed enzymatically to recover protein. Protein hydrolysates from several marine species have been analyzed for their nutritional and functional properties, and researches have mainly explored the possibility of obtaining biologically active peptides (Benkajul and Morrissey, 1997). Moreover, skipjack tuna muscle (Kohama et al., 1988), sardine muscle (Bougatef et al., 2008), and shark meat (Wu et al., 2008) have been used to separate potential peptides. 

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. 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino acids, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forma an easily identifiable phenylthvobydantoin (PTH) derivative of the N-terminal amino acid. A series of sequential Edman degradations allows the sequencing of a peptide chain up to 50 residues in length. 

Glycosaminoglycans are solubilized from stromal or other tissues by extracting the source tissue with dilute acid or alkali. Hyaluronan is electrostatically bound to specific proteins called hyaladherins, which possess a structural domain of -100 amino acids termed a link module. Other glycosaminoglycans are O-linked to serine and threonine residues of polypeptides and these bonds hydrolyze before the rest of the polysaccharide. The protein moiety precipitates when trichloroacetic acid or ammonium sulfate is added to the cooled mixture. The composition of the GAGs (including hyaluronan) was identified by chromatographic separation of the purified polysaccharides, followed by their hydrolysis in boiling 1.0 M HC1 for 2 1 h and identification of the individual monosaccharide components. 

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