Proteases

PROTEASES, enzymes that produce the hydrolytic cleavage of protein substances, or proteolysis. Corresponding to the predominant way of linking amino acids in proteins, the action of P. consists of the addition of water at the site of the peptide bond, with the formation of free carboxyls and amino groups: R-CO-NH-R' + H₂O = R-COOH + NH₂-R'. Until quite recently it was assumed that among P. there exist enzymes that do not cause actual hydrolysis, do not break peptide bonds, but merely disaggregate large colloidal complexes of protein particles into smaller aggregates. However, at present almost all authors accept that all P. in essence of their action are "peptidases," i.e., that they break peptide bonds. Special enzymes that split compounds of the diketo-piperazine type (see Proteins, chemical structure of proteins) have not yet been discovered [according to Abderhalden's data the diketo-piperazine ring can be split by P. (trypsin) if certain amino acids are attached to it by a peptide bond]. Complete cleavage of protein into amino acids requires the sequential action of several P., and the classification of the latter is based on at what stage of protein breakdown their action begins and to what final products it leads. The classification and nomenclature of individual P. over the very last years, especially since 1929, have undergone very significant, sometimes fundamental changes, mainly as a result of the research of the Willstätter school and its employees (Waldschmidt-Leitz, Grassmann, etc.). In particular, these changes concern the ideas about the character and nature of P. of natural digestive juices. Since this area is being developed very intensively at present, one can give only a schematic presentation of the main views. All P. can be divided into two main groups: proteases acting on native proteins, and peptidases acting on the products of protein cleavage, i.e., on more or less high-molecular-weight polypeptides formed from native proteins under the influence of proteases or obtained synthetically. The action of proteases on any synthetic products has never yet been observed. Proteases can in turn be divided into three main groups: 1) pepsinases, of the pepsin type, with an optimum of action in a strongly acidic medium, at pH about 1.4–2.5; act only on cations of protein (Northrop*); 2) trypsinases, optimum of action in an alkaline medium; react only with the anion of protein; the most important representatives are P. of the pancreas and intestinal juice; 3) tissue and cellular proteases: papain in plants, cathepsin in animal cells. The pH optimum of their action coincides with the isoelectric point of the substrate used, from which it follows that these proteases act on the isoelectric protein. With the exception of pepsin, other proteases require the activation by certain activators for their action. For trypsinase of the pancreas and intestinal juice such an activator is enterokinase, for papain and cathepsin—hydrocyanic acid or compounds containing a sulfhydryl group (SH)—for example H₂S, cysteine, glutathione; the latter is the natural kinase of cathepsin in animal tissues. Pepsinases split protein into relatively large complexes, papainases (papain and cathepsin)—into peptides of lower molecular weight, trypsinases already give a certain amount of free amino acids. Peptidases can apparently be divided mainly into the following groups: 1) dipeptidases, acting only on dipeptides built from amino acids occurring in nature; pH optimum about 7.8; 2) polypeptidases: a) aminopolypeptidases; act only on peptides containing at least three residues of amino acids, but do not act on proteins; detach from the polypeptide amino acids carrying a free amino group; substitution of this group makes the substrate inaccessible to the action of aminopolypeptidases; optimum of action about pH = 7.0; b) carboxypolypeptidases; detach amino acids from that end of the polypeptide which carries a free carboxyl group. Pancreatic juice carboxypolypeptidase (trypsinic), like the corresponding protease (trypsin), is activated by enterokinase. Activation is expressed in an intensification of the action and in an expansion of the circle of substrates subject to cleavage. In contrast to it, tissue carboxypolypeptidase (catheptic), like the corresponding protease (cathepsin), is activated by sulfhydryl compounds and HCN. Of the P. considered until recently as biochemical individuals (gastric juice pepsin, pancreatic trypsin, intestinal juice erepsin, tissue and cellular P.), at present only pepsin can be considered homogeneous from an enzymological point of view, being a typical protease. In all other cases there is a more or less complex mixture of different P.-proteases and peptidases. On the basis of the classification given, which became possible thanks to the systematic application of the methods of separation and isolation of enzymes developed by the Willstätter school by selective adsorption, one can give the following scheme of the composition of proteolytic systems of the digestive tract and tissues. Gastric juice—proteases (pepsinases): pepsin and chymosin; the enzymological independence of the latter is still not finally established. Pancreatic juice: 1) pancreatic protease, trypsinase (trypsin or trypsinogen); 2) carboxypolypeptidase (trypsinic); 3) aminopolypeptidase; 4) dipeptidase. Intestinal juice contains a mixture of enzymes, receiving the name erepsin, by its composition corresponds to pancreatic juice, but the amount of protease recedes far into the background and peptidases predominate; in the juice of the intestinal loop isolated by Thiry and Vella, proteases are not contained; it is possible that they simply come from the pancreatic juice. Tissues and cells: 1) protease-cathepsin; 2) dipeptidase; 3) aminopolypeptidase; 4) catheptic carboxypolypeptidase. Biological significance of proteases. P. of digestive juices, by splitting food proteins into amino acids and simple peptides capable of diffusion, make them available for absorption and open the possibility for the synthesis of specific tissue proteins of the given animal organism from non-specific fragments of foreign food protein. The construction of tissue protein is probably carried out under the influence of the synthesizing action of tissue proteases. These latter also produce the cleavage of the own proteins of cells in proportion to their wear and death, as well as in those cases when there is mobilization of amino acids of tissue protein for energetic purposes (during starvation) or for the construction of specific substances (formation of hormones, sexual products, milk casein and other specific secretions). The action of P. of tissue cells and leukocytes plays an important role in physiological and pathological processes of resorption (tissue elements during metamorphosis, resorption of inflammatory exudates, autolytic melting of tissues in suppurations, necrosis, etc.). The coagulating enzyme (chymosin) producing partial hydrolysis of milk casein participates in the coagulation of milk. According to Waldschmidt-Leitz, thrombin, which causes blood coagulation, is also a P., related to trypsin, and its action consists in the partial hydrolysis of fibrinogen with the formation of insoluble fibrin. --Methods for determining P. are based either on measuring the amount of substrate remaining unsplit (methods of Gross and Fuld, Folin, etc.), or on determining the soluble non-protein nitrogen liberated by the action of P. (according to Kjeldahl), or finally on determining the forming free carboxyl and amino groups. The latter methods are the best; they include titration of carboxyl groups in the presence of formaldehyde (according to Sörensen) or ethyl alcohol (according to Willstätter and Waldschmidt-Leitz), further determination of amino groups—gasometric according to Van Slyke and colorimetric—according to Folin (see Amino nitrogen). The Willstätter school has developed standard units in which the activity of preparations of various P. is measured.