HISTAMINE AND PROTEOLYTIC ENZYMES LIBERATION OF HISTAMINE BY PAPAIN BY M. ROCHA E SILVA AND SYLVIA 0. ANDRADE (From the Department of Biochemistry and Pharmacodynamics, Instituto Biologico, &io Paulo, Brazil) (Received for publication, March 31, 1943) Previous papers have shown that trypsin is very active in releasing histamine from the living tissues (1, 2). This would suggest, as indicated before (3), that histamine is bound to cells forming peptide linkages with the amino acid chain of cell proteins. On the other hand, the fact that chymotrypsin is very ineffective in releasing the histamine, would indicate that the linkages of this substance with the cell proteins display a certain specificity toward proteolytic ferments. Among the innumerable peptides tested by Bergmann and his associates (4,5), only a few are split by trypsin: benzoyllargininamide, benzoylllysinamide, hippuryllargininamide, and hippurylzlysinamide. Trypsin is a proteinase which attacks a peptide linkage involving the carboxyl group of a basic amino acid. Consequently, we might conclude that the linkage of histamine with the cell proteins is of the amide type, the next amino acid residue being either arginine or lysine. A further step presented in this report has been to fractionate papain and to show that its histamineliberating activity at ph 7.3 to 7.5 runs parallel to its benzoylzargininamidesplitting component. The methods developed by Bergmann and his associates (G), which have been used in the following experiments, proved rather successful, despite the fact that the bindings of histamine with the cell constituents are labile and by no means realize the conditions of simple stable substrates, such as those used in the study of proteolytic enzymes. Furthermore, the amounts of histamine to be liberated are so small that one has to use a biological test to detect them; consequently, one has to compare results obtained with purely chemical methods with those obtained in biological experiments. The results presented in this paper, however, show that quite clear information can be gathered from the application of methods of enzymatic chemistry to the solution of problems of liberation of biologically active substances. Material and Methods The papain preparation used was obtained by purification of a crude dried latex, according to the procedure described by Irving, Fruton, and Bergmann (7). A stock solution of papain was prepared by dissolving 1 Rocha e Silva, M., unpublished results. 9
10 HISTAMINE ASD PROTEOLYTIC ENZYMES 200 mg. of purified papain in 25 cc. of saline. The protein nitrogen content, of this preparation was 2.6 mg. per cc. From this solution, 2.5 cc. were transferred to a 5 cc. volumetric flask and 1 cc. of cysteine solution (0.5 M) f 1.5 cc. of phosphate (ph above 6) or citrate (ph below 6) buffer was added. The enzymatic experiments were performed in 2.5 cc. volumetric flasks containing 0.5 to 1 cc. of the papaincysteine solution, 1.25 mm of substrate, and 0.3 cc. of the corresponding buffer solution. The final concentration of cysteine in the solution was 0.04 rnm per cc. The flasks were incubated at 39 and samples of 0.2 cc. were taken at varying intervals for estimation of the free carboxyl groups by the GrassmannHeyde method (8). From each analysis the first order constant K = l/t log a/a. x: was determined and the average K of three or four determinations with the same substrate was used to calculate the respective proteolytic coefficient CC BAA, CoIG, CL*, etc.). The value of CX was calculated by dividing the average constant K by the concentration of the enzyme, expressed in mg. of protein nitrogen contained in 1 cc. of the solution (for more details, see (6)). The histamine experiments were performed according to the met,hod previously described (2). Into each of three or four flasks were put 1 cc. of papain solution and 1 cc. of saline. Samples of 7 to 8 cc. of heparinized rabbit blood, obtained by heart puncture, were added to each flask. After standing 5 to 10 minutes at room temperature and 3 minutes at 38, the samples were centrifuged and 2 cc. of the supernatant plasma added to 4 cc. of trichloroacetic acid. A 2 cc. sample of total blood was also taken. The histamine extraction and estimation were performed according to Code s method (9). Results Tissue cathepsins and papain are complex mixtures of proteolytic enzymes which split innumerable peptides of the most varied type (10). To study t.he possibility of applying to the histamine problem the methods of enzymatic chemistry, we have chosen papain as a representative of this group of enzymes. As is well known, tissue cathepsins are almost inactive above ph 6., being a vegetable cathepsin and having many of the components of animal cathepsins, is still fairly active at ph 7 to 7.5. As shown in Fig. 1, papaincysteine has two definite optima, one around ph 5 and the other around ph 6.8 to 7. At ph 7.3 to 7.5, papaincysteine splits benzoylzargininamide very quickly, carbobenzoxyisoglutamine, 2 Most of the substrates used in the following experiments were prepared by one of us in Dr. M. Bergmann s laboratory at The Rockefeller Institute for Medical Research, New York.
