Peptides hydrolysates

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

B Gustavsson, I Betner. Fully automated amino acid analysis for protein and peptide hydrolysates by precolumn derivatization with 9-fluorenylmethylchloroformate and 1-aminoadamantane. J Chromatogr 507 67-77, 1990. 

The major nutraceutical application of marine-derived bioactive peptides has been ACE inhibition, and a partial list of identified bioactive peptides is given in Table 27.1. Fish protein has been reported to be an advantageous starting material for preparation of ACE inhibitory peptide hydrolysates (Curtis et al., 2002). Marine-derived protein hydrolysate tends to contain high concentrations of cysteine, methionine, and arginine, all of which help reduce hypertension (Kristinsson, 2007). Such marine-derived peptides could be used as potent functional food additives and represent a healthier and natural alternative to ACE inhibitor drugs (Li et aL, 2004). Currently, the bioactive oligopeptides from dried bonito and sardine muscle have been approved as Foods for Specified Health Use by the Ministry of Health and Welfare in Japan. 

Figure 4. Chromatographic separation of amino acids after derivatization with phenylisothiocyanate (PITC)using standardard conditions A. Separation of 200 picomole standard amino acid mix H (17 amino acids) and Norleucine. B. Separation of a protein hydrolysate, bovine serum albumin (BSA) C. Separation of a peptide hydrolysate bradykinin.

Due to an increased interest in analysis of physiological samples, we wanted to establish analyzer methods which would allow us to choose between our standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids, to include the most important free amino acids found in physiological samples. A study of common analysis requirements in our facility indicated that only a limited number of the possible free physiological amino acids is needed for most unknown samples. These additional amino acids of interest are a-amino butyric acid, citrulline, y-amino butyric acid (GABA), hydroxyproline, hydroxylysine, ornithine, taurine, and tryptophan. Other amino acids of interest to us are phosphoserine, phosphothreonine, phosphotyrosine and carboxy-amino acids since they are released from glycoprotein or glycopeptide hydrolysates. 

Current methodology sequencing by partial acid hydrolysis, followed by direct MS analysis of peptide hydrolysates... 

Figure 3.16 Determination of amino acid composition. Different amino acids in a peptide hydrolysate can be separated by ion-exchange chromatography on a sulfonated polystyrene resin (such as Dowex-SO). Buffers (in this case, sodium citrate) of increasing pH are used to elute the amino acids from the column. The amount of each amino acid present is determined from ihe absorbance. Aspartate, which has an acidic side chain, is first to emerge, whereas arginine, which has a basic side chain, is the last. The original peptide is revealed to be composed of one aspartate, one alanine, one phenylalanine, one arginine, and two glycine residues.

R. H. Buck and K. Krummen, High-performance liquid chromatographic determination of enantiomeric amino acids and amino alcohols after derivatization with o-phthaldialdehyde and various chiral mercaptans Application to peptide hydrolysates,/. Chromatogr., A, 1987,357, 255-265. 

The solutions are dried in vacuo, and 100/il of 6.1 M HCl is added to each tube. The tubes are sealed and hydrolysis is effected for 6-18h at 105 °C. The HCl is removed in vacuo over NaOH pellets. Most of the material in the peptide hydrolysates is a mixture of sodium chloride and Dns-OH with small amounts of Dns-amino acids and Dns-NH2. Two extractions with water-saturated ethyl acetate (100/rl) will remove the neutral Dns-amino adds almost completely, Dns-aspartate and Dns-glutamate by approximately 80%, moderate amounts of mono-Dns-lysine and O-Dns-tyrosine, and only traces of Dns-histidine, Dns-arginine and Dns-cysteic acid. 

Protein source Preparation Peptide/Hydrolysates References... 

Different amino acids in a peptide hydrolysate are separated on an ion-exchange resin. Buffers with different pH s elute the amino acids from the column. Each amino acid is identified by comparing the peaks with the standard elution profile shown near the bottom on the figure. The amount of each amino acid is proportional to the area under its peak. This sample contains eight amino acids aspartic acid, glutamic acid, glycine, alanine, cysteine, methionine, phenylalanine, and arginine. 

Proteins can be modified by a group of peptide hydrolyses (peptidases) commonly called proteases (or proteinoses). Based on their ability to hydrolyze specific proteins, proteases are classified as collagenase, keratinase, elastase, etc. On the basis of the pH range over which they are active, they are classified as either acidic, neutral, or alkaline. However, according to their mechanism of action, the Enzyme Commission classifies proteases into the four distinct classes of serine, cysteine, aspartyl, and metalloproteases. Serine proteases, for example, always contain serine residue at their catalytic center, which is essential for the action of proteolysis. 

It is reasonable to inquire whether recognition of such a concept might apply to mechanistic ideas about enzyme reactions. It now appears that the stereoelectronic theory that Deslongchamps and co-workers have developed is specifically applicable to peptide hydrolyses by serine proteases. Hydrolysis of simple esters will be used first to illustrate the approach. 

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