K. GEORGE FALK AND RIARSTON I,.
210
HAAILIS.
0.62j 5 gram substance in I r .90 grams naphthalene caused a depression of 0.685 O in the freezing point of the solvent. Calculated for &H3,06: Xol. wt., 618.3. Found: 530.
This ether apkars to be somewhat more readily hydrolyzed to the carbinol than the ethers previously described. But even so, it is not afYected by water and only very slowly by dilute alkalies. __
The facts presented above were withheld until now from publication in THISJOURNAL, in the hope that the synthesis of the much disputed p-hydroxy-triphenyl-methyl oxide would also be realized. The appearance, however, of the paper by Schlenk in the last number of Liebig’s Annulen? makes it desirable that the results obtained in this laboratory thus far be published now. Schlenk, too, attempted to prepare triphenylmethyl oxide by the use of various reactions. His results proved entirely negativ, and he concluded that this oxide is in all likelihood incapable of existence. We have recently worked out a still different method for the preparation of triarylmethyl oxides. When triphenylmethyl carbonate (m. p. 205-1o0) is heated under definit conditions it loses carbon dioxide and yields almost quantitatively triphenylmethyl oxide. The details of this reaction and the work relative to the oxides described by Decker,4 and by Barbier5 will be given in a subsequent paper. In connection with the oxides the study of the triarylmethylsulfides has also been in progress. Here, apparently, lead sulfide promises to give the best results. 4 ~ i ARBOR, s ~IICIIIGAX
STUDIES OR ESZTMS ACTION. 111. THE A G T M OF #LWAWXJS SULFATE OW CA§TOR BEAN LIPASE. B Y K.G ~ o ~ FALK o g AND MARSTON L.HAMLIX Received November 19, 1912.
In the first pagere of this series an experiment was described in which it was shown that the cantinued passage of an electric current through a suspension in water of the castor bean lipase preparation resulted in the pxoduction of a l&mlytically active substance a t the anode. As stated there, this substance was probably formed by some oxidation reaction of the preparation. Hoyer7 found that small amounts of manSee footnote on title page of this paper. Ann., 394, 178 (1912). ’ Ber., 34, 330s (1901) J . prakt. Chem., 84, 232 (1911). ’ i l n n . chzm., [SI 7, 507 (1876). THISJOLIRNAL, 34, 7\35 (1912). 7 pitiszol Chcin 50, 414 (1907). Cf. also Tanaka, Orzg. Corn. 8th Intern. Congr. .3p$/
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STUDIES ON ENZYME ACTION.
111.
211
ganous sulfate increased the activity of lipase to a greater extent than any other salt studied by him. In view of these facts, it appears reasonable to suggest the hypothesis that the activating action of manganous sulfate is based upon the property of the manganous salt of acting as an “oxygen carrier”’ and in this way causing the production of a lipolytically active substance from the castor bean preparation by an oxidation reaction which would not take place (or only very slowly) in its absence. This hypothesis was tested in a series of experiments to be described in this paper. Experimental. The experiments may be divided into four groups, namely: ( I ) Those in which the activity of the lipase preparation was tested after varying preliminary treatment, which consisted either in letting the mixture stand, or in passipg air through the mixture in water, in the presence and absence of manganous sulfate, or in extracting and testing the filtrate and residue. ( 2 ) A similar set of experiments with a lipase preparation which had been rendered lipolytically inactive by heat. (3) A set of experiments similar to ( I ) and (2) but titrated by the.formo1 method. (4) Two comparative experiments (with and without heating) with somewhat larger amounts of lipase preparation and manganous sulfate. The lipase preparation was obtained from castor beans, oil-free and finely ground as described in the former article. It was there found on testing with different esters, that the action on methyl acetate was not varied much by external influences, but that with ethyl butyrate, differences in action were observed which depended upon the conditions of the experiments and that a t the same time sufficient action was obtained for purposes of comparison under the different conditions. Ethyl butyrate was, therefore, used to test the activity of the preparations in the experiments which follow. In every test, I cc. ester was used and the lipolytic activity determined a t 38’ for the lengths of time indicated in the tables. The acid formed was determined by titrating with 0.1114 N sodium hydroxide solution, using 5 drops of a 1% solution of phenolphthalein in 95% alcohol as indicator; the results of the action are given, in every case, in cc. of the sodium hydroxide solution. Table I contains the results for the experiments in which the lipase preparation was used without preliminary heating in solution. These results as well as those in the other tables are each based updn two or more duplicate determinations not reported separately. Column I shows the number of the experiment, column 2 , the weight of lipase preparation used, and columns 3, 4 and 5, the treatment preliminary to testing the Engler and Weissberg, “Vorgange der Autcuridation,” 1904, p.
