VOL. 6, Nos. 7 AND 8
TABPURIPICATION 0s PEPSIN
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THE PURIFICATION OF PEPSIN J. C. FORBES, MEDICALCOLLEGEOF VIRGINIA,RICHMOND, VIRGINIA
Pepsin is the proteolytic enzyme of the gastric juice. It possesses the power of peptonizing typical proteins, i. e., of hydrolyzing them to proteoses and peptones. Gastric digestion was first considered as a purely physical action, and was explained by the friction of food particles under the influence of pressure exerted by the walls of the stomach. Reaumur' (1683-1757) was the first to demonstrate the influence of the stomachal juice in the phenomenon of digestion. His experiments consisted in causing falcons to swallow tubes pierced with holes and filled with food. He found that after a certain time these substances, although protected from friction, were completely digested. Spallanzani2a few years later demonstrated that gastric juice retained its power for dissolving flesh even after its removal from the stomach. To procure gastric juice Spallanzani caused a tame eagle to swallow a sponge fastened to the end of a cord. By drawing the sponge out and squeezing it he obtained the juice sought. However, the very clear data of Reaumur and Spallanzani were not accepted by the scientificworld of the period. Experiments, the results of which contradicted each other, left the question still in doubt, so that physiologists for a long time disagreed on the subject of the mechanism of digestion. Some attributed it to effect of vital and nervous force, others assumed the intervention of the gastric juice, although it was then considered only salivary juice which had undergone acidification. The study of digestion, abandoned for some time, was again actively taken up between 1820 and 1840. Tiedmann, Gmelin, Leuret, and Lassaigne' showed anew that digestion is due to the gastric juice. They further established that gastric juice is not to be confounded with saliva, hut is, on the contrary, secreted by the glands of the stomach. In 1836 Schwann4showed that the digestion was due to an enzyme presented in the gastric juice and to this active principle he ascribed the name pepsin, which means "digestion." Pepsin is secreted by the glands of the stomach in an inactive form, pepsinogen. This pepsinogen is converted into pepsin by the hydrochloric acid of the gastric juice. Like all other enzymes pepsin is very sensitive to high temperatures and variations in the hydrogen-ion concentration of. the solution. The optimum hydrogen-ion concentration for its action is
' Reaumur, Me. de 2'Acad. des Science,
1752, p. 461.
SpaUaneani, "Vers. ub. d . Verdauungsgescheft," Deutsch von Michaelis, Leipzig, 1785.
JLeuret and Lassaigne, "Recherches Physiologiques et chimiques $our semi" a l'hista're de h digestion," Paris, Mme. Huzard, 1825. "hwann. Muller's A~chiu., 1836, p. 90.
1278
JOURNAL 011 CABMICAL EDUCATION
Jay-Auo., 1929
that of about 0.2 per cent hydrochloric acid but it varies to some extent with the protein used. It is very rapidly destroyed by alkaline solutions and it is fairly rapidly inactivated even a t a pH of 5 ( N ~ r t h r u p ) . ~ Many attempts have been made to isolate this enzyme in a pure form and to determine its chemical nature and its ultimate physiological properties. In spite of these efforts the problem remains unsolved. However a discussion of those publications to which we owe our present knowledge of the chemical nature of the enzyme may be of interest. Pekelharing,= by dialyzing, a t low temperature, against distilled water, gastric juice from a dog with a gastric fistula, till the concentration of the hydrochloric acid in the juice was decreased to about 0.02 per cent, precipitated the pepsin thereby as a fine suspension. The suspension was separated by centrifuging and after washing with water dried in a desiccator. From the clear fluid left after centrifuging he precipitated, by half saturation with ammonium sulfate, more pepsin which was then freed from this salt by dialysis. Hydrochloric acid was then added to the extent of 0.2 per cent and the solution dialyzed as before against distilled water until an additional quantity of pepsin precipitated out. The average composition of the pepsin so prepared was, C = 51.99, H = 7.07, N = 14.44, S = 1.63, C1 = 0.49, and P = 0.01 per cent. Pekelharing regarded the phosphorus as due to traces of impurities. Ringer,7 by using oxalic acid instead of hydrochloric acid, obtained a preparation by Pekelharing's method which was free from chlorides and which had a digestive action equal to that prepared with hydrochloric acid. Hammar~ten,~ by the use of methods which gave him pepsin preparations as active as those of Pekelharing, but of which he made no elementary analysis, generalized from his observations that pepsin is always an enzyme-protein complex. Dezanis obtained a very active preparation which contained only 12.02 per cent nitrogen; 16.30 per cent of the total nitrogen was present as basic nitrogen, that is, as arginine, lysine, histidine, and ammonia. The preparation gave the characteristic protein color reactions. Davis and Merkerlo investigated the change in composition of pepsin preparations during purification. They showed that commercial pepsin 1:3000 U. S. P. contained a considerable quantity of secondary protein derivatives as shown by their high amino nitrogen content, while their purest preparation, which had an activity of 1:40,000 U. S. P., had an amino nitrogen content of the same magnitude as native proteins. Their purest
' Northrup, J. H., J. Gen. Pl~yysiol.,2 , 465
(1920).
' Pekelharing, C. A., Z. plzysiol. Chem., 22, 233 (1896),
35, 8 (1902).
