SOME OBSERVATIONS ON THE ACTION OF ALKALI UPON CYSTINE AND CYSTEINE.* BY H. T. CLARKE AND J. M. INOUYE. (From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York.) (Received for publication, July 26, 1930.) The decomposition of cystine by alkaline reagents has received much study, particularly in recent years by Gortner and his col- laborators (1) and by Andrews (2). Consideration of the results obtained by these workers leads to the conclusion that the process is far from simple, apparently consisting of a series of successive reactions, possibly accompanied by simultaneous side reactions. The experiments here reported are, in the main, of an orientating character only, having been carried out in the hope of disclosing fresh lines of attack on the general problem. Fate of the Organic Portion of the Molecule.-Investigation of the alkaline decomposition products of cystine and of certain of its derivatives led Baumann and others (3) to the conclusion that the initial product is pyruvic acid,1 which subsequently breaks down under the influence of the alkaline reagent. We have been able to confirm this by the isolation of pyruvic acid p-carboxyphenyl- hydrazone (which is stable towards alkali) in yields of over 60 per cent from cystine by heating with alkaline plumbite in the presence of sodium phenylhydrazine-p-carboxylate. The same product is obtained in somewhat better yield from cysteine under similar conditions. The phenylhydrazone of pyruvic acid can also be * This work was aided by the Research Grant from the Chemical Foun- lation to this Department. 1 It is interesting to note that Dewar and Gamgee (4) reported the forma- tion of pyruvic acid by the action of nitrous acid upon cystine. Since we have been able to show that pyruvic acid gives rise to a small but definite &mount of nitrogen in the Van Slyke amino nitrogen procedure, this may ‘urnish a possible explanation of the well known fact that cystine gives tbnormally high results in the Van Slyke process. 399 by guest on February 11, 2018 http://www.jbc.org/ Downloaded from
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SOME OBSERVATIONS ON THE ACTION OF ALKALI UPON CYSTINE AND CYSTEINE.*
BY H. T. CLARKE AND J. M. INOUYE.
(From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York.)
(Received for publication, July 26, 1930.)
The decomposition of cystine by alkaline reagents has received much study, particularly in recent years by Gortner and his col- laborators (1) and by Andrews (2). Consideration of the results obtained by these workers leads to the conclusion that the process is far from simple, apparently consisting of a series of successive reactions, possibly accompanied by simultaneous side reactions. The experiments here reported are, in the main, of an orientating character only, having been carried out in the hope of disclosing fresh lines of attack on the general problem.
Fate of the Organic Portion of the Molecule.-Investigation of the alkaline decomposition products of cystine and of certain of its derivatives led Baumann and others (3) to the conclusion that the initial product is pyruvic acid,1 which subsequently breaks down under the influence of the alkaline reagent. We have been able to confirm this by the isolation of pyruvic acid p-carboxyphenyl- hydrazone (which is stable towards alkali) in yields of over 60 per cent from cystine by heating with alkaline plumbite in the presence of sodium phenylhydrazine-p-carboxylate. The same product is obtained in somewhat better yield from cysteine under similar conditions. The phenylhydrazone of pyruvic acid can also be
* This work was aided by the Research Grant from the Chemical Foun- lation to this Department.
1 It is interesting to note that Dewar and Gamgee (4) reported the forma- tion of pyruvic acid by the action of nitrous acid upon cystine. Since we have been able to show that pyruvic acid gives rise to a small but definite &mount of nitrogen in the Van Slyke amino nitrogen procedure, this may ‘urnish a possible explanation of the well known fact that cystine gives tbnormally high results in the Van Slyke process.
obt’ained, though less readily, when the reaction is carried out in the presence of phenylhydrazine in the place of its carboxylic acid, Repeated attempts to isolate hydrazones of pyruvic acid from reaction mixtures containing cystine with calcium hydroxide in the place of sodium hydroxide have consistently failed; cysteine, on the other hand, under these conditions furnishes a high yield of pyruvic acid p-carboxyphenylhydrazone.
Further evidence of the production of pyruvic acid by the alka- line decomposition of cystine, both with and without plumbite, is furnished by the development of an intense orange-yellow color when the reaction is carried out in the presence of salicylaldehyde. This test, which is delicate though not specific for pyruvic acid, undoubtedly depends on a reaction analogous to the condensations of salicylaldehyde with acetone (5) and of benzaldehyde with pyruvic acid (6). The product, which is soluble in water, is orange-yellow in alkalies, almost colorless in weak or dilute acids, and lemon-yellow in concentrated hydrochloric acid. A faintly acid solution readily gives up the substance to ether, from which it may again be extracted by dilute alkali and by concentrated hydrochloric acid. The reaction can be followed calorimetrically if the match be made through a light filter which transmits only a narrow band of the spectrum around 495 rnp, at which wave- length the absorption of alkaline solutions of salicylaldehyde is negligible. The method, however, apfiears unreliable for quanti- tative purposes.
