Catalyzed hydrolysis of amide and peptide bonds in proteins

Abstract

The rates of hydrolysis by dilute acids of both a dissolved protein (egg albumin) and an insoluble protein (wool) are shown to depend not only on the temperature and acidity but also on t he acid used. When hydrolyzed at 65° C by certain strong monobasic acids of high molecular weight, the amide and the peptide bonds are broken over 100 times as fast as when they are hydrolyzed with hydrochloric acid. Even among the common mineral acids, large differences appear. These differences in hydrolytic effectiveness parallel differences in the affinities of the anions of the acids for protein. A further reason for attributing t his effect to the anions is the attainment, with anions of high affinity, of a maximum rate of amide hydrolysis at r elatively low concentrations, stoichiometrically equivalent to the sum of the amino plus the amide groups. A similar limiting anion concentration or maximum rate of hydrolysis of t he much more numerous peptide groups is not observed. On the basis of details of t he dependence of the rate of hydrolysis on concentration of effective anions and hydrogen ions, a m echanism which involves combination of the groups hydrolyzed with hydrogen ions, is proposed.At low concentrations of effective anions, amide hydrolysis is catalyzed more strongly than peptide hydrolysis. By keeping the concentration slightly below stoichiometric equivalence to the sum of the amino plus amide groups the amide groups may be rapidly hydrolyzed without extensive hydrolysis of the peptide bonds in the protein. Practical applications are suggested.

The terminal peptides of insulin

“…It was found that the specificity of this hydrolytic agent was very different from that of concentrated HCl, and that the smaller DNP-peptides were produced in much better yields. It seems probable that the hydrolysis of these oxidation products in dilute acid is analogous to the hydrolyses catalysed by long-chain sulphonic acids (Steinhardt & Fugitt, 1942), the peptide chains containing -SO3H groups acting as long-chain anions and catalysing a particular type of hydrolysis which is characterized by the increased lability for the amide groups and no doubt a corresponding difference in specificity towards other peptide bonds.…”

The irreversible combination of formaldehyde with proteins

“…This is confirmed by the fact that deaminated, as well as untreated fibres, which have been boiled in formaldehyde at pH 1, fail to supercontract on boiling in bisulphite solution (Stoves, 1944). On deamidation by the Steinhardt & Fugitt (1942) method (residual amide N, 0-4 %), it is noticed that formaldehyde treatment confers no stability to supercontraction, although it does prevent the hair from being immediately dissolved in the boiling bisulphite solution. From these results, it is suggested that the values obtained in column 4 are due to cross linkages formed between the amide groups of glutamine and the guanidino groups of arginine (Wormell & Kaye, 1945;Fraenkel-Conrat et al 1945b.…”

“…Deamidation. Proteins were partly deamidated by the following techniques: (i) in a solution of cetylsulphuric acid and HCI (Steinhardt & Fugitt, 1942); (ii) in a solution of 1% NaOH for 40 hr. at 450 (Wormell & Kaye, 1945);…”

A longitudinal proteomic assessment of peptide degradation and loss under acidic storage conditions

“…Some of the possible routes by which peptides can be chemically degraded are through (i) hydrolysis [1], (ii) deamidation [2,3], (iii) oxidation [4], and (iv) 2,5-diketopiperazine and pyroglutamic acid formation [5]. Hydrolysis can occur in peptides containing aspartic acid, whereby the amino acid can dehydrate to form a cyclic intermediate.…”