'Ringer, W. E., Z. physiol. Chent., 95, 195 (1915). Hammarsten, 0..2. physiol. Chzm., 121, 240 (1922). * Dezani, S., Atli. accod. sci., Torino, 48, 194 (1910). 'O Davis, L., and Merker, H. M., J. Am. Chem. Soc., 41, 221 (1919).
preparation contained 13.77 per cent nitrogen and gave the ordinary protein color tests as well as avery marked Molischreaction for carbohydrates. The authorLL in 1927 published the results of a fairly extensive investigation on the purification of pepsin and the chemical constitution of the preparations thus obtained. The most active preparation was obtained by precipitating the enzyme from water solution with safranine after which the precipitate was dissolved in slightly acidified water containing about 20 per cent ethyl alcohol. The dye was then removed by extraction with iso-amyl alcohol, traces of iso-amyl alcohol removed with ether, and the pepsin precipitated by the addition of two volumes of a solution containing equal volumes of ethyl alcohol and ether. The optimum pH for precipitation was approximately 2.5 and corresponds to the determined isoelectric point. After standing for a short time the precipitated enzyme was centrifuged offand dried in a desiccator. The most active material obtained had a digestive power of 1:64,000 U. S. P. Chemical analysis calculated on an ash-free basis showed the following composition, C = 45.24, H = 6.19, N = 11.50, S = 1.79 per cent. Ash was 1.19 per cent. The preparation gave positive Millon's, xanthoproteic, biuret, Hopkins-Cole, and Molisch's reactions for carbohydrates. The Ninhydrin reaction for amino nitrogen was negative although the test was positive after slight hydrolysis with acids. FengerL2and co-workers prepared pepsin of very high proteolytic activity by precipitating it from solution by dialysis at low temperatures against solutions of pH 2.4-2.5. Such preparations showed a maximum digestive action of 1:65,000 U. S. P. Pepsin precipitated from solutions of higher pH, namely 3.0 and 3.8, respectively, though still possessing marked digestive action, was considerably weaker than that precipitated at pH 2.5. Chemical analysis of the various preparations showed some marked differences, e. g., the percentage of phosphorus increased with proteolytic activity while that of sulfur decreased, and chlorine was completely absent in the one precipitated at the isoelectric point, namely pH 2.5. The most active preparation had the following chemical composition C = 53.15, H = 7.20, N = 14.43, P = 0.13, C1 = 0 per cent. All their preparations gave positive Millon's, xanthoproteic, and biuret reactions. Pepsin is readily adsorbed from solution by various substances such as aluminum hydroxide, charcoal, fibrin, Lloyd's reagent, and albumin. Aluminum hydroxide was used by the author with fair success in the separation of pepsin from some of the accompanying impurities present in artificial gastric juice, and commercial pepsin. McMeekinsLS used egg albumin for the same purpose with considerable success. His most active prepara-
" Forbes, J.
C., I. B i d . Chem., 71, 559 (1927). Fenger, F., and Andrews, R. H., J . Bid. Chem., 73, 371 (1927). l 3 McMeekins, T. LeRoy, Science Series,Univ. of Chicago, 4, 391 (1928). l2
tion had a digestive action of 1:52,000U. S. P. Maximum adsorption was obtained a t pH 3 and the adsorbed enzyme was removed from the albumin by using NaHC08 or NazHPOa solutions of suitable pH. Solutions of Na2HP04of a pH of 6.1 to 6.2 were found the most effective. The purified material contained 12.8-13.8 per cent nitrogen. It gave positive biuret, xanthoproteic, and diazo reactions as well as tests for phosphorus and a reducing substance. In view of the differences in the chemical analysis of the various pepsin preparations obtained by different authors, using different methods, i t seems fairly evident that the isolation of pure pepsin is left for the future. Pepsin is very readily adsorbed by proteins and it is generally believed that its digestion of proteins is through mass action promoted by its adsorption on the substrate. I t is therefore very probable that in ordinary solutions of it most of the pepsin is present in such association with the protein and these consequently will tend to be precipitated with it. The variations in the chemical constitution of the preparitions obtained by different authors may then consequently be explained on the assumption that an enzyme-protein adsorption compound was obtained in each case and that the protein part of the complex was not always the same. The methods employed by Fenger and by Pekelharing are practically the same, the pepsin being precipitated in both cases by dialyzing the solution against distilled water of suitable hydrogen-ion concentration until the pepsin precipitated out. The chemical analyses of their preparations also agree very closely and correspond to that of an albumin. The preparations of Dezani, McMeekms, and the author were obtained by different methods and have a much lower nitrogen content than those of Pekelharing and of Fenger. Their chemical analysis, especially that of the author's, corresponds fairly closely to that of some glycoproteins. The glycoprotein character of the author's preparation is further emphasized by the strong Molisch test and the liberation of a reducing substance on hydrolysis with acids. One of the main difficulties encountered in the purification of pepsin is the ease with which it is inactivated, especially in the purer state. Fenger has shown that at pH 3.8 and above the enzyme disintegrates so that the particles will pass through parchment or animal membranes with a gradual decrease in the proteolytic activity of the solution. The decrease in proteolytic activity seems to go hand in hand with a decrease in the protein characteristics. It is indeed possible that pepsin cannot exist as such except in a protein-enzyme complex and that the enzyme is inactivated if freed from such combination. The enzyme is very sensitive to the action of alkali, heat, or strong acids, it being immediately inactivated in alkaline solutions or by boiling. The ultimate explanation of the phenomenon of gastric digestion must be
V04.6, Nos. 7 AND 8
TEEPURFICATION OP PEPSW
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preceded by a thorough understanding of the structure of the enzyme and of its combination with the substrate on which it acts. It is hoped that the day is not far distant when this important physiological phenomenon will be explained, as a thorough understanding of the action of even one enzyme might throw a great deal of light on the various physiological actions which make up what we understand as life.