The succession of reactions whereby pyruvic acid is formed dur- ing the decomposition of cystine and cysteine by alkali, outlined by Bergmann and his collaborators (7), is in complete accord with our results. The observation that sulfur and nitrogen appear to be eliminated in approximately equivalent amounts suggests that the second stage (hydrolysis of a-aminoacrylic acid) proceeds with much higher velocity than the first, which therefore determines the rate of the reaction as a whole.
CHzSNa CH2 I II
CHNHz + NaOH = CNHz + Na& + Hz0 (Slow reaction) I I
This mechanism finds a partial analogy in the well known con- version by alkali of a!, fl-dibromopropionic acid into Lu-bromoacrylic acid.
CHzBr CHz I II
CHBr + NaOH = CBr [+ NaBr + Hz0 I
COsNa COtNa
When the dibromo acid is boiled with salicylaldehyde in dilute alkali, the solution acquires the orange-yellow color characteristic of the reaction product of salicylaldehyde with pyruvic acid. This can be explained by the hydrolysis of cY-bromoacrylic acid.
CH?
Fr YHa
+ NaOH = 00 + NaBr
COsNa COzNa
The intensity of the color so formed amounts to less than 7 per cent of that obtainable from cysteine under similar conditions, so that the formation of pyruvic acid constitutes merely a side reac- tion in the alkaline hydrolysis of the dibromopropionic acid.2
Availability of Labile Sulfur in Cyst&e and Cysteine.-Some doubt arises, after reviewing the literature, as to the proportion of the sulfur in cystine and in cysteine which can be obtained in the form of lead sulfide. Suter (9) stated that 83 per cent could be so
2 p-Chlorolactic acid, in the form of its ethyl ester (8), gives a pure yellow color, the intensity of which is equivalent to about 12 per cent of the maxi- mum obtainable from cysteine. With glycerolor-chlorohydrin, on the other hand, a dull yellow color of very low intensity is developed only after many hours at 100”. dl-Serine (a sample of which was kindly furnished by Dr. H. D. Dakin) develops the yellow color slowly, but no color is obtained with glyceric acid.
No attempt has yet been made to characterize the yellow substance pro- duced in these reactions, beyond noting that it forms a colorless lead salt which is insoluble in dilute acetic acid.
obtained from cystine; Schulz (10) reported a similar figure but stated that only 52 per cent of the sulfur can be obtained from cysteine. Morner (11) reported that 75 per cent, on the average, of the total sulfur of cystine can be recovered as lead sulfide,
We find that cystine can yield 75 to 80 per cent of its sulfur in the form of lead sulfide, the balance appearing mainly to take the form of sodium thiosulfate;3 the sulfur of cystine thus seems to be initially eliminated as disulfide. On this interpretation the amount theoretically obtainable as lead sulfide is 75 per cent.
Cysteine, on the other hand, furnishes a yield of lead sulfide equivalent to its total sulfur content. The same result is ob- tained on decomposing cyst.ine with alkaline plumbite in the presence of a suitable reducing agent, such as arsenite or stannite.
The observation that more than 75 per cent of lead sulfide is produced under certain circumstances from cystine can be ex- plained by assuming a partial reduction of the disulfide linkage by the organic moiety of the cystine molecule or by added reducing substances. This effect is particularly noticeable in the presence of salicylaldehyde (see below), when over 90 per cent of the sulfur appears as lead sulfide.
Rate of Elimination of Nitrogen and Sulfur.-A series of measure- ments was made on the rate of liberation of ammonia at the boiling temperature, employing 0.025 M solutions of cystine in 1 N sodium hydroxide and in suspensions of calcium hydroxide, with and without the addition of lead oxide or acetate. The operation was carried out in a current of hydrogen, thereby excluding air and carrying off the ammonia as fast as it formed. The results of typical experiments are shown in Fig. 1. These confirm the observation of Gortner and Hoffman (1) that deamination is much more rapid with calcium hydroxide than with sodium hydroxide. They also indicate that in both cases the rate is increased (though to different extents) by the addition of lead hydroxide, as has been demonstrated by Andrews (2) for the case of sodium hydroxide.
3 We have not attempted to prove the formation of sodium thiosulfate by actual isolation, but have been satisfied to observe the amounts of sulfur, sulfur dioxide, and sulfuric acid formed on acidifying the filtrate from the lead sulfide.
