2378
GEORGE 13. I1OLH AND ROSS M K R N ~~R~~~~
I . ~ ~ ~ e n y ~has a rbeen s~~ eonitlensed e with ~ ~ t y r a ~ and ~ e ~h ~~ ~ ~~ e~ ‘lnyde to give compounds consisting oi t,wo moles of aldehyde arid one of arsine and probably having the fdpI1owing structure, ti i as(^^^^^^^^^. 2 The ~ ~ ~ ~ products e n are s ~stable ~ t~o i ~o ~ dilute d~ . ~ acid, alkali a.rd water.
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U R B A N A , ILLINOIS. _ _ . _ I _
[6ONTR.IF3PrTION’ FXOM
THE
DIVISION OF AGRICULTURhr, ~
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AGRICULTURAL .@XPBRIIM?2NT STATXQW.
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YS BY GBORGGE. Ho Recei ed Aug, 28, 1920.
i n previous articles of this series2we have sl-rown ““tat the formation of black acid insoluble humin in a normal protein hydrolysate is dependent upon the presence of tryptophane in that protein molec~le.’~While tryptophan e reacts readily with aldehydes to give a black insoluble product, it alm ergoes some umknown reaction even when dlawed to .st.and in an acid t h . This was noted by * ~ ~ d ~who r ~ fou.nd a ~thatd the ~ mother ~ ~ liquor from a tryptophane preparation, which had been allowed to stand for a long time, slowly darkened, lost its ability to absorb bromine, gave no glyoxylic test, and finally deposited a dark brown produet, soluble in acids and alkalies, and which when heate gave a strong indole odor. Harries arid I,angheld* studied the effect, of ozone upon ~ y ~ r ~ ~ ~ ~ awith n eQ Z O K ~ ~ products of proteins and found that ~ r ~ ~ t o kre.ated colored very dark, gave no y loxylie reaction, reduced PehU ng’s ~ ~ ~ in the cold, was precipitat by baririm hydroxide, lead acetate, basic lead acetate, and liberated ammonia .tvY~entreated with sodium ~ y ~ Van Slyke states5 “ ~ ~ y p ~ o ips ~known ~ n e to be precipitated ~ ~ ~ ~ t ~ by p ~ o s ~ h o acid ~ ~even ~ i from ~ s ~fairly ~ ~dilute solution. When it is boiled with mineral. acids, Iiowever, it is, to a Barge extent, a t least destroyed, the nature and fate of the pro duet.^ being un1cnown.” In to ascertain the behavior of tryptophane under the conditions of p 1 Published with the approval of the Director as Paper No. 208, ’Jomiial Series of the Minnesota Agricultural Experiment Station. Presented before the Bioiiogical Division of the Rinerican Clzen~icalSociety at the Spring Meeting, St. Lollis, WpriS
11-16, 1920.
Gortner and Rlish, THISJOURNAL, 3y1 1630-36 (191.5);Gortner, J . Biol. Cheherr,., (1916); Gortner and Holm, TEIS JOWR~M,, 39, 2477-2501 (~9x7);.#a, 82a-827 ( 1 9 2 0 ) ; Holm and Gortner, ibid., 42,632-40 (rgzo). Abderhalden, Z . physiol. Cirern., 78, rgy-160 (‘1912). Harries and Langheld, ibid., s a , 371-383 (1907). Van Slyke, S. B i d . CAem., IO, 39 (1911). 2
, 177-204
EFFECT OF ACID HYDROLYSIS ON TRYPTOPEIANE.