XII.
R. GEORGE FALK AND MARSTOR‘ I,. HAMLIN.
212
activity. The preparation was made up with 2 5 cc. water and ‘ I 2 to I cc. toluene, and tested directly, experiment I , tested directly after addition of manganous sulfate (shown in column 5 ) , experiment 4, or allowed to stand stoppered a certain number of hours before being tested (shown in column 3 ) or a slow current of air was passed first through a flask containing toluene and then through the solutions for the lengths of time indicated (column 4), experiment 3. If more than one of these treatments was applied, the Roman numerals preceding the numbers in the column show their order, thus in experiment 6, manganous sulfate was added and the solutions allowed to stand for 48 hours before testing their activity; in experiment 8 , they were allowed to stand for 17 hours and air was passed through for 8 hours after manganous sulfate had been added, before the activity was tested, in experiment j , manganous sulfate was added just before the activity test, the solutions having stood and air having been passed through for the lengths of time indicated. TAB1.S
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STUDIES ON ENZYME
ACTION. 111.
213
preparation was treated in-the same way preliminary to the activity test in the tests in which ethyl butyrate was subsequently added and in those in which it was not. For experiments p-15, the lipase preparation was ground with water, filtered through coarse filter paper, and the clear, yellow filtrate diluted so that 25 cc. portions corresponded to 0.2gram of the original perparation. These were used for experiments 9-11. The residue after liltration was dried on a porous plate and 0.2 gram portions used for each of the experiments 12-15. Experiments 1-8 show that the activity of the lipase preparation is decreased after standing in solution, but that if manganous sulfate is present the activity after standing or passing air through the solutions, while not as a rule greater than when tested immediately, tended to approach the value in magnitude. Experiments 9-11 show that the slight activity found in the filtrate from the preparation after extraction with water, disappeared on standing, even when manganous sulfate was present, while experiments 12-15 show that the residue contained material which was lipolytically active, Znd that increased activity was shown after the action of manganous sulfate. In Table I1 the results obtained by testing the lipase preparation after it had been inactivated by heat and then subjected to varying treatment are given. Requisit portions of the substance were heated with 25 cc. TABLE II.-THE ACTIONOF CASTOR BEANLIPASEON ETHYL BUTYRATE AFTER HEATING.
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a 14
K. GEORGE FALK AND XARSTON L. HAMLIN.
water in test tubes, in a boiling water-bath, foy two hours. The mktures were then transferred to small flasks, the amount of water being increased to 40-jo cc. during the transfer, i / - I cc toluene added and then the portions treated as indicated in the tables The various columns in Table I1 have the same significance as in Table I. The results of experiments 16-21 with 0.2 gram lipase preparation show that if no manganous sulfate is present, no activity is observed, but that if manganous sulfate is added, a distinct lipolytic action is apparent. With experiments 2 2 - 2 7 , using 0.j gram of the lipase preparation, there is apparently a very slight activity shown if no manganous sulfate is present, but much more marked activity if it is present. In experiments 28-31, the lipase preparation was boiled for an hour with water, filtered, the residue dried on a porous plate and 0 . 2 gram portions used. Here no activity was shown unless manganous sulfate was added and air passed through for a considerable length of time. In the experiments shown in Table 11, the end point in titrating was taken to be the pink color which was permanent for at least 30 seconds. I n some of the titrations the pink color faded in a few seconds and as there may have been a question as to the end paints in these cases, a series of experiments was carried out in which the formol method of titrating was employed. The results are given in Table 111. The treatment preliminary to the activity tests and until the titrations did not differ from the former experiments and the data concerning these, are shown in columns 1-5. The first part of the titrations was carried out like the titrations reported in Table I1 ; alkali being added with phenolphthalein as indicator until a distinct pink was obtained; these results are given in columns 6-9 headed "Direct." Column 9 gives the amounts of action on direct titration ilfter the end point had been attained, 5-10 cc. of T ~ B LI11 E -THE ACTIOXOF CASTOR Bcas LIPASEo s ETHYLRr TYRATI:
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STUDIES ON ENZYME ACTION.