The deamination of cysteine (Fig. 2) by boiling alkaline solu- tions takes place with considerably lower velocities than those observed with cystine under similar conditions, an effect, recently recorded by Andrews (2), which will be discussed from a theo- retical standpoint in a later communication. The slight augmenta- tion of the initial velocity of ammonia formation may be ascribed to the alkaline decomposition of some of the cystine before the
Hours
FIG. 1. Curve I, 0.025 M cystine in boiling 1 N sodium hydroxide; Curve II, 0.025 M cystine in a boiling 5 per cent suspension of lime; Curve III, 0.025 M cystine + 0.073 M litharge in boiling 1 N sodium hydroxide; Curve IV, 0.025 M cystine + 0.1 M litharge in a boiling 5 per cent suspension of lime.
reduction by stannite is complete, or of cystine present in the sample of cysteine hydrochloride employed. Of particular interest is the character of the curve depicting the elimination of ammonia from cysteine with a boiling suspension of lime; the reaction in- creases in velocity during its progress, furnishing an S-shaped type of curve resembling those of autocatalytic reactions.
Addition of salicylaldehyde to the sodium hydroxide greatly increases the rate of deamination (Fig. 3). The nature of the
reactionleadingto this enhanced reactivity remains to be explained; it appears to be due to the presence of the aldehydic group since, as will be shown below, analogous effects can be induced by the addition of other carbonyl compounds to the reaction mixture. Salicylaldehyde exerts at least two interesting effects on the decom-
FIQ. 2. Curve I, 0.025 M cystine f 0.05 M sodium stannite in boiling 1 N
sodium hydroxide; Curve II, 0.05 M cysteine in boiling 1 N sodium hydroxide; Curve III, 0.05 M oysteine in a boiling 5 per cent suspension of lime; Curve IV, 0.05 M cysteine + 0.073 M litharge in boiling 1 N sodium hydroxide; Curve V, 0.05 M cysteine + 0.073 M litharge in a boiling 5 per cent suspension of lime.
position of cystine, apart from increasing the velocity of ammonia formation and combining with the resulting pyruvic acid to form a yellow product. Thus, it permits the liberation of all of the nitrogen as ammonia, apparently inhibiting side reactions wherein the amino group rearranges into more stable forms of combination.
It also reacts with cystine in an acid medium, yielding products from which hydrogen sulfide is readily liberated by warming, even in the absence of lead acetate.
In all of the experiments on the elimination of ammonia at the boiling temperature, except those with mixtures containing salicyl- aldehyde, the formation of ammonia proceeds very much more slowly towards the end of the reaction. The level at which the curves become nearly horizontal depends roughly upon the initial
0 //ours 2
FIG. 3. Curve I, 0.025 M cystine + 0.1 M salicylaldehyde in boiling 1 N sodium hydroxide; Curve II, 0.05 M cysteine + 0.1 M salicylaldehyde in boiling 1 N sodium hydroxide.
velocity, being the highest with the more rapid reactions. This behavior may be regarded as evidence of the formation of second- ary decomposition products which break down more slowly than the original cystine at rates apparently uninfluenced by the pres- ence of lime or of lead oxide.
Attempts to correlate the elimination of the sulfur with that of the nitrogen at the boiling temperature were ineffectual owing, on the one hand, to the high speed of the reaction in the presence of plumbite, and on the other, to the loss of hydrogen sulfide from
the boiling alkaline solutions containing no lead. The indications were, however, that the sulfur is split off at rates at least equal to those at which ammonia is evolved. The decomposition was accordingly studied at 25”, at which temperature the formation of lead sulfide proceeds with measurable velocity. As can be seen in Fig. 4, the formation of lead sulfide follows a course not unlike that of an autocatalytic reaction; since the low initial velocity is
91
FIG. 4. Curve I, 0.025 M cystine + 0.073 M litharge in 1 N sodium hydrox- ide at 25”; Curve II, 0.025 M cystine + 0.073 M litharge -I- 0.144 M sodium pyruvate in 1 N sodium hydroxide at 25”; Curve III, 0.025 M cystine + 0.073 M litharge + 0.7 M ammonia in 1 N sodium hydroxide at 25”; Curve IV, 0.025 ti cystine + 0.073 M litharge + 0.13 M sodium p-hydrazinobenzoate in 1 N sodium hydroxide at 25”.
maintained without increase in the presence of sodium p-hydra- xinobenzoate and the maximum velocity may be induced by the addition of sodium pyruvate, it appears highly probable that the accelerating agent is the pyruvate formed in the reaction. The ammonia produced can have little effect on the velocity, since in- clusion of 14 equivalents of ammonia in the mixture only slightly depresses the rate of reaction, the “autocatalytic” character of
which remains unimpaired. An effect similar to that of sodium pyruvate can also be induced by the addition of benxaldehyde, and even more markedly by salicylaldehyde (Fig. 5). Formaldehyde has a small but appreciable accelerating effect on the reaction in its initial stages but appears to suppress the subsequent increase in rate of precipitation of lead sulfide.
