Hydrolysis with Proteolytic Enzymes

The action of proteolytic enzymes on proteins and synthetic peptides has been extensively studied and reviewed (see Bergmann and Fruton, 1941 Bergmann, 1942 Neurath and Schwert, 1950 Linderstr0m-Lang, 1949) so that the subject will not be dealt with in great detail here. 

In a study of the action of proteolytic enzymes on an oxidation product (fraction B, p. 54) of insulin, Sanger and Tuppy (1951b) found that other bonds besides those adjacent to aromatic residues were split by pepsin, including those of Leu-Val, Ala-Leu, and Glu(—NH2)-His. It thus seems that in the case of pepsin at least there is much to be learnt about its specificity when proteins act as substrate. Trypsin and chymotrypsin were found to split oxidized insulin with the same specificity as was found for synthetic peptides, and it seems probable that this specificity may be shown in their action on other proteins. Clearly a knowledge of the exact mode of action of these enzymes would greatly help in the elucidation of protein structure just as advances in our knowledge of protein structure must throw light on the behavior of the endopeptidases. 

The possibility of rearrangement of sequences of amino acids under the action of proteolytic enzymes has already been mentioned (p. 15) but this danger would not seem sufficiently great to offset the advantages to be gained by using them. Nevertheless, the results must be interpreted with caution. 

It has already been emphasized (p. 14) that the best stage of hydrolysis at which to attempt the fractionation of peptides is at the point where enzyme action will proceed no farther or when there is a very sharp break in the hydrolysis curve. The disadvantage of a long incubation period is of course that the danger of rearrangements increases (see Linder-str0m-Lang and Ottesen, 1949). 

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Enzyme hydrolysis of peanut flour also altered the physical characteristics of baked cookies (60). With the exception of the bromelain hydrolysate, the use of peanut flour in cookies resulted in increased specific volume when compared to the 100% wheat flour control. Untreated peanut flour substitution reduced the diameter and increased the height of cookies however, treatment with proteolytic enzymes reversed the behavior. As evidenced by substantial increases in spread ratios, the diameter of cookies containing treated flours increased proportionately more than did the height. These data promote the feasibility of decreasing or increasing the spread of cookies through the addition of various amounts of untreated or enzyme-treated peanut flour. 

Let us now consider the retarding action of proteolytic enzymes on hydrolysis. Attention should be drawn to the fact that the carbonyl absorption band splits into two parts as a result of interaction of the cured KL-3 with proteolytic enzymes and kidney extract. Evidently it is associated with the specific interaction of the enzyme and urethane group in the polymer, the structure of which resembles the peptide group of a protein molecule. Owing to the specific action of the enzyme, this interaction does not accelerate the hydrolysis of urethane groups but even retards it owing to the shielding effect of the enzyme protein molecule. 

Hydrolysis of Protein. The radioactive, proteinaceous residues from the sulfate-injected insects were separated into equal fractions, one of which was hydrolyzed with acid and the other with proteolytic enzyme. Analyses of unlabeled protein were made after acid or alkaline hydrolysis. 

The Role of Enzymatic Hydrolysis with Proteolytic and Nonproteolytic Enzymes in Sample Preparation Methods... 

In common with most laboratories engaged in fundamental research on proteins, our laboratory has studied the denaturation and renaturation of proteins. Many of these studies have been with the two related homologous iron-binding proteins, human serum transferrin and chicken ovotransferrin. Earlier studies showed that on the binding of iron these proteins were greatly stabilized against denaturation by a variety of environmental stresses as well as to chemical scission of their disulfide bonds and to hydrolysis by proteolytic enzymes (8,9j. Such a seemingly simple question as to why these iron complexes, as well as some other proteins, are much more stable than others is still impossible to answer with presently available information. 

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).

Biotin in the diet is largely protein bound, and digestion of these proteins by gastrointestinal enzymes produces biotinyl peptides, which may be further hydrolyzed by intestinal bio-tinidase to release biotin. Avidin, a protein found in raw egg whites, binds biotin tightly and prevents its absorption. The peptide biocytin (e-N-biotinyl lysine) is resistant to hydrolysis by proteolytic enzymes in the intestinal tract but together with biotin is readily absorbed. A biotin carrier, the sodium-dependent multivitamin transporter (SMVT)... 

Chitin is isolated by dissolving away the calcium carbonate with 5% cold hydrochloric acid. After filtering and washing, the proteins are removed either with boiling 4% caustic soda or with proteolytic enzymes. The chitin recovered after bleaching is insoluble in water, dilute acids and bases, as well as in organic solvents. It dissolves with hydrolysis in formic acid and in concentrated mineral acids. 

After reaction with the labeled reagents, the protein is treated with proteolytic enzymes thermolysin, chymotrypsin, and trypsin have been used for proteolysis of the labeled macromolecule. The resulting peptides are separated by high-pressure liquid chromatography (HPLC), and the amount of radioactivity of each eluted peak is measured. Then the labeled peptides are analyzed after acid hydrolysis and the amount of radioactive label incorporated into individual amino acid side chains is measured. 

Proteins have been hydrolyzed by treatment with sulfuric acid, hydrochloric acid, barium hydroxide, proteolytic enzymes, and other hydrolytic reagents, but no condition has been found which avoids some destruction or incomplete liberation of tryptophan, cystine, and some other amino acids. The early work on this problem has been reviewed by Mitchell and Hamilton (194). The literature and their own excellent experiments on the hydrolysis problem in relation to the liberation and destruction of tryptophan have been presented recently by Spies and Chambers (269). 

These proteolytic enzymes are all endopeptidases, which hydrolyse links in the middle of polypeptide chains. The products of the action of these proteolytic enzymes are a series of peptides of various sizes. These are degraded further by the action of several peptidases (exopeptidases) that remove terminal amino acids. Carboxypeptidases hydrolyse amino acids sequentially from the carboxyl end of peptides. They are secreted by the pancreas in proenzyme form and are each activated by the hydrolysis of one peptide bond, catalysed by trypsin. Aminopeptidases, which are secreted by the absorptive cells of the small intestine, hydrolyse amino acids sequentially from the amino end of peptides. In addition, dipeptidases, which are structurally associated with the glycocalyx of the entero-cytes, hydrolyse dipeptides into their component amino acids. 

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

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