111.
215
40% formaldehyde solution (neutralized toward phendpbthaieh) was added and the titration continued until a distitact pink &r w&s produced in the solution. In no case was any difficulty experienced in determining this end point. The total amounts of dkali required (2.. e., the awwnts used in direct titration plus the amounts required after the addition of formaldehyde) are in columns headed “Form01 Total.” In experiments 32-35, the lipase preparations were not heated with water preliminary to the treatments; in experiments 36-39, they were. A comparison of experiments 32-35 with the results given in Table I shows a minor digerence in the action of manganous sulfate in experiment 35, the action not being quite as great as wwld have been expected. The results given in Table I for this mixture are based upon a greater number of experinnab tltan the result given in experiment 35, and this small difference need not be further eoasidered. Experiments 36-39 show again a distinct increase in activity of the preparations in the presence of manganous sulfate after these had been inactivated by heat. The important conclusion to be drawn from the results in Table I11 is that the same action is obtained by direct titration and by the total formol titration, as is shown by a comparison of the amounts of acid formed, indicated for each experiment in the last column. The difference between these two sets is less than 0.10cc. in every case, a result within the experimental error of the titrations. This fact increases the probability that the results in Table I1 are conect, as the end points there and in the direct titrations of Table I11 were similar. A number of 0.5 gram portions of lipase preparation were titrated to neutrality directly and then again after neutralized formaldehyde solution had been added. The mean values of the cc. 0.1114N sodium hydroxide solution used were 0.45 cc. for the direct titration and 0.52 cc. additional after formaldehyde had been added. The same amount of lipase preparation heated to boiling with water for different lengths of time (1-4 hours) required slightly varying amounts of alkali in the direct titrations, but gave remarkably constant results for the additional amount of alkali required for neutralization after formaldehyde had been added, these results varying only between 0.52 and 0.55 cc. 0.I I 14 N sodium hydroxide solution. A similar result is shown in experiments 36-39. The amount of alkali required after addition of formaldehyde varied from 0.48 cc. to 0.57 cc. in the experiments in which no ester was present, and from 0.49 cc. to 0.59 cc. when ester was present, the presence or absence of manganous sulfate and the preliminary treatments exerting practically no effect. The experiments with 0.2 gram lipase preparation, not heated, showed a mean formol titration value of 0.23 cc. 0.1114 N sodium hydroxide solution. There is then no change in the number of amino and carboxyl groups neutralizing each other as shown by the formol titration results
2 16
R. GEORGE FALK AND XARSTON I,. HAMLIN.
when the lipase preparation was rendered inactive by heat or when it was activated again by the manganous sulfate. In order to test the action of manganous sulfate and the lipase preparation in somewhat larger amounts, the experiments shown in Table IV were carried out. In experiment 40, a paper extraction thimble was placed in the mixture of 5 grams of lipase preparation, 2 5 0 cc. water and ‘5 grams of manganous sulfate. Activity tests on ethyl butyrate were made in the usual way with I O cc. portions (slightly opalescent) taken from within the thimble, called “Solution” in the table, and with IO cc. portions of the mixture outside the thimble, called “Mixtue” in the table. After each successive removal of material for testing sufficient water was added to keep the total volume at 2 5 0 cc. and a current of air was passed through the increasingly dilute mixtures outside &e thimble for:the total lengths of time indicated. Columns 4-6 in the first part of the table show the treatment preliminary to testing the activities, and columns 2-9 under the heading “Activity Tested” give the results of the activity tests. The headings of the columns indicate, in sufficient detail, the significance of the results. TABLE11’ --ACTION OF CASTOR BBANLIPASE O K ETHYLRrTVRATE Wt. of
Iipase Grams
Bxpt. No
Not heated
.....
40
Time of acWater as tion of air. solvent. (Total) Cc. hours.
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