Hydrolysis of a protein

A general feature of optimum sample preparation is that maximum recovery of the analyte is observed. Consider a graph of recovery vs. variation in one experimental condition. Figure 5 shows such a graph, with temperature as the experimental variable. The curve exhibits a maximum and a decline on either side of the maximum. The assay will be most reproducible at the point of zero slope, i.e., at the maximum recovery, because small variations in conditions will not affect the result. In hydrolysis of a protein to its constituent amino acids, for example, it will be found that at very high temperatures or long hydrolysis times, degradation of the product amino acids occurs, while at low temperatures or short hydrolysis times, the protein... 

Our example is the sequencing of a peptide (P) derived from partial hydrolysis of a protein which, on complete acid hydrolysis, gave Ala, 3 Gly, Glu, His, 3 Lys, Phe, Tyr, 2 Val, and one molar equivalent of ammonia. 

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. 

Usually, in food technology, the hydrolysis of a protein product is not intended to proceed to the production of free amino acids, because extended hydrolysis yields a product with poor functional properties. Therefore, it is important to know the amount of free amino acids generated by the hydrolysis. The TNBS reaction (see Alternate Protocol 1) allows a useful tool to quantify the amount of free amino acids. 

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). 

Acid hydrolysis of a protein or polypeptide to yield amino acids. 

Fingerprinting. The characteristic two-dimensional paper chromatogram obtained from the partial hydrolysis of a protein or a nucleic acid. 

The method consists of the complete hydrolysis of a protein or peptide to release its component amino acids. These may be separated by reverse phase high performance liquid chromatography (HPLC), and quantified with fluorometric detection after derivatization with o-phthaldehyde (Larsen and West, 1981). The method serves to determine both the amino acid composition and the total quantity of protein present in the sample. 

Hydrolysis is a chemical reaction in which water reacts with a molecule and decomposes it. There are many such reactions involving hydrolysis in the area of chemistry, but the most significant biological reaction is the hydrolysis of a protein. 

Table XVI gives a partial list of native proteins that have been hydrolyzed with proteolytic enzymes. A discussion of the interpretation of each example listed is beyond the scope of this review, but a few comments concerning certain features of proteolysis are ivarranted. The mechanism of enzymatic hydrolysis of native proteins was studied in detail by Tiselius and Eriksson-Quensel (1939), who examined the action of pepsin on ovalbumin. Two mechanisms of proteolysis were considered by these workers. In the first mechanism the enzyme hydrolyzes all susceptible peptide bonds in one substrate molecule before hydrolysis of a second molecule begins. This type of mechanism has been described by Lmderstrpm-Lang (1952) as the all or none type. In the second mechanism, the enzyme hydrolyzes the single, most susceptible bond in all substrate molecules before hydrolysis of other bonds occurs. This mechanism is called the zipper type. Hydrolysis of a protein can proceed by either of the two mechanisms or by a mechanism which has features of both types. General aspects of the problem have been reviewed and theoretical equations which describe the kinetics of ea( h mechanism have been derived (Linderstr0m-Lang, 1952, 1953).

We can easily calculate that, due to different arrangements of the four units, there are eight possible isomeric tetra-peptides containing these same four amino acids of the same stereo-isomeric forms. One of these eight isomers may prove to be actually identical with the hydrolytic tetra-peptide but it may be necessary to wait until these eight isomers have all been synthesized before we can state positively that a synthetic poly-peptide is identical with a poly-peptide obtained by the hydrolysis of a protein. The probability is that one of these possible synthetic compounds will be found to be identical with the hydrolytic. When this is done we shall have proven that proteins are poly-peptide combinations... 

Partial hydrolysis of a protein yielded a number of polypeptides. One of them was purified. Deduce the sequence of amino acids in this polypeptide from the following information ... 

In 1891, Kossel reported the first study of the hydrolysis of a protein-free nucleic acid. Mainly as a result of his work, it was recognized that there are two nucleic acids, similarly constituted but differing in certain components. The nucleic acid readily isolated from yeast is of one type that from thymus gland and fish sperm is of the other. It is possible that other nucleic acids exist in fact, there have been indications from time to time that there are other nucleic acids, but only the two types under discussion have been adequately characterized. 

Hydrolysis of a protein into peptides can be accomplished by group-specific chemical and enzymatic reagents (Table 3-2). N-Bromosuccinimide and cyanogen bromide hydrolyze proteins at tryptophan and methionine (Figure 3-12) residues, respectively. Trypsin hydrolyzes... 

Hydrolyzed vegetable protein (HVP) is one of the earliest known forms of thermal reaction or process flavors (7,2). HVPs can been produced by acid (HCl) or enzyme (proteolytic) hydrolysis of a protein source (usually of plant origin) to form principally amino acids (7,5-5), which, themselves, can impart taste (e.g. monosodium glutamate) or participate in subsequent thermal reactions, e.g. Maillard reaction, to form aroma compounds (6,7). Among the numerous process parameters involved in the production of HVP, the substrate or protein source material may have a great in5)act on the resulting amino acid profile and flavor characteristics of the final product (7,5). 

General Characteristics of Enz3unatic Hydrolysis. As earlier reported (2), a limited hydrolysis of a protein product may improve certain functional properties such as whipping and emulsifying capacity. 

The nutritional quality of a protein can be increased by the plastein reaction (46). Following partial hydrolysis of a protein by pepsin, the ethyl ester of a limiting amino acid such as methionine or cystine, or a partial hydrolysate of another protein which is limiting in another amino acid residue, can be added to the hydrolysate and covalently linked through plastein formation. 

Goto et al. 1998). None of them probably reflects properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a trans-esterification reaction that produces thiophenol as one of the products (Han et al. 2004) in this case, a method based on a reaction leading to a fluorescent adduct of thiophenol is a good system to assess the transesterification potential of such proteases and is to be preferred to a conventional protease assay based on the hydrolysis of a protein (Gupta et al. 1999 Priolo et al. 2000) or a model peptide (Klein et al. 1989). 

Peptones are small polypeptides that are intermediate products in the hydrolysis of proteins. The term is often used for any partial hydrolysate of proteins, e.g., bacteriological peptone, which is used as a medium for the growth of microorganisms. Peptones are water-soluble protein derivatives obtained by the partial hydrolysis of a protein by an acid or enzyme during digestion. 

The molecular weights of all of the peptides in a mixture obtained by the enzyme-catalyzed hydrolysis of a protein are determined simultaneously by mass spectrometry using matrix-assisted laser desorption ionization (MALDI). 

Enzymatic techniques can be used to endow proteins with surface-active functionality. An enzymatic technique that has shown promise in enhancing surface properties of proteins is a modified version of the classical plastein reaction. The plastein reaction is known to be a protease-catalyzed reverse process in which a peptide-peptide condensation reaction [11,12] proceeds through the peptidyl-enzyme intermediate formation [13]. It is essentially a two-step process enzymatic hydrolysis of a protein and plastein formation from the hydrolysate peptides. A novel one-step process was developed as a modified type of the plastein reaction by Yamashita et al. [14,15], which... 

---