FIG. 5. Curve I, 0.025 M cystine + 0.073 M litharge + 0.05 M benzaldehyde in 1 N sodium hydroxide at 25”; Curve II, 0.025 M cystine + 0.073 M litharge. -I- 0.05 M salicylaldehyde in 1 N sodium hydroxide at 25”; Curve 111, 0.025 M cystine + 0.073 M litharge + 0.25 M formaldehyde in 1 N sodium hydroxide at 25”.
The deamination experiments carried out at 25” differ from those hitherto considered in that the ammonia originally produced remained in the reaction mixture until decomposition was checked by the addition of acid. During the early stages of the reaction the amount of ammonia liberated corresponds closely to the amount of lead sulfide precipitated; later, the quantity present becomes stationary at a level below that reached by the sulfide, and in most cases subsequently falls (Fig. 6). This points to the occurrence of a recombination of ammonia with some decomposi-
tion product of the cystine. The resultant product is apparently decomposed by alkali at a very slow rate, which is not appreciable at 25” and just measurable at 100”. Pyruvic acid appears to play only a minor role in this reabsorption of ammonia, since the addi- tion of sodium pyruvate has little effect on the ratio of nitrogen to sulfur mobilized, the reaction proceeding from the outset at its maximum velocity with respect to both elements.
90 .
FIG. 6. Curve I, PbS from 0.025 M cystine + 0.073 ~litharge in 1 N sodium hydroxide; Curve II, NH3 from 0.025 M cystine + 0.073 M litharge in 1 N
sodium hydroxide; Curve III, PbS from 0.025 M cystine + 0.073 M litharge + 0.144 M sodium pyruvate in 1 N sodium hydroxide; Curve IV, NH, from 0.025 M cystine + 0.073 M litharge -j- 0.144 M sodium pyruvate in 1 N sodium hydroxide.
In the course of experiments on the action of alkali alone at 25” the unexpected observation was made that during the first stage of the decomposition none of the sulfur appears as sulfide ion. If at any time during the first 2 to 3 days lead acetate or sodium plumbite solution be added, a perfectly clear solution results which, however, soon becomes turbid. If this clear solution be acidified with acetic acid, an amorphous, reddish brown precipitate sepa-
rates. This can be collected by centrifuging; on treatment with alkali it rapidly turns black owing to the formation of lead sulfide, the alkaline solution being colored light yellow by a soluble organic compound. A similar change occurs more slowly in the absence of alkali. It has so far been found impracticable to study this compound, owing to its instability, and no attempt will here be made to offer an explanation. It is of interest, however, to note that the rate at which the sulfur is thus mobilized in cystine by 1 N sodium hydroxide alone is nearly equivalent to that at which ammonia is liberated in the same solution.
As in the other cases studied, reduction of the disulfide linkage by alkaline stannite causes a pronounced decrease in .velocity of decomposition.
EXPERIMENTAL.
Pyruvic Acid p-Carboxyphenylhydrazone.
From Cystine.-A mixture of 8 gm. of lead acetate in 300 cc. of water, 200 cc. of I N sodium hydroxide, 3.1 gm. (0.02 mol) of p- hydrazinobenzoic acid, and 2.4 gm. (0.01 mol) of cystine was boiled under reflux for 16 hours. At the end of that time the lead sulfide was removed and the filtrate acidified with 6 cc. of glacial acetic acid, again filtered, treated with 10 cc.of 28 per cent ammonia, and filtered a third time. The clear solution so obtained was evaporated to dryness on the water bath, redissolved in boiling ammonia, filtered, and strongly acidified with hydrochloric acid. The yellow precipitate was collected, well washed, and dried; weight, 2.8 gm. or 63 per cent of the theoretical amount. On recrystallization from 50 cc. of hot 95 per cent ethyl alcohol it separated as light yellow leaflets which melted at 257” (corrected) with evolution of gas but without darkening. On dissolving in dilute ammonia and acidifying the hot solution with acetic acid, it separated slowly in pale orange leaflets which melted with decom- position at 259” (corrected).
From Cysteine.-To a boiling mixture of 8 gm. of lead acetate, 220 cc. of 1 N sodium hydroxide, 275 cc. of water, and 3.1 gm. (0.02 mol) of p-hydrazinobenzoic acid were added 3.3 gm. (0.02 mol based on the analysis) of cysteine hydrochloride. After boiling under reflux for 18 hours the crude product was isolated in the manner above described; the yield was 3.8 gm. or 86 per cent of
the theoretical amount. Recrystallization was effected by acidify- ing with acetic acid a hot solution in dilute ammonia; yield, 3.1 gm. The properties were identical with those of the substance described above.
Analysis.-4.679 mg. gave 0.153 cc. of nitrogen at 27” and 765 mm., whence N = 12.56 per cent. Neutral equivalent = 110.2. CloHloOdNn requires N = 12.61 per cent; neutral equivalent = 111.