4 u 7 FIG. 1. Influence of ph on the splitting of benaoylzargininamide (continuous line) and carbobenzoxyisoglutamine (dotted line) by papaincysteine. At the ordinates the proteolytic coefficients are shown (CBA* and Cero), and at the abscissas the ph. TABLE Splitting of Di$erent Substrates by Cysteine, at ph 7.3 to 7.6 Enzyme concentration, 0.13 mg. of protein N per cc. Benzoyllargininamide Carbobenzoxyisoglutamine Hippurylamide ILeucinamide Glycylglycine Substrate CarbobenzoxyIglutamylItyrosine GlycylZleucine Carbobenzoxylleucylglycine ChloroacetylItyrosine 11 I Time min. Hydrolysis gcr cent 30 62 40 77 120 91 80 29 120 40 150 49 75 14 120 22 155 6 275 12 110 1 140 3 1440 0 1440 0 1440 0 1440 0 Proteol~t~ ;o$cients c BAA = 1200 ccig = 140 cha = 70 CL.4 = 15 cgg = 0.7 CCGT = 0 CGL =o cclg = 0 COT = 0
12 HISTAMINE AND PROTEOLYTIC ENZYMES hippurylamide, and Zleucinamide less quickly, and glycylglycine very slightly (Table I). CarbobenzoxySglutamylltyrosine, carbobenzoxylleucylglycine, and glycylzleucine are not split by papain above ph 7. Liberation of Histamine from Rabbit Blood Cells by A was prepared directly from a fresh stock solution of papain. B was prepared by treating 4 cc. of the stock sohrtion of papain with 0.5 cc. of N NaOH. After 2 hours of contact, the solution was neutralized with N HCl and the cysteine solution and the phosphate buffer added. C was prepared from a stock solution of papain which had been heated for 12 to 15 minutes at 52 or 2 hours at 39 (Experiment 8). ExpiYi?t 1 0.11 2 0.11 3 0.12 4 0.11 5 0.12 6 0.10 7 0.12 8 0.12 A CBAA B 0.12 0.10 0.09 0.10 C Histamine content 1 Plasma + I Plasma + Plasma + A I B C I l y per cc. y per cc. y per cc. y per cc. y per cc. 0.50 1.15 1.25 1.10 2.20 1.15 2.00 1.00 2.00 1.20 2.00 0.42 2.00 0.40 0.42 0.25 0.90 0.27 0.90 1.00 0.30 1.40 0.60 1.30 0.94 0.25 0.50 0.50 0.75 0.25 0.50 0.50 0.75 TABLE III Liberation of Histamine jrom Rabbit Blood Cells by Dialyzed A was prepared from a fresh stock solution of papain. B was prepared by dialyzing the solution of papain for 20 hours against distilled water in the ice box. CBAA Histamine content Plasma + A Plasma + B A B Total blood I _ y per GC. y per cc. y per cc. y per cc. 0.11 0.20 0.75 0.62 0.11 0.10 0.75 0.12 0.50 0.60 0.60 0.08 0.08 1.00 2.00 2.00 1.25 0.11 0.06* 0.42 2.00 0.80 1.20 0.10 0.10 0.85 1.20 1.15 1.40 ~~ * Dialyzed 48 hours in the ice box; t,he CBAA of this solution of papain was abnormally low. As the experiments on histamine liberation ought to be done at a ph range (7.3 to 7.5) in which the curve of activity of papaincysteine begins to decline, a certain variation in the values of the proteolytic coefficient
M. ROCHA E SILVA AND S. 0. ANDRADE 13 CBA,, should be expected. This would explain small variations of the proteolytic coefficients as shown in Tables II and III. The results presented in Tables II, III, and IV definitely show that the ability of papain to liberate histamine from rabbit blood cells runs parallel with the activity of its benzoylzargininamidesplitting component. This component is fairly resistant to heat; as shown in Table II, heating papain at 52 for 12 to 15 minutes does not change the activity of the enzyme upon benaoylzargininamide or its histamineliberating capacity. On the TABLE Effect of Treatment of with Isopropyl Alcohol on Iiistanrine Liberation from Rabbit Blood Cells and BenzoyllargininamideSplitting Activity A was prepared directly from a stock solution of purified papain containing 200 mg. of papain per 25 cc. of saline. From this solution 2.5 cc. were transferred to a 5 cc. volumetric flask and 1 cc. of cysteinc solution + 1.5 cc. of phosphate buffer was added. B was prepared by treating 10 cc. of the stock solution of papain with 20 cc. of chilled isopropyl alcohol; after 15 minutes at 4, the precipitate was collected by centrifugation and redissolved in saline. After two or three precipitations with isopropyl alcohol, the volume was made up to the volume of the original stock solution. From this solution, 2.5 cc. were transferred to a 5 cc. volumetric flask and 1 cc. of cysteine solution + 1.5 cc. of phosphate buffer was added. Temperature of the bath, 39 ; final ph, 7.3 to 7.5. CBAA Experiment No. I A : 6 Averages CIB,AA/C,AAA. 0.120 0.055 0.081 0.066 0.120 0.095 0.088 0.056 0.096 