Peptides breakdown

By the application of the methods described in this review the structure of a pure polypeptide containing thirty residues has been determined (p. 54) and there seems no reason why it should not be possible to work out the complete amino acid sequence in proteins which are as simple as insulin. How far it will be possible to apply these techniques to more complex proteins is difficult to say. The larger the polypeptide chains in a protein, the greater the necessity of isolating larger peptide breakdown products. Probably the chief need in this field is for techniques for the specific breakdown of proteins into larger peptides and for the fractionation of such peptides. Most of the more commonly studied proteins contain more than 300 residues but it is possible that some of them, when studied in greater detail may be found to have a simpler structure than is at present believed. The relative simplicity of insulin may be merely apparent as insulin has been studied in more detail than have other proteins. 

Enzymes have also been used in the preparation of protein ingredients from animal wastes. Mohr (1980) described the use of proteolytic enzymes in the preparation of protein concentrates from fish offal. Fish protein waste was ground with protease and incubated to yield a concentrated fish protein, which was then dried. Generally, the contractile and connective tissue proteins are hydrolysed most efficiently. Sarcoplasmic proteins tend to aggregate and resist enzyme attack. The proteins are broken down into peptides and individual amino acids, and the longer the hydrolysis continues the higher the yield of hydrolysates but the greater the peptide breakdown. For many food applications peptide breakdown needs to be carefully controlled. 

CGRP has a wide distribution in the nervous system (19) and was the first peptide to be localized to motoneurons (124). It is also found in primary sensory neurons where it is colocalized with substance P (125). CGRP is derived from a precursor stmcturaHy related to the calcitonin precursor. The latter precursor produces two products, calcitonin itself and katacalcin, while the CGRP precursor produces one copy of CGRP (123). Like other peptides, CGRP is cleaved from its precursor by tryptic breakdown between double basic amino acid residues. 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

Breakdown of the amide dihydrate occurs by a mechanism similar to its formation. The ionized aspartate carboxyl (Asp in Figure 16.27) acts as a general base to accept a proton from one of the hydroxyl groups of the amide dihydrate, while the protonated carboxyl of the other asparate (Asp in this case) simultaneously acts as a general acid to donate a proton to the nitrogen atom of one of the departing peptide products. 

Figure 7-6. Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows Indicate directions of electron movement. Aspartate X acts as a base to activate a water molecule by abstracting a proton. The activated water molecule attacks the peptide bond, forming a transient tetrahedral Intermediate. Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .

Penetration enhancers are low molecular weight compounds that can increase the absorption of poorly absorbed hydrophilic drugs such as peptides and proteins from the nasal, buccal, oral, rectal, and vaginal routes of administration [186], Chelators, bile salts, surfactants, and fatty acids are some examples of penetration enhancers that have been widely tested [186], The precise mechanisms by which these enhancers increase drug penetration are largely unknown. Bile salts, for instance, have been shown to increase the transport of lipophilic cholesterol [187] as well as the pore size of the epithelium [188], indicating enhancement in both transcellular and paracellular transport. Bile salts are known to break down mucus [189], form micelles [190], extract membrane proteins [191], and chelate ions [192], While breakdown of mucus, formation of micelles, and lipid extraction may have contributed predominantly to the bile salt-induced enhancement of transcellular transport, chelation of ions possibly accounts for their effect on the paracellular pathway. In addition to their lack of specificity in enhancing mem-... 

Strange and Dark demonstrated the presence of a hexosamine containing peptide in the spore coats of B. megaterium and B. subtilis. The breakdown of an insoluble peptide complex might well be one of the first steps of the germination process. It was believed that the release of the hexosamine-amino acid complex was the result of the action of lysozyme present in the spores. 

The tripeptides in Fig. 6.17 underwent a few breakdown reactions (N-ter-minus elimination, Qm formation, peptide bond hydrolysis), some of which will be considered later in this section. Of relevance here was that, of the two amidated tripeptides, the amide at the C-terminus underwent deamidation predominantly (Fig. 6.17, Reaction a), which, perhaps, explains the somewhat lesser stability compared to the free carboxylic acid forms. While the hexapeptide (6.52, Fig. 6.17) followed a different pattern of decomposition [76b], deamidation was also a predominant hydrolytic reaction at all pH values. Thus, the procedure to extrapolate results from small model peptides to larger medicinal peptides appears to be an uncertain one, since small modifications in structure can cause large differences in reactivity. 

This example illustrates that the reactivity of peptide bonds involving serine residues is not due solely to the presence of serine other, as yet poorly understood factors must also play a role. Threonine residues also show this particular type of reactivity [9]. Here, we examine the case of cyclosporin A (CsA), a cyclic undecapeptide that contains some unusual amino acids (6.57, Fig. 6.23), whose major breakdown reaction is hydrolytic cleavage at a threonine analogue. Position 1 is occupied by 4-[( )-but-2-enyl]-A,4-dimethyl-threonine, whose OH group reacts as a nucleophile at the carbonyl C-atom of the adjacent A-methylvalinc (position 11) [86],... 

Further insights into the influence of pH on the reactivity at aspartic acid residues are provided by a study of the model peptide Val-Tyr-Pro-Asp-Gly-Ala (Fig. 6.28,a) [93], At pH 1 and 37°, the tm value for degradation was ca. 450 h, with cleavage of the Asp-Gly bond predominating approximately fourfold over formation of the succinimidyl hexapeptide. At pH 4 and 37°, the tm value was ca. 260 h due to the rapid formation of the succinimidyl hexapeptide, which was slowly replaced by the iso-aspartyl hexapeptide. Cleavage of the Asp-Gly bond was a minor route. At pH 10 and 37°, the tm value was ca. 1700 h, and the iso-aspartyl hexapeptide was the only breakdown product seen. In Sect. 6.3.3.2, we will compare this peptide with three analogues to evaluate the influence of flanking residues. 

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