2379
hydxolysis he boiled pure tryptophane for 12 hours with 20% hydrochloric acid and studied the solution with regard to melanin (humin nitrogen), ammonia, amino nitrogen and total nitrogen, and also with regard to tbe amounts precipitated by phosphotungstic acid. He draws the lollowing conclusions. I . Tryptophane is responsible for none of the nitrogen estimated as ammonia, arginine or melanin. 2 . Boiling with 20y0 hydrochloric acid does not alter the ratio 2 : I of total nitrogen to amino nitroen in tryptophane. 3. It appears improbable that tryptophane affects the composition of the phosphotungstic acid precipitate under the usual conditions of analysis, but it is advisable, in the latter, as a precaution, to test a few drops of the solution of the bases for tryptophane. Homer1 later studied the effect of acid hydrolysis upon tryptophane and found that after 42 hours’ hydrolysis with 25% sulfuric acid almost 8 complete recovery of the tryptophane was possible, but under the same conditions in a 21-24 hours’ hydrolysis and in the presence of ferrous sulfate there was pigmentation and no recovery of tryptophane. She therefore, concludes that hydrolysis changes tryptophane to a compound which absorbs bromine, but does not precipitate with mercuric sulfate. Johns and Jones2 in studying the quantitative estimation of tyrosine by use of the Folin-Denis phenol reagent state “it is well known that tryptophane is decomposed by acid hydrolysis” and “the tryptophane was completely decomposed and its decomposition products gave no blue color with the reagent of Folin and Denis,” The evidence which they submit in substantiation of the latter statement we have recently shown3to be in error, due to the fact that they decolorized their hydrolysate with ‘‘Norit” and that carbon adsorbs tyrosine, tryptophane and tryptophane decomposition products. In the present study our interest is confined primarily to the “acid insoluble” and “acid soluble” humins formed on protein hydrolysis. Our previous studies4 have shown that aldehydes, when present during the acid hydrolysis of a protein greatly increase the amount o€ acid-insoluble humin which may be formed, the amount of acid-insoluble humin rapidly increasing to a maximum, at which point the tryptophane is entirely converted into acid-insoluble humin. When the hydrolysis is conducted in the presence of an optimum amount of formaldehyde the nitrogen present in the acid-insoluble humin may be taken as a quantitative measure of the tryptophane nitrogen of the protein. Certain of our experiments have been taken as evidence that proteins contain some unknown component which reacts with tryptophane to form the small Homer, J . Biol. Chem., 23,382-85 (1915). 36, 319-322 (1918). a Gortner and Holm, THISJOURNAL, 42, 1678-1692 (1920). koc. cit
* Johns and Jones, ibid.,
amount of insoluble liumin whicli is present in a f-lomal protein hydrolysafe.’ We therelore, wished to a c w Gain if the tryplopham molsczilc contributes to the “acid insoluble” or “acid” solnbk huiairns when no aidehydes or oi-ler ~eactirag;con:l;olinds are present, ’$his paper reports thc resdts 01 a qtudy of t h e effect of prohinged boiling with ?o(;l,Iiydrochloric acid upon the trypkqihane m o l e ~ ~ lwith e , especial I efea-ence t o the nitrogen distribution ~
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ethod.--Five hundred milligrams of tryptophane was added to exactly loo cc. of 2 ~ ” 7 chydroclslorjc acid. ian aliquot of z cc. was then removed with a pipet and amino nitrogen d e t e m h e d upon this sample by the Van Slyke method. One cc was also removed, diluted to 2 5 cc tdunze, and a colorimetric determination of the tryptopliane content made upon 5 cc. (representing orie mg, of tryptophane), using the phenol reagent of Polin and D e n i ~ . ~The , ~ flask was then weighed, fiLted with a reflux condenser and heated to boiling U ~ O O .a sand-bath. A t intervals 01 12, 2 4 , 36, 48, 96 and 144 houiq, the flask mas allowed t o cool and wa5 weighed. In case of a loss of weight, Z Q % hydrochloric acid was added at each interval to make up exactly for the acid lost by evaporation tkcreby insuring correct aliquots in each sample taken. *I‘lxe samples wejte taken at intervals as designated rnntii the ’nojlnng had continued for 144 hours. The contents were the71 filtered, the residue washed thoroughly and nitrogen determined upon the residue by the Kjeldahl method. T h e filtrate was evaporated to dryness z?iz VL’I~C%Oand “solrzbk 17p1n&a9’ and “ammonia” determined in the usual iaanner. The iiltrate €~QIII these determinations was acidified, concentrated to about 35 cc and precipitated by phosphotiiogstic acid in the usual manner. After a separation el “filtrates” and “bases” had been made, both “amino” and ‘ total n1t1-o en” determinations were made upon cach fraction. This method enabled us to follow the rate of deanirrization and also gave the amounts of tryptophane converted into the black “insolubic humin” and “acid-soluble humin.” The amount of “acid-insoiubk humin” formed is Ailso indicated by the decrease in the tryptophane a5 1
40
For our evidence on this point, see Holm a d Gortner,
$PIS
JOURNAL,
42)632 -
(1990)
J Baol Chem, 12, 239-43 (1912) We haFe recently (THEJOURXAL, 42, x678-rG92 ( ~ 9 2 0 )studied )~ the colorlmerrtc vahes obtained by adding the phenol reagent to tryptophane solul~oris,a d have 4 o m that ~ the reagent cannot be used on protein hydrolysates to obtain re1:able data because or the fact that more than one reactive eomoound rnay be presmt Oui crihrmn, of the use of this reagent €or quantitative work do nat apply ir. the present imtaiice, because all of t h e color drveloped I“ these solutions cqii be definitely ascribcd i‘a the rtldole nricleiiq 2
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EFFECT OF ACID HYDROLYSIS ON TRYPTOPIIANE.
