THE SIGNIFICANCE OF CYANIC ACID IN THE UREA- TJRJXASE SYSTEM.

A COLOR TEST FOR CYANIC ACID.

BY WILLIAM ROBERT FEARON.

(From the Department of Physiology, Trinity College, Dublin, Ireland.)

(Received for publication, July 20, 1926.)

The close chemical relationship between cyanic acid and urea suggests a corresponding biochemical relationship whenever urea is synthesized or decomposed in animal or vegetable tissues.

For this reason the zymolysis of urea was studied by the writer (1) to see if cyanic acid could be detected among the products of the reaction, which would indicate that the enzyme decomposition of urea proceeds along the same lines as the normal decomposition of urea in solution by acids, alkalies, or heat.

The data obtained led to the conclusion that cyanic acid occurs in the urea-urease system. Mack and Villars (6) also claim to have detected cyanic acid under similar conditions, although they do not ascribe any particular significance to its presence.

Quite recently Sumner (ll), working with an extremely active preparation of jack bean urease, has failed to find any indication ,of the formation of cyanic acid during the zymolysis of urea. Sumner has employed the same analytical technique as the writer, and his results raise the question of the accuracy of the present methods of detecting cyanic acid in complex mixtures, and the validity of the assumption that the cyanic acid comes from the action of the enzyme on the substrate, and not from some other source.

Detection of Cyanic Acid in Complex Mixtures.

Three types of method are available: (1) Conversion of the cyanie acid into urea by incubation with an excess of ammonium chloride; the increase in urea being determined by the xanthydrol method of Fosse (3). (2) Precipitation and separation of the

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cyanate as a silver salt. (3) Color reactions of varying degrees of specificity.

The first of these methods is suitable for the detection of cyanic acid in protein solutions free from urease and excess of urea. It has been used by Fosse to demonstrate the formation of cyanates during the oxidation of proteins and amino acids. The method is not applicable to the urea-urease system.

The second method is the most direct. The silver cyanate can be separated from such associated compounds as carbamic acid, carbonic acid, and ammonium bicarbonate, since the silver salt is more resistant to the action of dilute acids. After separation, the cyanate may be converted into ammonia, and estimated.

This method, on account of its simplicity, was applied to the analysis of the urea solutions. There still remains, however, the possibility that other materials associated with the crude enzyme, precipit,ated by the silver nitrate, may yield ammonia during the subsequent treatment, and thus obscure or mislead the conclusion.

A realization of the time consumed, if not wasted, by the appli- cation of inadequate methods to micro analysis made it desirable that the presence of the cyanate, indicated by the silver method, should be confirmed by an independent reaction. The various color tests for cyanic acid were examined to see if any could be applied directly to the urea-urease system. Such a test has not yet been obtained, but a color reaction of considerable delicacy has been devised which will show the presence of cyanate in the silver precipitates obtained from the zymolysis of urea.

Having occasion to test the purity of some specimens of potas- sium cyanate (prepared by the hydrolysis of urethane), a test for cyanide was made by the Schijnbein method.

On addition of about 1 mg. of purified KCNO to 3 ml. of water containing 3 drops of 3 per cent guaiacum tincture and 3 drops of 2 per cent CuS04, an intense blue color developed similar to the color obtained with pure KCN. The method by which the KCNO had been prepared made it most unlikely that any cyanide was present. Nor did it seem likely that cyanide was formed during the progress of the test, since cyanates are very resistant to reduction.

The specimen of KCNO was found to give a negative response to the Prussian blue test, the thiocyanate test, and the picric acid

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test for cyanides as employed by Wailer (12). Gottlieb (4), in discussing the toxicity of cyanates, observes that his preparations “gave no positive reaction for cyanide,” but omits to mention what tests were employed, nor does he comment on the positive result with the guaiacum reagent.

