Glutamine, destruction

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

An enzyme can deactivate irreversibly for two kinds of reasons (i) conformational processes, such as aggregation (intermolecular), or incorrect structure formation (intramolecular), such as scrambled disulfide bond formation between wrong side chains, and (ii) covalent processes, such as reduction and thus destruction of disulfide bonds, deamidation of asparagine (Asn) or glutamine (Gin) side chains, or hydrolysis of (usually) labile asp-X bonds in the protein sequence. 

In alkaline solution, proteins are known to undergo the following types of reactions (a) denaturation, (b) hydrolysis of some peptide bonds, (c) hydrolysis of amides (asparagine and glutamine), (d) hydrolysis of arginine, (e) some destruction of amino acids, (f) e elimination and racemization, (g) formation of double bonds and (h) formation of new amino acids. 

This was first foimd by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et al. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. In addition, this reaction can occur when an internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced as the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962). 

Figure 11. Time course of the destruction of heterocysts and solubilization of the activities of glutamine synthetase and glutamate synthase during sonic cavitation of N2 -groton A. cylindrica the number of...

The principal disadvantages of acid hydrolysis are the destruction of some amino acids, notably serine, threonine, cystine, and tryptophan, and the slow release of amino acids from some dipeptide combinations, notably those involving isoleucine and valine (Hill, 1965). For most of these, timed analyses allow the extrapolations that give excellent estimations of the original amino acid content of the sample. Cystine can be readily estimated as one of several derivatives vide supra), and tryptophan can be either analyzed after alternative hydrolysis procedures or determined directly on the intact protein or peptide by spectrophotometric techniques (Edelhoch, 1967). Two amino acids, glutamine and asparagine, are quantitatively destroyed and can be determined only on enzymatic hydrolysates. 

A representative analysis of bovine serum albumin electroblotted onto PVDF is given in Table I. The corresponding chromatogram is shown in Fig. 2. Under the standard conditions given, asparagine and glutamine are completely converted to aspartic acid and glutamic acid, respectively. Destruction of serine (up to 50%) and threonine (up to 30%) is usually expected. Cysteine and tryptophan are destroyed... 

Ironically, while water is critical in maintaining molecular shape, the aqueous state is not one in which proteins are long resistant to denaturation. A variety of environmental changes such as temperature, pH, salts and solvents can cause protein inactivation in the aqueous state, and the mechanisms of irreversible protein inactivation often follow conunon pathways. These include cystein destruction, thiol-catalyzed disulfide interchange, oxidation of cystein residues, deamidation of asparagine and glutamine residues and hydrolysis of peptide bonds at aspartic acid residues. 

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