In another experiment a solution of 3.1 gm. of p-hydrazino- benzoic acid in 20 cc. of water containing just enough sodium hydroxide for complete solution was added to a boiling suspension of 10 gm. of calcium oxide and 5 gm. of purified litharge in 180 cc. of water. A solution of 3.3 gm. of cysteine hydrochloride in 50 cc. of water was then added. The mixture immediately became pasty, apparently owing to the separation of an insoluble complex. After boiling under reflux for 24 hours this original precipitate had become entirely replaced by lead sulfide, which was filtered off and washed with dilute potassium carbonate solution. The filtrates were concentrated and treated with a slight excess of potassium carbonate; the calcium carbonate was removed and the clear filtrate concentrated to 100 cc. This was allowed to cool and was then acidified with hydrochloric acid. The yellow precipitate was collected and washed successively with dilute hydrochloric acid, water, and a little cold alcohol. The yield was 4.5 gm. of a very pale yellow solid which melted at 251” (corrected) with decomposi- tion. On treating with dilute ammonia, 0.7 gm. of lead oxide remained undissolved. The filtrate was acidified with acetic acid, when typical pale yellow leaflets slowly separated in a yield of 3.0 gm. The product melted at 253” (corrected) and showed a neutral equivalent of 109.5.
From Pyruvic Acid.-The same compound was obtained on adding 1.5 cc. of pyruvic acid to a solution of 3.1 gm. of p-hydrazino- benzoic acid in 50 cc. of water and 85 cc. of 1 N sodium hydroxide and warming at 90-95” for 90 minutes. It melted at 258.5” (corrected).
Pyruvic Acid Phenylhyclrazone.
A mixture of 6 gm. of lead acetate, 85 cc. of 1.0 N sodium hydrox- ide, 5.4 gm. of phenylhydrazine, 2.4 gm. of cystine, and a few drops
of butyl alcohol (to prevent foaming) was boiled for 48 hours under reflux. The odor of benzene developed during the reaction. The lead sulfide was removed and the filtrate acidified with acetic acid. The yellow crystalline precipitate was collected and recrystallized from alcohol, from which the product separated in long, yellow needles which melted with decomposition at 191” (corrected). The yield was 1.4 gm. A further small quantity of the sa.me crystals was secured from the mother liquor, together with a yellow amor- phous by-product and traces of an oil possessing an odor like that of acetophenone.
Behavior of Pyruvic Acid, towar& Nitrous Acid.
Solutions of different amounts of pyruvic acid in 2 cc. of water were treated in the Van Slyke amino nitrogen apparatus according to the standard procedure.
Pyruvic acid.
mg. 10 10 40 40 40
Time of reaction. Apparent amino nitrogen.
5 min. 2 hrs. 1 hr. 2.5 hrs. 4.5 “
ml. 0.208 0.205 0.490 1.050 1.005
Color Test for Pyruvic Acid.
A solution of 1.06 cc. (0.01 mol) of salicylaldehyde and 1 cc. of pyruvic acid (Eastman Kodak Company) in 50 cc. of 2 N sodium hydroxide was warmed on the steam bath for 2 hours. At the end of this time examination of a small sample showed the presence of unchanged salicylaldehyde. A further 1.6 cc. of pyruvic acid was therefore added and heating continued for a half hour longer, when the reaction appeared to be complete. 1 cc. of the resulting solution was diluted to 50 cc. and its color intensity was found to be 85 per cent of that of a 0.01 M potassium dichromate solution, using a Wratten light filter No. 75, whence, on a molar basis, the color intensity is equivalent to that of 2.12 M dichromate. Repe- tition of this experiment with twice the quantity of pyruvic acid gave a color of considerably lower intensity, indicating the prob- ability that side reactions occur and render the test unsuitable for accurate quantitative measurements.
From Cystine.-A solution of 0.600 gm. (0.0025 mol) of cystine and 1.06 cc. (0.01 mol) of salicylaldehyde in 100 cc. of 1 N sodium hydroxide was allowed to stand at 25”. At the end of 20 minutes the color had already changed appreciably, and a 10 mm. layer matched 20.8 mm. of 0.01 M potassium dichromate. At the end of 19 hours 1 cc. of this solution was diluted to 25 cc.; a 20 mm. layer matched 21.4 mm. of 0.01. M dichromate, whence the molar inten- sity was equivalent to 5.35 M dichromate.
A similar mixture after boiling for 4 hours showed a color intensity, on a molar basis, corresponding to 6.175 M dichromate.
From Cysteine.-A similar solution containing a corresponding quantity of cysteine hydrochloride, after being boiled for 5 hours, showed a molar color intensity equal to that of 6.7 M dichromate; in a duplicate experiment, however, a color intensity of only 4.74 was obtained.