2381
determined colorimetrically, The effect of acid hydrolysis upon the precipitability of tryptophane by phosphotungstic acid is also shown. The Experimental Data.-The color changes observed in the solution were as follows, before the boiling began the solution of the tryptophane in t h e acid 'was perfectly colorless; a t the end of 12 hours' boiling the solution was transparent but dark red-brown; a t the end of 24 hours' boiling the solution was a IOO deep brownish-black and was no longer transparent 95 (depth of liquid about 4 em.) ; while the formation of dark, amorphous par- 90 tides floating in the liquid was first noted after 48 hours' boiling. 85
The changes in amino nitrogen due to the pro- 8 0 longed boiling with hydrochloric acid are s h o w n 75 graphically in the upper curve of Fig. I. This curve shows that boiling 7 0 with 20% hydrochloric acid causes a gradual decrease 6j In amino nitrogen, more rapid a t first than during 6o later periods. X colorimetric d e t e r mination at each interval 24 48 96 I44 serves to i n d i c a t e the Hours boiling with 20% HCl. a m o u n t of tryptophane Fig. 1.-Showing (upper curve) the decrease in 'which been destroyed amino nitrogen (deaminization) in percentage of o* the amount Or Portion of original amino nitrogen, and (lower curve) the dethe indole nucleus in which crease in color values of a tryptophane solution with the a-gositionl has 50 been increasing length of boiling with ~ 0 % hydrochloric changed or combined that acid. no oxidation is possible. It may indicate even to a greater extent than bumin formation or deaminization the total changes in the indole nucleus. The lower curve in Fig. I shows the colorimetric values a t the different ~
That it is the a-position of the indole nucleus of tryptophane which reacts with the phenol reagent is indicated by the fact that a,P-di-phenyl indole did not produce the blue color when added to the phenol reagent. Of course, the failure to produce the blue color with the diphenyl compound may have been due tQthe insolubility of the indole.
Ptartiou of total nitroNitrogen. gen. NitroKen. Mv. 70. Mgr.
Fraction.
Total.. . . . . . . . . . . . . . . . . . . . “Acid-insoluble” humin N. . Ammonia Pa.. . . . . . . . . . . . . . “”Acid-soluble” humin N . . . . “Pliosphotungstic acid humin’ N ...................... Total N in “basei”. . . . . . . . . Amino N in “bases”. ....... Total N in “filt. from bases. ’ I Amino Ti in “filt. from bases” ~
TrJTpta,phaue a5 Tryptotyroeine phane Fraction equiva- actually present of lents (eolori(calc. total nitrogen. metric). from N.) ?&. ?Vlsr. I\agr.
..... 8.38 25.55 40.04 O.9Y
ro.66
..... ..... 7 .40
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..... 71.43 . “ . I .
86 .Y‘!
..... 12.78
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36.44 .
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........... 98.32 In order to verify these results and also to investigate more closely the nature of the “soluble humin” in its relation to color formation with thc phiexxol reagent, we boiled 250 mg, of ~ r y p t o p ~ ~ for a n e144 hours with 20% ~ y d r ~ ~ h acid. ~ o r ~Act the end of that time the solution was filtered, concentrated and the cseparations made as .krt the earlier experiment with the ~ o ~ ~ exceptions: o ~ ~ n gthe soluble humin was filtered off and, after t h ~ ~ ~ uwashing, gh was dissolved in dii. hydrochloric acid a ICO cc. volume. Both a total nitrogen determination and ~ e ~ ~ ~ m were ~ ~ made ~ a ~ upon i o nthis dark sohtion. Colorimetric de. terminations were also made upon the “bases” and “filtrates from the bases ’’ Table I: (Sample 2 ) shows the results vsihich were obtaiined. Total recovered.