The delicacy of the reaction between the guaiacum and the cyan- ate led to a study of the behavior of the cyanate with the other oxidase tests: aloin, benzidine, phenolphthalin, and a-naphthol, small quantities of a copper salt being added to each test. With phenolphthalin no reaction was observed. With aloin and with a-naphthol the characteristic oxidation colors were obtained. With benzidine, instead of the usual blue color, a dense sepia purple precipitate was obtained. This soon flocculated and separ- ated from the solution. From this observation the following test was devised.

Color Test for Cyanates.

To 5 ml. of water add 2 to 5 drops of a 6 per cent alcoholic solu- .tion of benzidine, and a couple of drops of a 6 per cent solution of copper acetate. Mix. Add the cyanate solution, which should be neutral or faintly acid. A purple color develops and rapidly changes into a sepia precipitate, which soon separates from the solution.

Notes on the Test.

1. The test is definitely positive with 1 to 2 drops of a 0.1 per cent KCNO solution (i.e. less than 0.1 mg. KCNO), but is capable of a much greater degree of delicacy.

2. Acids interfere with the test by decomposing the compound and hydrolyzing the cyanate. Alkalies inhibit the formation of the colored compound.

3. Cyanides, thiocyanates, thiosulfates, iodides, and bromides interfere with the reaction by forming dense colored precipitates with the benzidine-copper reagent. Sulfates interfere by precipitat- ing the benzidine; for this reason a solution of copper acetate is used.

4. The test is capable of a gravimetric application, as the pre- cipitate can be filtered, washed with water and alcohol, and dried. The compound ‘is sparingly soluble in the commoner neutral or-

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ganic solvents. It dissolves with decomposition in pyridine and in acetic acid. It is readily hydrolyzed by dilute mineral acids, the cyanate being converted into the ammonium salt of the acid.

Analysis of the products of the hydrolysis indicates that the compound consists of equimolecular proportions of copper and benzidine with 2 molecules of cyanic acid, which corresponds to the formula: Cu(OCN)2Bz, and belongs to the same class of body as the benzidine iodide compound recently isolated by Spacu (9). The test also resembles the cyanide reaction described by Pertusi and Gastaldi (8). However, the color given by the cyanate is quite distinct from the dark blue given by most of the other reactants, such as this cyanate (2).

By means of the above test is was found possible to detect small quantities of cyanate in presence of urea and ammonium carbon- ate, and also in presence of albumin. But on applying the test to the urea-urease system at the early, middle, and later stages of the zymolysis no positive result could be obtained.

Addition of small quantities of cyanate to the carefully neu- tralized mixture could not be recognized, hence it was concluded that something was present which interfered with the test. This was found to be the enzyme preparation employed. Compara- tively large amounts of cyanate can be added to the soy urease solution before a positive response can be obtained to the cyanate test. The urease is precipitated by, and removes, copper from the mixture, but this is not sufhcient to inhibit the test, which will appear if sufhcient cyanate be added. Addition of more of the urease solution masks the added cyanate.

The benzidine test is not given by silver cyanate, although a faint color will appear after some time. If, however, the mixture of benzidine, copper, and silver cyanate be carefully acidified with ~/lo HCl the liberated cyanic acid will react to produce the color and precipitate. Excess of acid discharges the color, which returns on neutralization unless the acid has been sufficiently strong to destroy the cyanate.

When the benzidine test is applied to the precipitate obtained by the addition of excess of ~/l AgNOs to a ~/lo urea solution, after 10 hours incubation at 12°C. with 0.1 per cent Dunning urease, a positive cyanate reaction is obtained on careful neutral- ization of the mixture. This evidence, although it is indirect,

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W. R. Fearon

supports the belief that the precipitate obtained by the action of silver nitrate on the urea-urease system contains a cyanate.

Origin of Cyanic Acid in Urea-Urease System.

The cyanic acid detected by the previous methods may arise from four sources: (a) An impurity in the urease preparation. (b) A product of the action of the alkali or the urea on the urease. (c) The zymolysis of the urea. (d) The spontaneous dissociation of urea in aqueous solution. The last of these need not be con- sidered further. It can be clearly proved by means of control experiments that the urea undergoes no measurable spontaneous dissociation in solution under the time and temperature conditions of the experiments.

