Peptide liberated

To exert their physiological effects in vivo, bioactive peptides must be released during intestinal digestion and then reach their target sites at the luminal side of the intestinal tract or, after absorption, in the peripheral organs. Figure 1 presents a scheme of the intestinal assimilation of protein and the routes of bioactive peptide liberation. 

Gray [192] proposed a method in which by acetylating the initial protein the terminal peptide liberated by specific enzymic degradation was isolated and its permethylated derivative sequenced by mass spectrometry. 

Ammonia is the smallest noncharged nucleophile for peptide liberation from polymer phase. Cleavage of the benzyl ester link to polystyrene by ammonolysis with the formation of peptide amides, however, liberates on the average only 60—80% of the synthetic product from the support. The best conditions for this detachment yielding directly the important class of peptide amides can be seen in the utilization of dimethylformamide/ methanol 4 1 (v v) saturated with ammonia at 0 °C, in which the peptide-on-polymer is suspended and stored for at least 3 days in a pressure bottle at room temperature. 

An alternative method for C-terminus identification reduces the carboxyl group (—COOH) of a peptide to an alcohol (—CH2OH) with lithium aluminum hydride (LiAlH4 see Chapter 19, Section 19.2.1). Subsequently, acid hydrolysis of the peptide liberates all of the amino acids, but one amino alcohol was also generated by reduction of the C-terminal amino acid residue. If tripeptide 190 is reduced with LiAlH4 and then hydrolyzed, for example, the products are alanine (53), vahne (54), and leucinol (195). 

By incubation of porcine, bovine, canine, or chicken pepsinogens and calf prochymosin with pepstatin at pH 2.5, the first active protein generated on activation is trapped in an inactive complex. The first activation peptide liberated from porcine pepsinogen has been identified as residues 1-16 whereas that from prochymosin is derived from residues 1-27. This suggests that pepsin and chymosin are not formed by one-step conversions from their zymogens, but by (different) sequential, activation mechanisms. 

Acid hydrolysis of these peptides liberates D-proline, whereas alkaline fiydrolysis liberates L-proline (564). Stoll et al. (566) showed that in these peptides, L-proline is actually present. The transformation from the l to the d form in acid hydrolysis is explained by the instability of the asymmetric carbon of the carboxylic group of proline. 

In the last few years a further type of modified flavocoenzyme has beeen discovered which is structurally related to the coenzymes of succinate dehydrogenase and monoamine oxidase, but differs considerably in its chemical properties. Early literature reports indicated the presence of a flavin in cytochrome C552 from Chromatium which could not be extracted with trichloroacetic acid or acidic ammonium sulfate (7), but could be released, for example by trypsin digestion or by incubation with saturated urea solutions (2). Absorption, fluorescence and ESR behaviour were closely similar to those of 8a-cysteinyl-ribo-flavin (12) and indicated the presence of a covalent link to the protein, through position 8a (185). Strong acid hydrolysis of these peptides liberated the flavin as mixture of riboflavin derivatives oxidation with per-formic acid and acid dephosphorylation yielded a homogeneous riboflavin derivative, which was identical with 8-nor-8-carboxy-riboflavin (185). 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

During the cleavage of the Fmoc group with base, dibenzofulvene is liberated and must be scavenged to prevent its reaction with the liberated peptides during... 

The parent alcohol can be obtained in a straightforward manner from chloromethyltrimethylsilane (Chapter 16). The protection afforded is naturally related to the functionality involved, and liberation is achieved using either fluoride ion or a Lewis acid. For example, 2-trimethylsilylethyl esters (Chapter 16) are stable to a wide variety of conditions such as those used in peptide synthesis, but are readily cleaved by fluoride ion (14,15). 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

The determination of amino nitrogen before and after acid hydrolysis of urine has frequently been used for the quantitative estimation of the amount of urinary peptides (H5, M4). The number of liberated a-amino groups represents, in fact, the whole of formerly combined amino groups, not necessarily attached to a second amino acid partner. Besides, considerable losses connected with decomposition of some amino acids occur in the course of hydrolysis thus limiting the true quantitative value of this procedure. 

According to Stein (S8), 1761-2459 mg of amino adds appears in 24 hours urine collection from normal subjects after its acid hydrolysis. The estimations performed by Muting (M4) showed that the average content of amino acids in 24-hour specimens of urine derived from 20 normal males and the same number of normal female subjects was 2221 and 2288 mg, respectively. The results of these two authors do not, however, represent exclusively the amount of peptide-bound amino acids, but rather the whole of these compounds liberated as a result of the acid... 

Amino Acids Liberated in the Course of the Hydrolysis of Cehtain Peptide Fractions Isolated from Urine... 

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