WhiIe the figiires for the n ogen distribution as shown in Table 1 are not in exact agreement, they indicate clearly that the nitrogen distribution of tryytophane is altered very markedly by boiling the amino acid with myohydrochloric acid. Moreover, the Iact that the 2 s a ~ ~ ~ p l c s were boiled. with 2oY0 hydrochloric acid for equal periods of time and yet exbibit a difference in nitrogen distribution, indicates the ease with rhe hydrolysate of a protein containing tryptophane may be d t e r slightly varying the conditions ~f hy&olysis.
EFFECT OP ACID HYDROLYSIS ON ”FtYPTOPHANE.
2383
We are undecided as to whether or not some of the changes which take place are due to oxidizing reactions. The work of Abderhalden and others would tend to indicate this. Our experimental conditions were such that this could easily be the case, for all hydrolysates were freely open to air. e have also statedl that “the vigor of boiling seems to influence bo ammonia and insoluble humin formation.” This difference (since the a samples were not boiled a t the same time) might, and we believe does, account for the differences in the nitrogen distribution. However, as stated above, we are interested in possible changes in the nitrogen distribution of tryptophane under conditions comparable with those obtained im the acid hydrolysis of a protein over extended periods of time, whether they be due to oxidizing effects, or directly due to the effect of the acid, or to both. The data for the acid-insoluble humin show that no detectable amount was formed during the first 24 hours. At the end of that time a few black amorphous particles could be seen in the dark reddish-brown liquid. A t 11.8 hours there were a great number to be seen and these increased in amount with prolonged boiling. Our figures show that, a t the end of 144 hours’ boiling, from 4 to 8% of the tryptophane nitrogen appeared in this fraction. The figures for the ammonia fraction substantiate the evidence that we have already put forth in a former publication2 where we state “a part of this (ammonia fraction) must be due to the breaking up of tryptophane.” Fig. I indicates that dcaminization proceeds quite rapidly a t first as is the case in a protein h y d r ~ l y s a t e . ~This rate diminishes as the concentration of unaltered tryptophane in the solution decreases, The concordant results obtained for ammonia in each of the 2 hydrolysates appear to indicate that acid concentration and length of hydrolysis are alone responsible for the deaminization. The ‘‘soluble humin” nitrogen in these experiments is probably an intermediate product in “acid-insoluble” humin formation. No tyrosine is present and eonsequently tyrosine could not contribute to this fraction. This would appear to conflict with some of our earlier conclusions and it may be possible that some of the nitrogen in the soluble humin fraction of a normal protein hydrolysate is derived from tryptophane. We have already shown that traces of the tryptophane may appear in this f r a ~ t i o n . ~However, the experiments referred to show conclusively that only a small fraction of the tryptophane is converted into “soluble” humin in a 24-hour hydrolysate ( 0 . 2 0 mg. of soluble humin N from 100mg. of tryptophane). In the same experiments no “insoluble humin” N was found. I
4
Gortner and Holm, THISJOURNAL, 39, 2738 (1917). dbid., 39, 2742 (1917). Cf. Fig. I, &id., 39, 2736-2745 (1917). Gortner atid Holm, ?%id.,42, 821-827 (1920).
!”s cdorinletrie detcrinination upon the soluble Irumirn lractiopl also that i t still retains some of the y r o p ~ t i eof~the, ~ t r y p t o p h a e molet it gives d o r with the pl~enolreagent A separatiou of chipi
into a “”basic”
izid
‘“fion-basic”
atid indicaks, a5 we h a w Eoxnnedy s no ~ I ~ a r - c isepxainon it of tryglopbme into tbc trate from the l~ase:,.” The result in case of a
alysis wilI be that, if tryptophane is present, all of the nonamino nitrogen of a 51tra tc cannot with certaiiitj- be cde~vlatrdz$ proline n31ogen and, in fact, might with equal certainty be calculated as EO^ansino tryptophane nitrogen, The h ~ s e s ,on the other hard, ~ 1 1 1be in error just to the extent of precipitated tryptopbanc which, as we have &eaily suggested, will appear and be calculakd largely as histidine. The colorimetric determinations indicate that the “bases” and the ‘%Itrate from the bases” contain substances, which, if they are not unchanged tryptophane, are, a t a q - rate, tryptophane which has been o d y slightly altered. The color produced by solutions of the bases is approxi:i?:Ite_ly equal (82% of & tyrosine equivalent) to that given by unaltered ~ ~ (85-90% of” a tyrosine~ equivalent)p vide the~ subsknses~ in Lhe fikrate from the bases gives a somewhat lower color value, indicating 3 :uiaewhat greater alteration. W e ictend in the near fut..ire t o make a more exact study of some of the h c tors involved in tryptophane decomposition.