From (Y ,&Dibromopropionic Acid.-A mixture of 2.3 gm. (0;Ol mol) of a$-dibromopropionic acid and 1.06 cc. (0.01 mol) of salicylaldehyde in 30 cc. of 2 N sodium hydroxide and 20 cc. of 1 N sodium hydroxide was boiled for 5 hours. The color intensity was equivalent to that of 0.092 M dichromate, corresponding, on the molecular basis, to 0.46 M dichromate.
From p-Chlorolactic Acid.-A mixture of 1.525 gm. (0.01 mol) of crystalline ethyl @-chlorolactate (8) and 1.06 cc. (0.01 mol) of salicylaldehyde in 20 cc. of 2 N sodium hydroxide and 30 cc. of 1 N
sodium hydroxide was boiled under a reflux. Samples were with- drawn at intervals, suitably diluted, and matched against dichro- mate.
After 1 hour the color was equal to that of 0.062 M dichromate; after 2 hours, 0.0825 M; after 4.5 hours, 0.079 M. The color intensity thus passes through a maximum corresponding, on a molar basis, to 0.412 M dichromate.
Determination of Labile Sulfur.
The decomposition of the cystine and cysteine was effected by boiling under reflux a standard sodium hydroxide solution or a suspension of lime, to which had been added more than a sufficient quantity of lead acetate solution, in a current of hydrogen until free of air, then adding the accurately weighed sample, washing it in with freshly boiled distilled water. In order to avoid cont.am-
ination by rubber stoppers, ground glass joints were exclusively employed in the apparatus. Boiling was continued until the formation of lead sulfide had ceased for some hours; the mixture was allowed to cool in an atmosphere of hydrogen.
The lead sulfide was collected, thoroughly washed, and replaced in the reaction vessel, together with freshly boiled water. This suspension was then boiled in a current of hydrogen until all air had been displaced; 20 per cent hydrochloric acid was then added slowly through the dropping funnel, and the hydrogen sulfide thus liberated was absorbed in a solution of pure sodium hydroxide. The alkaline sodium sulfide was oxidized by means of bromine, followed by potassium chlorate and hydrochloric acid, and the resulting sulfate estimated as barium sulfate. In almost all cases it was found impossible entirely to avoid oxidation of lead sulfide during collection and washing, with the result that small amounts of sulfur remained in the decomposing flask. The acid residue was therefore boiled with about one-third of its volume of ethylene chloride; this readily dissolved the sulfur. The oily layer was oxidized with bromine, chlorate, and hydrochloric acid, and the resulting sulfate estimated as barium sulfate.
The filtrate from the lead sulfide was now placed in the appara- tus, concentrated to a small volume in a current of hydrogen, and acidified with hydrochloric acid after restoring the cold water to the reflux condenser. Sulfur dioxide was evolved and collected in sodium hydroxide solution. It was oxidized by bromine and estimated as barium sulfate. Sulfur separated from the residual liquid and deposited to some extent in the lower part of the con- denser. It was likewise dissolved in boiling ethylene chloride and oxidized. The residual acid solutions contained small quantities of sulfate, which also was estimated as the barium salt.
In certain cases the sulfur was collected together with the sulfur dioxide, by taking advantage of its volatility with steam.
Cystine with Lime.--To a boiling mixture of 5 gm. of calcium oxide, 100 cc. of water and 35 cc. of a 10 per cent solution of crystallized lead acetate, there was added 1.0000 gm. of cystine in 17 cc. of 0.75 N sodium hydroxide, followed by three 10 cc. portions of water. After 6 hours boiling, the ammonia evolved amounted to 88.9 per centof the theoretical amount, and after a total of 14 hours this figure had increased to 89.06 per cent. A further 0.55
per cent was obtained on concentrating the filtrate from the lead sulfide, making 89.2 per cent in all.
The distribution of the sulfur was as follows: from lead sulfide, 84.5 per cent; from sulfur dioxide and sulfur, 4.6 per cent; as sul- fate in the residue, 1.4 per cent.
In a similar experiment, 94.3 per cent of the theoretical amount of ammonia was evolved during 29 hours boiling, and 5.2 per cent more was recovered (by Kjeldahl digestion) from the filtrate after estimating the various sulfur fractions. These were distributed as follows: hydrogen sulfide from lead sulfide, 82.9 per cent; sulfur with lead sulfide, 1.9 per cent; sulfur dioxide from filtrate, 1.1 per cent; sulfur from filtrate, 0.3 per cent; sulfuric acid from filtrate, 0.3 per cent.
