Hydroxide ions, peptide hydrolysis

The strongly acidic conditions used for hydrolysis destroy all the tryptophan residues because the indole ring is unstable in acid (Section 21.9). The tryptophan content of the protein can be determined by hydroxide-ion-promoted hydrolysis of the protein. This is not a general method for peptide bond hydrolysis because the strongly basic conditions destroy several other amino acid residues. 

A similar situation arises when the metal-ion-catalysed base hydrolyses of aminoacids and peptides are studied. Such hydrolyses contrast with the base hydrolyses of simple esters, which are not catalysed by metal ions. Again, metal ion complexes with the aminoacid esters or the peptides are involved in the catalyses. The presence of the metal ion increases the electron-withdrawing power of the carbonyl group and helps attack by the hydroxide ion, leading to efficient hydrolysis. 

To understand the role of metal ions in hydrolysis reactions, it is useful to first consider the background hydrolysis reactions. Table 6.1 lists the second-order rate constants for hydroxide-catalyzed hydrolysis of various substrates. The reactivity of methyl acetate (first entry in Table 6.1) [16] is comparable to those of other unactivated esters found in nature (e.g. acetyl choline and carboxyl esters in phospholipids). The reactivity of N-methylacetamide (second entry in Table 6.1) [17] is comparable to those of typical peptides (1.1 x 10 6 m-1 s 1) [18] and that of dimethyl phosphate (P-O bond... 

The work described above suggests that carbonyl-bonded amides and peptides when coordinated to Co Rh and Ir will undergo base hydrolysis ca. 10 times faster than the free ligand, the rate acceleration arising primarily from a more positive value of AS. At high pH, amide deprotonation occurs (Scheme 8) leading to catalytically inactive complexes. Much higher rate accelerations can be obtained if intramolecular attack by coordinated water or hydroxide ion can take place (ca. 10 -10"-fold). 

The base hydrolysis of the carbonyl bonded amides and peptides display a first order dependence on the hydroxide ion concentration up to a pH of ca. 10, but then become independent of the hydroxide ion concentration due to the formation of the unreactive deprotonated amide (pK = 11 to 12). Some typical kinetic data for these reactions are summarised in Table 7.6. The p2-Co(trien) complexes have the configuration shown in (7.12). Similar studies have been carried out with complexes of the general type trans-[Co(dien)X(peptideOR)] (7.13). Typical kinetic results... 

The Ce + ion is one of the most active catalysts for peptide hydrolysis. Its activity is much higher than that of the trivalent lanthanide ions and other transition metal ions. In particular, Ce + is far superior to other tetravalent ions like Zr" or Hf +. Yashiro et al. (1994) reported that dipeptides and tripeptides were efficiently hydrolyzed under neutral conditions by the y-cyclodextrin complex of cerium(IV). Komiyama and coworkers (Takarada et al., 2000) studied the catalytic hydrolysis of oligopeptides by cerium(IV) salts. The hydrolysis is fast, especially when the oligopeptides contain no metal-coordinating side-chains. The hydrolysis rates of the dipeptides, tripeptides and tetrapeptides is similar. The hydrolysis reaction was performed at pH 7 and 50 °C and under these conditions, the half-life of the amide bond was only a few hours. The authors found that ammonium hexanitratocerate(IV) is more active than other cerium(IV) compounds like ammonium cerium(IV) sulfate, cerium(IV) sulfate and cerium(IV) hydroxide. The lower reactivity of ammonium cerium(IV) sulfate is ascribed to the competitive inhibition by sulfate ions, while the low reactivity of cerium(IV) sulfate and cerium(IV) hydroxide can be explained by their poor solubility in water. However, in the reaction mixtures at the given reaction conditions, most of the cerium(IV) consists in a gel of cerium(IV) hydroxides. No oxidative cleavage has been observed. 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

Ioffe and Sorokin (1954) investigated a novel procedure for the hydrolysis of elastin using copper sulfate and 0.4 N barium hydroxide at 37°C for 60 hr. The first product of hydrolysis was a protein which resembled a-elastin in that it showed reversible coacervation on raising the temperature. This substance was subsequently degraded further to yield a soluble fraction and a fraction containing peptides. Alkaline hydrolysis was much more rapid in the presence than in the absence of copper ion. 

Adsorption in this manner may also account for the increased reactivity of wool cystine disulfide bonds to attack by alkali in the presence of cationic surfactants and their decreased reactivity in the presence of anionics (Meichelbeck, 1971). The adsorption of cationic surfactants onto the wool surface, which is negatively charged in an alkaline medium, can impart a positive charge to the surface, thus increasing its attraction for hydroxide and sulfite ions, with consequent increase in its rate of reaction with these ions. In analogous fashion the acid hydrolysis of peptide bonds in the wool is increased by the presence of anionic surfactants (which... 

Earlier experiments with Dowex-50 in 0.05N hydrochloric acid [16] had shown that aspartic acid, serine and threonine are hberated very rapidly but valine and isoleucine more slowly in comparison to hydrolysis in 6N hydrochloric acid. Peptide bonds with cystine and cysteic acid are said to be very resistant to this type of hydrolysis. About 25% of the glutamic acid present is converted into pyrrolidone carboxylic acid. Basic amino acids are only incompletely removed from the ion exchange resins by means of dilute ammonium hydroxide solution. 

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