Protein hydrolysis side reactions

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

The lability of peptides and proteins to acidic conditions was first reported in 1920 by Dakin,12031 who found that acid hydrolysis of peptides or proteins that contain consecutive N-alkyl amino acids leads to the formation of piperazine-2,5-diones (DKP) this side reaction lowered their yield during amino acid analysis. For example, the piperazine-2,5-dione c[-Hyp-Pro-] was isolated from the hydrolyzate of gelatine. 

Maleic acid is a linear four-carbon molecule with carboxylate groups on both ends and a double bond between the central carbon atoms. The anhydride of maleic acid is a cyclic molecule containing five atoms. Although the reactivity of maleic anhydride is similar to that of other cyclic anhydrides, the products of maleylation are much more unstable toward hydrolysis, and the site of unsaturation lends itself to additional side reactions. Acylation products of amino groups with maleic anhydride are stable at neutral pH and above, but they readily hydrolyze at acid pH values around 3.5 (Buder et al., 1967). Maleylation of sulfhydryls and the phenolate of tyrosine are even more sensitive to hydrolysis. Thus, maleic anhydride is an excellent reversible blocker of amino groups to mask them temporarily from reactivity while another reaction is being done. For additional information and a protocol for the modification of proteins with this reagent, see Section 4.2. 

With respect to the hydrolysis step, it can be accomplished by acid, by enzymatic, or by direct microbial attack. Microbial hydrolysis results primarily in the production of cellular biomass or single-cell protein. Acid hydrolysis, while simple and direct, results in a sugar syrup with considerable contamination from the side reaction products. Enzymatic hydrolysis is usually the cleanest hydrolysis process. Unfortunately, it is the most costly of the three to operate. 

A complex procedure for determining the content of asparagine and glutamine separately in proteins has been described (see Chibnall et al. 1958). This procedure makes use of esterification of the protein, reduction of the resulting esters of aspartic acid and glutamic acid with lithium borohydride and hydrolysis of asparagine and glutamine to the acids in which form they are analyzed. Despite the side reactions for which corrections must be made, this method, in conjunction with total enzymic hydrolysis, may be useful to those who must quantitatively estimate these 4 amino acids. 

There are numerous protocols for protein hydrolysis, involving minor variants of the standard procedure, that are intended to minimise the destruction of particular amino acids (tryptophan and cysteine/cystine in particular) through the sensitivity of their side-chains to the reaction conditions, especially when access of oxygen is not prevented. Tryptophan largely survives alkaline hydrolysis (but other coded amino acids, particularly serine and threonine, but also arginine and cysteine, do not). 

Dansyl chloride (DNS-Cl) (l-dimethylaminonaphthalene-5-sulphonyl-chloride). This reagent was originally introduced into protein chemistry for end-group analysis over twenty years ago (Gray and Hartley, 1963) and has been widely used because of the simplicity of the reaction and its ability to react with both primary and secondary amines, unlike OPA and fluram. Furthermore, in contrast to other fluorescent reagents, the dansyl derivatives are stable to acid hydrolysis, and can therefore be used in N-group labelling before hydrolysis. HPLC separations of dansyl derivatives have recently been published (Tapuhi et al., 1981). Sensitivity of detection is at the low picomole level. The sensitivity is limited because of the side-reactions which can occur with lysine and, to a lesser extent, histidine and tyrosine. 

The wool fiber consists predominantly of proteins therefore, the reactions of wool are the reactions of the protein backbone (e.g., peptide bond hydrolysis) and the reactions of the side-chains of the 21 different types of amino acid residues of which wool is composed. Six of these amino acids (glycine, alanine, valine, leucine, isoleucine, and proline) are essentially chemically inert so are not normally available for chemical reaction. The reactions of the... 

Very trace amount of water or nearly anhydrous state renders practically no enzymatic reaction. In this context, it should be reminded that commercially available enzyme preparations, or the enzymes even after lyophilization or other drying procedures, contain some water bound to the enzyme proteins. Whereas, excess water in the reaction system results in hydrolysis of the substrate, which is often unfavorable side reaction, giving rise to lower yield of product. Thus, there exist usually the optimal water content for each enzymatic reaction of concern. 

These compounds thus appeared to be good candidates for protein heavy atom labeling because they may target particular protein residues and form stable covalent adducts. Moreover their rate of aminolysis was shown to be much higher that their rate of hydrolysis [75]. Reaction of 40 with a series of a-aminoesters always afforded the expected aminocarbene adducts even in the presence of a competing nucleophile on the side chain. Reaction of 40 or 41 with the protein BSA in solution yielded conjugates with spectral characteristics similar to those of aminocarbenes [76]. 

Over the past three decades, an increasing concern was put on application of nonaqueous solvents to facilitate biocatalytic reactions where several industrially attractive advantages are presented, such as increased solubility of nonpolar substrates, reversal of hydrolysis reactions, alternation of enzyme selectivity, and suppression of water-dependent side reactions. However, there are some inherent problems and technical challenges, including inactivation of biocatalysts, potentially reduced protein stability and lowered reaction rates due to mass-transfer limitations, and/or the increased rigidity of protein structure. 

The lability of tryptophan under the conditions usually employed for acid hydrolysis of proteins (6N HCl, 24 hrs, in sealed tubes under vacuum) is not due to instability of the indole nucleus under such conditions, but to side reactions involving non-proteinaceus material, like carbohydrates, or to the presence of particular amino acids in the acidic hydrolysis mixture. In fact, gramicidin A, a pentadecapeptide containing, apart from tryptophan, only purely aliphatic amino acids like glycine, alanine, leucine and valine, gives a quantitative recovery of tryptophan after acid hydrolysis in 6N HCl (l20). 

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