Cyst&e with Lime with p-Hydrazinobenzoic Acid.-This was carried out similarly to the above, except that 18.1 cc. of 0.5 M
sodium p-hydrazinobenzoate were included. After 37 hours boiling, 100.0 per cent of the calculated amount of ammonia had been evolved. The distribution of the sulfur was as follows: hydrogen sulfide from lead sulfide, 81.9 per cent; sulfur with lead sulfide, 6.0 per cent; sulfur dioxide from filtrate, 2.4 per cent; sulfur from filtrate, 0.9 per cent; sulfuric acid from filtrate, 0.5 per cent.
Cystine with Sodium Hydroxide and Xtannite.-To a boiling solution of 2.0 gm. of crystallized stannous chloride in 100 cc. of 1 N sodium hydroxide was added a solution of 1.0002 gm. of cystine in 15 cc. of 1 N sodium hydroxide, followed by 15 cc. of 1 N
sodium hydroxide. After 45 minutes boiling, a suspension of 4.0 gm. of pure litharge in 25 cc. of 1 N sodium hydroxide was added, and the mixture was heated for 96 hours in boiling water.4 The lead sulfide gave no free sulfur on decomposition with hydrochloric acid; 99.1 per cent of the calculated amount of hydrogen sulfide was obtained. The acid solution contained metallic lead, formed by the reduction of plumbite by the unoxidized excess of stannite. In a parallel experiment, heated for 120 hours, 98.5 per cent of the calculated amount of hydrogen sulfide was obtained; a minute amount of sulfur was observed to have collected in the condenser.
4 This method of heating was necessitated by the violent bumping observed on boiling mixtures of cystine with sodium plumbite solution. Suspensions of lime do not show this tendency.
Cystine with Lime, Sodium Hydroxide, and Arsenite -A solution of 3.0 gm. of arsenious acid and 0.3330 gm. of cystine in 100 cc. of 0.5 N sodium hydroxide was boiled for 45 minutes, whereupon a suspension of 2 gm. of lime and 2 gm. of litharge in 135 cc. of 0.5 N sodium hydroxide was added and washed in with 15 cc. of water. The mixture was boiled in a current of hydrogen for 24 hours, dur- ing which time 96.6 per cent of the calculat,ed quantity of ammonia was evolved. The mixed precipitate was collected and thoroughly washed with dilute alkali in order to remove arsenic (which inter- feres with the liberation of hydrogen sulfide) as completely as possible. On decomposit.ion with hydrochloric acid, 93.4 per cent of the calculated quantity of hydrogen sulfide was liberated and 1.2 per cent of sulfur was recovered from the condenser, giving a total of 94.6 per cent.
Cysteine with Lime.-The cysteine hydrochloride employed contained 19.35 per cent (95.4 per cent of the theoretical amount) of sulfur and 25.5 per cent of chlorine. On titration with iodine, 81 per cent of the sulfur present was found to be in the sulfhydryl form. A sample of this material weighing 0.9324 gm. was decom- posed by boiling for 3 hours with a suspension of 5 gm. of lime in 120 cc. of water containing 2 gm. of lead acetate. The ammonia evolved amounted to 74 per cent of the calculated quantity; 93.2 per cent of the sulfur was recovered as hydrogen sulfide and 0.23 per cent as sulfur dioxide.
Decomposition of Thiosui$ate.-Known quantities (25 cc. of 0.06067 N) of sodium thiosulfate solution were treated in the man- ner described above for the filtrates. The distribution of the sulfur, in a typical experiment, was found to be: as sulfur dioxide,, 46.4 per cent; as sulfur, 48.1 per cent; as sulfuric acid, 1.3 per cent.
Rate of Evolution of Ammonia at the Boiling Temperature.
The apparatus consisted of a 2-necked flask to which were attached a dropping funnel, an inlet tube for hydrogen, and an efficient reflux condenser, from the top of which a tube led to an absorption vessel containing standard acid. All connections were of glass. In carrying out a run the flask was charged with all of the reagents except the cystine or cysteine. The mixture was boiled and all of the air carefully swept out with hydrogen, where- upon the cystine was added to the boiling mixture through the
dropping funnel, a definite quantity of boiling water being em- ployed for washing it down. The current of hydrogen was regu- lated at 2 to 3 bubbles per second and the absorbing acid was removed and replaced at definite intervals.
The total volume of liquid was so adjusted that the cystine was always present in a concentration of 0.025 M and the cysteine in a corresponding concentration, 0.05 M. The concentration of the
TABLE I.
Total Percentages of Theoretically Obtainable Ammonia.
0.025 M cystine + 0.1 M lead acetate in a boiling 5 per cent suspension of lime.
sodium hydroxide was always 1 N, suitable adjustment being made when necessary for the acidity of any added substances and for the hydrochloric acid present in the cysteine hydrochloride. In the experiments with calcium hydroxide an ample excess of lime in suspension was employed.
The experimental results are expressed as total percentages of the theoretically obtainable amounts of ammonia, It may be added that in all cases in which the reaction mixture contained no lead
oxide, hydrogen sulfide began to escape with the ammonia after the reaction had progressed to the extent of about 30 per cent.
