Hydrolysis peptide bond, measurement

An automated pH-stat method gives a direct measurement of the percentage of hydrolyzed peptide bonds, the degree of hydrolysis (DH). The DH is calculated using the equation ... 

In any quantitative work on protein hydrolysis, it is necessary to have a measure of the extent of the hydrolytic degradation. The measurement of the number of peptide bonds cleaved during a hydrolytic process is related to the activity of proteinolytic enzymes and the extent of hydrolysis. Various techniques that evaluate the progress of hydrolysis have been reported, such as the trichloroacetic acid (TCA) solubility index, which evaluates the percentage of nitrogen soluble in TCA after partial hydrolysis of the protein. 

A foodstuff (or other sample) obtained by hydrolysis of a protein material is called a protein hydrolysate. The degree of hydrolysis measures the percentage of peptide bonds hydrolyzed during protein hydrolysis (Adler-Nis-sen, 1976). An advantage of the DH concept is that for a given enzyme/substrate system the DH is independent of five variables substrate concentration, enzyme/substrate ratio, pH. temperature, and time (Adler-Nissen, 1982). 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Data are based on spectral measurement of FAD in the solutions used for amino acid analyses. Corrections have been made for losses in serine and threonine upon hydrolysis by extrapolation of data at various times of hydrolysis to zero time and for the slow hydrolysis of some peptide bonds involving valine and isoleucine by using only those values obtained at later times of hydrolysis. 

For historical reasons many pharmaceutical enzymes are assayed with physiological or biopolymeric substrates (proteins, polysaccharides, bacteria, oil emulsions), which causes a number of theoretical and practical problems. The interpretation of results is difficult when natural substrates are converted into products that are substrates themselves for the next enzymatic attack. Reaction rates often depend on the position of the scissile bonds in the molecule and the chemical nature of the moieties. Hydrolysis can proceed simultaneously on various bonds at various rates. In proteolysis it is assumed that some products are liberated only after denaturation and that during the reaction course new peptide bonds become accessible for hydrolysis. In these cases the enzymatic mechanisms become exceedingly complex, kinetic parameters are apparent values, and experimental results are strongly influenced by the reaction conditions. Reproducibility problems can occur upon assaying proteinases with a limited specificity for particular casein types. Bromelain and pancreatic proteinase, FEP pharmaceutical enzyme standards, are assayed with a casein substrate. The extent of soluble peptide release is a measure of proteolytic activity. However, due to limited specificity, some proteinases release peptides with a nonrandom aromatic amino acid composition. Contamination of casein preparations with protein and of test enzyme substances with other proteinases biases the assay results. Under these conditions, relative assay methods are indicated. 

The 100 e.v. yield for the radiolytic degradation of the peptide bond, as measured in terms of G( NH3) after mild hydrolysis, has been determined for a variety of aliphatic, aromatic and sulfur-containing amino acids in the N-acetyl form. These data are summarized in Table I. In the case of the aliphatic series, we note that the length of the side-chain has relatively little effect on the yield of main-chain degradation. The effect of the aromatic groups of acetylphenylalanine and of acetyltyrosine is to quench in part the yields of those reactions that lead to formation of amide ammonia. The sulfur moiety of methionine on the other hand appears to be relatively ineffective in quenching such reactions. 

Chulalaksananukul et al. measured the residual activity of a lipase from Mucor miehei after one day in SCCO2 at 40-100 °C at various water concentrations. As the temperature rises, the enzyme molecule at first unfolds reversibly and then undergoes one or more of the following reactions formation of incorrect or scrambled structures, cleavage of disulfide bonds, deamination of tryp-sine residues, and hydrolysis of peptide bonds. Each process requires water and is therefore accelerated with increasing water concentration [19]. The role of water on the performance of enzymes in SCFs is described in more detail in Section 4.9.4.3... 

Measurement of the amino acid composition can be achieved in two steps. First, all the peptide bonds in the protein are cleaved by either acidic, basic or enzymatic hydrolysis. Subsequently, the free amino acids are separated from each other and quantified. 

Furthermore, during esterification, hydrolysis of proteins occurred. This phenomenon was evaluated by measuring the amount of amino groups after reaction (Fig 3). An increase of amino groups indicates die hydrolysis of the peptide bonds. Indeed, the latter is the consequence of the acid attack of the protein at relatively high temperatures yielding lower molecular weight peptides. 

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