0.040 0.093 0.040 0.086 0.061 0.097 0.059 IV y per cc. Histamine Plasma + A y per cc. 0.04 0.46 0.25 0.81 0.30 1.25 0.12 1.25 0.26 0.56 0.26 0.65 0.35 3.12 0.23 1.16 = 0.61. Histamine B = 0.82 0.23 = 0.62. Histamine A 1.18 0.23 content I I ~_ Total blood y PEI cc. y per CC. 0.15 0.52 0.74 0.63 0.80 1.70 0.85 1.80 0.35 0.90 0.35 0.90 2.50 1.80 0.82 1.18 contrary, contact with N NaOH, for 4 to 1 hour, was enough to destroy the benzoylzargininamidesplitting component and at the same time its histamineliberating activity. Furthermore, we have verified that dialysis against distilled water does not interfere with the papa,incysteine component which splits benzoyllargininamide, but definitely lowers the activity of papain toward Zleucinamide, the CLA dropping from a value of 0.0015 to 0.0007. At the same time, the liberation of histamine by papain is not altered appreciably by dialysis against distilled water (Table III).
14 HISTAMINE AhD I ROTEOLYTIC ENZYMES That the liberation of hi&amine does not run parallel with the activity of the papain component which splits carbobenzoxyisoglutamine is shown in Table V, since treatment with isopropyl alcohol does not change very conspicuously the activity of papaincysteine toward carbobenzoxyisoglutamine. There is no correlation between the changes of the proteolytic coefficients toward benzoyllargininamide (C,,,) and carbobenzoxyisoglutamine (Co,,). This shows that the papain component which splits the former at ph 7.3 to 7.5 is not identical with the component which splits the latter. As shown in Table V, the quotient C$,/C& = 6.3 before TABLE Proteolytic Coeficients of Cysteine towards IArgininamide and Carbobenzoxyisoglutamine A was prcparcd from a fresh stock solution of papain, while B was prepared after three precipitations of the same solution by isopropyl alcohol, as indicated in Table IV. A 0.096 0.093 0.088 0.106 0.081 0.115 0.106 0.112 0.130 0.086 CBAA 0.040 0.040 0.056 0.055 0.066 0.061 0.060 0.053 0.065 0.061 Averages. 0.101 0.056 0.016 0.014 B V I A CCIG 0.020 0.013 0.013 0.016 0.011 0.014 0.020 0.015 0.011 0.014 0.020 0.015 0.015 0.012 B treatment with isopropyl alcohol; after three precipitations with isopropyl alcohol, the quotient CgAA/C&, = 4. The data presented in Table IV, however, show that treatment of papain with isopropyl alcohol lowers at the same rate (0.61 and 0.62) the activity of the enzyme both in splitting benzoylzargininamide and in releasing the histamine from rabbit blood cells, this being strong suggestive evidence that both activities depend on the same papain component. DISCUSSION There are substantial proofs that the histamine linkages with the cell constituents can be ruptured by proteolytic enzymes which display the same specificity as trypsin. The possibility that in several cases the nor
M. ROCIIA E S&VA AND S. 0. ANDRaDE 15 ma1 cellular cathepsins might be the agent of histamine liberation would induce one to study the histamineliberating activity of these complex proteolytic enzymes. Unfortunately, animal cathepsins are almost inactive at the ph of blood, which makes them unsuitable for experiments on histamine liberation from living tissues. The study of a vegetable cathepsin like papain gave satisfactory results. is a mixture of proteolytic enzymes which display specificities somewhat similar to those shown by animal cathepsins. has a ph optimum around 5, and needs activators such as HCN, cysteine, ascorbic acid, etc., exactly as the usual animal cathepsins do. It has been possible to identify in papain a proteolytic component similar to Cathepsin II (which splits benzoyllargininamide), another similar to Cathepsin III (which splits Zleucinamide), and another similar to Cathepsin V (which splits carbobenzoxyisoglutamine). is unable to split glycylzleucine, and under the conditions of our experiments it did split hippurylamide slightly and glycylglycine very slightly (at ph 7.3 to 7.5). The great advantage offered by papain over animal cathepsins is the fact that it still retains an appreciable part of its activity at ph 7.4. The splitting of benzoyllargininamide at this ph is fairly rapid, while animal cathepsins prepared according to the method of Anson (11) lose their activity almost entirely when the ph of the medium is maintained above 6. In