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Summary. %‘e Ira\-e studied certain changes produced by boiling tryptophane with mt% hydrochloric acid for various lengths of time and have reached the Tryptophane is slo.vvly altered and parts of the molecule are broken by long acid-hydrolysis. Tryptophane, ill the absence of aldehydes or other reactive C O ~ pounds, contributes but an insignificant fraction of its nitrogen to the “acid insoluble” humin. A mi tch larger amount of the tryptophane appedrs in the “soluble humin” after 144 houi-s’boiling with acid. Siiice, howewr, a normal protein hydrolysis rarely requires more than ‘4 hours’ boiling, it appears extremely improbable that the “‘lota;” humin of such a hydrdy sate is derived from tryptophane witbotlt the intervention of sorw other reactive compound, which we have postdated in ozr earlier papers to he of the nature of an aldehyde. j Tryptophane is relatively easily deaminized by boiling with 20% hydrochloric acid. Probably some of the ammonia d a normal protein li ydrolysate is derived from tryptophane instead of being entirely der iaed from amide groupings. 4. When tryptophane has been boxled with 20% hydrochloric acid the L .
do I’D 51.
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distribution of the nitrogen is such that errors may be introduced into both the “basic” nitrogen and the “non-basic nitrogen” fractions oi a Van Slyke determination. ST. PAUL,MINN
[COMMUNICATION PROM TEE CHEMICAI,LABORATORY OF JOHNS HOPKINSUNIVERSLW
E ACID OR THE BUTYL ETHER OF THIOGEYCOEIC ACID. BY YOSHISUKE UYEDAAND E. EMXFTREID. Received August 30, 1920.
Introduction. Sulfur, when present in an organic compound in the sulfide condition, CB-S-CH = , may have comparatively slight influence on the chemicai and physical properties of the compound while in some cases i t gives rise to peculiar properties, as in P,P’-dichloro-ethyl sulfide. In this remarkable compound the presence of the sulfur atom renders the chlorine atoms very reactive. It seemed of interest to prepare other compounds containing a sulfide grouping along with some other group with a distinctive function. The present investigation is a study of the acid, CH3CH&H2CH2- S - CH2GOOH, arid some of its derivatives. Thiophene has nearly the same Soiling point as benzece but a considerably higher density and the same may be said of ethyl sulfide as compared with hexane. In physical properties the above acid and its esters should resemble caprylic acid, CeHlaOs, and its esters to which they are nearest in molecular weight. This has heen found to be true in a general way) though the acid boils a t 2 8 2 . ~ ’ ~ lirhich is 45 O higher than caprylic. The sulfide acid forms salts readily, the sodium salt being very soluble in water while the barium and calcium salts are only moderately so. %%en mineral acids are added to water soltuions of its salts the acid separates readily as a heavy oil which would easily be mistaken for one of the fatty acids of about its molecular weight, even to the odor. It is very slightly soluble in water and distils without decomposition. The odors of its esters are somewhat like those of the higher aliphatic esters. The acid is readily prepared by the action of sodium chloro-acetate on the sodium salt of butyl mercaptan in water solution. Its esters and many of its salts are easily obtained by the usual methods. The esters give good results on quantitative saponification as the acid is accurately titrated with phenolphthalein. The remarkable fact about 8,P’-dichloro-ethyl sulfide is that the inAueace of the sulfur renders the chlorine atoms quite movable.’ This =
Xarshall, J . Am. Meed. Assoc.,
lsr684 (1919);C. A , , 13, zgzg
(1919).
