Hydrolysis partial, of peptides

The covalent structure of insulin was established by Frederick Sanger in 1953 after a 10-year effort. This was the first protein sequence determination.237 238 Sanger used partial hydrolysis of peptide chains whose amino groups had been labeled by reaction with 2,4-dinitrofluorobenzene239 to form shorter end-labeled fragments. These were analyzed for their amino acid composition and labeled and hydrolyzed again as necessary. Many peptides had to be analyzed to deduce the sequence of the 21-residue and 30-residue chains that are joined by disulfide linkages in insulin.237 238... 

M20. Miyaji, T., luchi, I., Yamamoto, K., Ohba, Y., and Shibata, S., Amino acid substitution of hemoglobin Ube 2 (ai /32) an example of successful application of partial hydrolysis of peptide with 5% acetic acid. Clin. Chim. Acta 16, 347-362 (1967). 

Peptide cleavage, partial hydrolysis of peptides and proteins by chemical ( end group analysis) or enzymatic methods (-> proteolysis) for sequence analysis. Limited peptide cleavage (limited proteolysis) is important for various metabolic processes. 

Proteins consist of large numbers of amino-acids joined by the p>eptide link —CO —NH — into chains, as shown in the diagram, where R and R" are amino-acid residues. These chains are called peptides and may be broken into smaller chains by partial hydrolysis (see peptides). Proteins may contain more than one peptide chain thus insulin consists of... 

Partial hydrolysis of a peptide can be carried out either chemically with aqueous acid or enzymatically. Acidic hydrolysis is unselective and leads to a more or less random mixture of small fragments, but enzymatic hydrolysis is quite specific. The enzyme trypsin, for instance, catalyzes hydrolysis of peptides only at the carboxyl side of the basic amino acids arginine and lysine chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan. 

In the above experiments with polyleucines it was also observed that the partial hydrolysis of mixtures of nonhomochiral leudne peptides led to the preferential hydrolysis of those components of the mixtures that were enantiomerically more random. This produced a corresponding e.e. enrichment in the unhydrolyzed polymer. [96] For example, when the above polyleudne sample having an i > d e.e. of 45.4% was 27% hydrolyzed, the residual unhydrolyzed polymer had an e.e. of 55.0%, representing an e.e. increase of 9.6%. 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

The chemistry of this reaction has been extensively studied by Use and Edman. Although the reaction velocity is different for various amino acids, the reaction is complete in 1 N HCl at 80 °C within 10 min, with virtually all amino acids. This treatment, however, would cause partial hydrolysis of the remaining peptide. The method was made amenable to automation when Edman suggested to separate the thiazolinone derivative from the remaining peptide by extraction of the liberated thiazolinone derivative into butyl chloride and to perform the conversion into the PTH-amino acid outside the reaction vessel. 

Solomons, C. G Peptides obtained from partial hydrolysis of decalcified human dentin collagen. Nature 185, 101 (1960)... 

From study of peptides formed by partial hydrolysis of the 32P-labeled chymotrypsin, the sequence of amino acids surrounding the reactive serine was established and serine 195 was identified as the residue whose side chain hydroxyl group became phosphorylated. The same sequence Gly-Asp-Ser-Gly was soon discovered around reactive serine residues in trypsin, thrombin, elastase, and in the trypsin-like cocoonase used by silkmoths to escape from their cocoons.198 We know now that these are only a few of the enzymes in a very large family of serine proteases, most of which have related active site sequences.199 200 Among these are thrombin and other enzymes of the blood-clotting cascade (Fig. 12-17), proteases of lysosomes, and secreted proteases. 

Our example is the sequencing of a peptide (P) derived from partial hydrolysis of a protein which, on complete acid hydrolysis, gave Ala, 3 Gly, Glu, His, 3 Lys, Phe, Tyr, 2 Val, and one molar equivalent of ammonia. 

Partial hydrolysis of the peptide (P) with trypsin gave a hexapeptide, a tetrapeptide, a dipeptide, and one molar equivalent of lysine. The peptides, which we will designate respectively as M, N, and O, were sequenced by Edman degradation and found to have structures ... 

Partial hydrolysis of the peptide P using chymotrypsin as catalyst gave three peptides, X, Y, and Z. These were not sequenced, but their amino-acid composition was determined ... 

In any quantitative work on protein hydrolysis, it is necessary to have a measure of the extent of the hydrolytic degradation. The measurement of the number of peptide bonds cleaved during a hydrolytic process is related to the activity of proteinolytic enzymes and the extent of hydrolysis. Various techniques that evaluate the progress of hydrolysis have been reported, such as the trichloroacetic acid (TCA) solubility index, which evaluates the percentage of nitrogen soluble in TCA after partial hydrolysis of the protein. 

Activation reactions catalyzed by serine proteases (including kallikreins) are an example of limited proteolysis in which the hydrolysis is limited to one or two particular peptide bonds. Hydrolysis of peptide bonds starts with the oxygen atom of the hydroxyl group of the serine residue that attacks the carbonyl carbon atom of the susceptible peptide bond. At the same time, the serine transfers a proton first to the histidine residue of the catalytic triad and then to the nitrogen atom of the susceptible peptide bond, which is then cleaved and released. The other part of the substrate is now covalently bound to the serine by an ester bond. The charge that develops at this stage is partially neutralized by the third (asparate) residue of the catalytic triad. This process is followed by deacylation, in which the histidine draws a... 

Enantiomeric enrichment of homochiral peptides was achieved during partial hydrolysis of polypeptides composed from polymers that contain blocks of both random and a-helix [177] or random and /1-sheet sequences [178]. Hydrolysis of the atactic part of the polypeptides was found to occur much faster than within the isotactic blocks of a-helix and /3-sheets. Based on these observations, Bonner suggested that environmental dry/wet cycles on the primitive Earth might have caused repeated polymerization/hydrolysis cycles that permitted the eventual evolution of optical purity from a small abiotic ee value for the amino acids [177,179]. 

Partial hydrolysis of the LF molecule by heat as well as by pepsin results in the formation of an antibacterial peptide referred to as lactoferricin, which exerts a much stronger antimicrobial effect than the intact molecule (Section III.A.5.). 

Schultz observed (Schultz et al., 1961) that on partial hydrolysis of a commercial preparation of ribonuclease with 0.03 N HCl for times varying up to 48 hr at 105°C, up to 14 out of the 15 bound aspartic residues were liberated. He recommends the method for rapid comparative fingerprinting of the resulting peptides from proteins such as human and animal sera, heme proteins, pepsin, and albumins. 

Partial Acid Hydrolysis of Peptides and Proteins Containing Disulfide Bonds... 

The three forms are interconvertible and were found in greatest yields when 12 N HCl at 100°C was employed. Short periods of hydrolysis at 100°C with 5.7 A HCl gave lower yields of the a,/3- and /8-forms. Longer periods of hydrolysis at 37°C with 5.7 A HCl did not produce detectable amounts of either the a,/ - or d-form. The latter method seems to be preferred for partial hydrolysis if the interconversions of aspartyl residues are to be avoided. Hydrolysis of the a,/3-imide at alkaline pH values leads predominantly to the /8-form (Swallow and Abraham, 1958 Bernhard, 1958). Naughton el al. (1960) noted that this type of reaction occurred with the peptide aspartyltyrosine on partial hydrolysis of A-chain of insulin (Sanger... 

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