consequence, papain can be used as a cathepsin model when the experiments must be performed at ph 7.4, as in those with rabbit blood. We have been able to show that the histamineliberat,ing activity of papain does not show any relationship to its capacity for splitting lleucinamide, glycylglycine, glycyllleucine, carbobenzoxylglutamylltyrosine, or carbobenzoxylleucylglycine. On the other hand we have shown that submission of papain to several treatments, such as dialysis against distilled water, heating to 52 for 15 minutes, and contact with N NaOH, destroys some of its proteolytic components and that its capacity for splitting benzoylzargininamide disappears at the same rate as its histamineliberating activity. This shows a definit,e parallelism between the activity of the enzyme in splitting this substrate and in liberating histamine from rabbit blood cells. Treatment of papain with isopropyl alcohol lowered its activity in splitting benzoylzargininamide, but did not markedly alter its activity in splitting carbobenzoxyisoglutamine; the effect of this treatment upon the histamineliberating activity of papain was of the same order of magnitude as its effect upon the capacity for splitting benzoyllargininamide. The problem presented in this paper, therefore, concerns the probable nature of the linkages holding histamine to the cell proteins. Besides the fact that the release of histamine runs definitely parallel to the activity of
16 HISTAMINE AND PROTEOLYTIC ENZYMES papain in splitting benzoyllargininamide, it is worthy of note that this substrate is a typical one for trypsin, which is one of the most active substances in releasing the histamine from living cells. Chymotrypsin, which does not split this substrate, does not release histamine in appreciable amounts. Among the very few simple substrates which trypsin has been shown to split are hippurylzlysinamide and benzoyl!lysinamide, which indicates that lysine, instead of arginine, might be the amino acid residue proximal to the peptide linkage ruptured by trypsin. In this sense, one might draw the provisional conclusion that histamine is bound either to arginine or to lysine in the amino acid chain of cell proteins. On the basis of this conclusion, one would expect that Cathepsin II would probably be the cellular cathepsin component responsible for the release of histamine in the event that an abnormal activation of cellular proteinases is the primum mooens of a discharge of this pharmacologically active substance. SUMMARY cysteine shows two optima of activity, at ph 5 and ph 6.8 to 7, when tested on synthetic substrates like benzoyllargininamide and carbobenzoxyisoglutamine. At ph 7.3 to 7.5 papaincysteine splits benzoyllargininamide, carbobenzoxyisoglutamine, hippurylamide, Ileucinamide, and, very slightly, glycylglycine. The other substrates tested, namely carbobenzoxylglutamylztyrosine, glycyllleucine, carbobenzoxylleucylglycine, and chloroacetylltyrosine, were not split by papaincysteine in that range of ph. was found to release histamine from rabbit blood cells to plasma. Dialysis against distilled water and heating at 52 for 12 to 15 minutes did not appreciably alter this effect of papain upon rabbit blood histamine. Treatment with NaOH definitely reduced to zero the ability of papain to release histamine. Several precipitations with isopropyl alcohol markedly reduced this capacity. The papaincysteine component which splits benzoyllargininamide was altered by the above treatment in a quite similar manner, which led to the conclusion that the histamineliberating activity of papain runs parallel with its capacity for attacking the arginineamide linkage. The components which split Ileucinamide and carbobenzoxyisoglutamine were altered differently by the same treatment. A provisional conclusion is drawn that histamine is present in the cell, forming an amide type of linkage, the proximal amino acid being either arginine or lysine.
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HISTAMINE AND PROTEOLYTIC ENZYMES: LIBERATION OF HISTAMINE BY PAPAIN M. Rocha e Silva and Sylvia O. Andrade J. Biol. Chem. 1943, 149:917. Access the most updated version of this article at http://www.jbc.org/content/149/1/9.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/149/1/9.citation.full.html #reflist1