Peptide partial hydrolysis

Additional structural information can be obtained by cleaving some, but not all, of the amide bonds in a peptide. Partial hydrolysis of a peptide with acid forms smaller fragments in a random fashion. Sequencing these peptides and identifying sites of overlap can be used to determine the sequence of the complete peptide, as shown in Sample Problem 28.2. 

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. 

The versatility of the reagent system in the assay of small peptides is nicely illustrated in Figures 3 through 5. Neurotensin, a polypeptide composed of 13 amino acids, and three fragments from partial hydrolysis were derivatized with NDA/CN and separated by... 

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%. 

The repetitive cycle to identify a sequence of N-terminal amino acids has been automated. In practice, it is limited to about 20-30 amino acids, since impurities build up and the reaction mixture becomes too complex to yield unequivocal results. The usual approach is to break the polypeptide chain into smaller fragments by partial hydrolysis, preferably at positions relating to specific amino acid residues in the peptide chain. There are ways of doing this chemically, and the enzymes chymotrypsin... 

Peptide bonds are cleaved in a nonselective, but not in a completely random manner. Based on anchimeric side-chain assistance, steric factors, and bond strains, acid-labile peptide bonds are predicted to include sites containing Asp, Glu, Ser, Thr, Asn, Gin, Gly, and ProJ22l The disulfide topologies of circulin B and cyclopsychotride, backbone-cyclized peptides with three disulfide bonds, were determined by partial hydrolysis for 5 hours.[22 Occasionally, the bond between adjacent half-cystine residues is cleaved due to the nonselective nature of the mechanism of partial acid hydrolysis.[21] By this procedure, in all cases, a complex mixture of peptide fragments is produced which requires careful chromatographic separation by RP-HPLC for subsequent analysis by mass spectrometry (see Section 6.1.6.2.7). 

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)... 

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... 

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. 

The subsequent paragraphs will focus on the preparation of C2-substituted malonyl derivatives for the incorporation into PMRI-peptides. In the classical method, C2 alkylation of a malonic acid diester is followed by partial hydrolysis to generate the C2-alkylated monoalkyl malonate (Scheme 7). The malonyl-derived topochemical replacements for Phe, Lys, Trp, Leu, Ala, His, and Met residues have been successfully prepared by this strat-egyJ48 57 ... 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

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. 

A-4. The carboxypeptidase-catalyzed hydrolysis of a pentapeptide yielded phenylalanine (Phe). One cycle of an Edman degradation gave a derivative of leucine (Leu). Partial hydrolysis yielded the fragments Leu-Val-Gly and Gly-Ala among others. Deduce the structure of the peptide. 

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... 

We cannot deduce, from the data given, the sequence of the amino acids in parentheses. To put them in order, we must seek peptides from partial hydrolysis that contain overlapping sequences of these amino acids. 

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.). 

Q Use information from terminal residue analysis and partial hydrolysis to determine the structure of an unknown peptide. 

E Breaking the Peptide into Shorter Chains Partial Hydrolysis... 

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. 

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