Table I relates to experiments the results of which have not been recorded graphically above.
Rate of Formation of Ammonia and Lead Xuljide at 25’.
As in the experiments at the boiling temperature, 0.025 M
solutions of cystine in 1 N sodium hydroxide were employed, 10 cc. portions being placed in stoppered centrifuge tubes and allowed to stand in a thermostat at 25“. These tubes were removed at
intervals and the lead sulfide collected by centrifuging and wash- ing. When it was desired to estimate the free ammonia present, the supernatant liquor was neutralized by the addition of 1 cc. of glacial acetic acid, allowed to stand for 24 hours in the refrigerator, and freed of any cystine that may have crystallized out. An aliquot portion of the clear solution was treated with an excess of solid potassium carbonate and the ammonia deterqined by aeration.
The sulfur in the lead sulfide was estimated in the following manner. The precipitate in the centrifuge tube, after being thoroughly washed with dilute alkali, was covered with an approxi-
mately equal volume of potassium chlorate crystals, then 6 to 8 cc. of a 10 per cent solution of bromine in 20 per cent hydrochloric acid were added. On cautiously agitating the precipitate with a glass rod the lead sulfide decomposed with liberation of sulfur; the latter could be readily oxidized by the addition of 2 cc. of concen- trated hydrochloric acid and gentle warming with agitation. The clear yellow solution was then washed into a beaker with distilled water and evaporated completely to dryness on the water bath. The residue, consisting of lead sulfate and potassium chloride, was dissolved in boiling 0.25 per cent hydrochloric acid and treated with barium chloride in the usual manner.
Table II supplies the records of experiments which do not appear in graphical form.
SUMMARY.
1. The formation of pyruvic acid as an intermediate product in the decomposition of cystine by alkaline plumbite has been con- firmed by the isolation of its p-carboxyphenylhydrazone when the reaction is carried out in the presence of a salt of p-hydrazino- benzoic acid. Cysteine yields the same product under similar conditions.
2. Pyruvic acid condenses in alkaline solution with salicylal- dehyde, producing a characteristic orange color. This same color is gradually developed on bringing together cystine (or cysteine) and salicylaldehyde in alkaline solution, either alone or in the presence of plumbite. Certain other compounds of analogous structure, notably a! ,/3-dibromopropionic acid and p-chlorolactic acid, respond qualitatively in the same way.
3. Cysteine is capable of yielding all of its sulfur in the form of lead sulfide on being subjected to the action of alkaline plumbite. Cystine, on the other hand, normally yields only 75 per cent, the remainder appearing largely as thiosulfate. Higher values are sometimes obtained, apparently through the reducing influence of decomposition products or of added substances. 9
4. In the alkaline decomposition of cystine, ammonia is formed in amounts not more, and frequently less, than the corresponding quantities of sulfide. Recombination of ammonia with decompo- sition products appears to occur, with the formation of substances which are relatively slowly broken up by alkali.
5. The decomposition.of cystine is accelerated by the addition of benzaldehyde, salicylaldehyde, and pyruvic acid. This effect is reflected in the S-shaped velocity curve representing the forma- tion of lead sulfide from cystine and alkaline plumbite. Addition of sodium p-hydrazinobenzoate prevents the increase in velocity, presumably by combining with the pyruvic acid as it forms.
6. Indications have been obtained that when cystine reacts with sodium hydroxide solution alone, an unstable product is primarily formed before sulfide ion makes its appearance.
It is a pleasure to acknowledge the valuable assistance rendered in the experimental work by Dr. Letha Davies Behr.
BIBLIOGRAPHY.
1. Hoffman, W. F., and Gortner, R. A., J. Am. Chem. Sot., 44, 341 (1922). Gortner, R. A., and Hoffman, W. F., J. Biol. Chem., 72, 433 (1927). Gortner, R. A., and Sinclair, W. B., J. Biol. Chem., 83,681 (1929).
2. Andrews, J. C., J. Biol. Chem., 80,191 (1928); 87,681 (1930). 3. Baumann, E., and Preusse, C., 2. physiol. Chem., 6, 309 (1881). Bau-
4. Dewar, J., and Gamgee, A., J. Anat. and Physiol., 6,142 (1870). 5. Harris, C. D., Ber. cheq. Ges., 24,318O (1891). 6. Claisen, L., and Claparede, A., Ber. them. Ges., 14,2472 (1881). 7. Bergmann, M., Miekeley, A., and Kann, E., 2. physiol. Chem., 146,247
(1926). Bergmann, M., and Stather, F., 2. physiol. Chem., 162, 189 (1926). Bergmann, M. and Grafe, K., .Z. physiol. Chem., 187, 183 (1930).
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