The possibility of the cyanate arising from the urease prepara- tion is indicated by Sumner’s results with the purer preparations. It was on account of this possible source of the cyanate that some of the writer’s earlier experiments were carried out with the enzyme solutions enclosed in collodion sacs (1). This method was not found satisfactory. In some experiments the activity of the en- zyme decreased considerably; whether owing to loss of a coenzyme or a promoter, or owing to the effect of the membrane, was not investigated. In other experiments the enzyme gradually escaped through a sac made permeable by the action of heat and am- monium carbonate. The later series of experiments, in which the silver nitrate was added directly to the enzyme, gave much more consistent results. The fact that control solutions of urease after inactivation by boiling give no cyanate reaction on incubation with urea is not necessarily decisive since the cyanate or its pre- cursor might be hydrolyzed during the boiling.

Note on Puri&ation of Soy Urease.

To aid in the location of the cyanate, a certain amount of work was done on the purification of the soy meal. This work was dis- continued on the appearance of Sumner’s paper describing the preparation of the much more active jack bean urease (10). How- ever, the following notes may be of interest to workers with soy urease.

The finely ground soy meal was repeatedly extracted with

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boiling ethyl ether, or with boiling light petroleum (petroleum ether, b.p. 40-6O”C.), in an improvised Soxhlet extractor. The defatted meal was then, in some experiments, thoroughly ex- tracted with alcohol. Incidentally, none of the various alcoholic extracts contained urea or yielded urea on incubation with excess of ammonium carbonate. The almost pure white product ob- tained by these methods was then freed from excess of protein by a process based on the work of Marshall. The meal was made up in 5 to 10 per cent suspensions and incubated for some hours at 40°C. to favor dispersion. The suspension, freed from the coarser particles, was then acidified by the addition of 20 per cent acetic acid in the proportion of 6 drops of the acid to 100 ml. of the 5 per cent suspension. This caused the formation of a dense curdy precipitate, which gradually subsided. The pale yellow supernatant liquid containing most of the enzyme was filtered off.

While this filtrate may be fractionated in various ways, such as by the addition of N/IO HCl, it is difficult to retain the active urealytic factor. The most successful results were obtained by the use of a 0.5 per cent aqueous solution of safranine (Griibler), which gives a bulky precipitate containing all the urease in the original solution. This was centrifuged, washed, freed from excess of the dye by repeated washings in alcohol, and then de- composed by ~/lo HCl, the liberated safranine being removed by extraction.

The safranine precipitate was inactive towards urea, but on being decomposed the urease was liberated.

Safranine was employed on account of the observation by Marston (7) that the azin and the azonium bases precipitate the proteoclastic enzymes, presumably owing to combination be- tween the enzyme and the nitrogen of the diketopiperazine ring.

It may be questioned if the method is as specific as Marston appears to believe, since safranine gives a precipitate with other protein preparations, such as egg albumin, so that the removal of the urease from the solution may be a general adsorption effect and not a specific reaction. Where an enzyme is associated with a protein “carrier ” any reagent capable of precipitating the carrier is likely to krecipitate the enzyme along with it. For this reason, it would be interesting to know if the azin dyestuffs will

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give precipitates with the purified urease preparations obtained by Sumner. The insoluble urease which he has prepared owes its insolubility, one presumes, to the removal of the appropriate colloid carrier.

Cyanic Acid as the Possible Intermediate in Urea-Urease System.

Both Kay (5) and Sumner (11) have objected to the hypothesis that cyanic acid is the intermediate product in the zymolysis of urea because the stability of cyanic acid in presence of ammonia is such that the enzyme decomposition of urea could not proceed at its normal velocity. This objection the writer fully realizes. If cyanic acid be the true intermediate product in the urea- urease system, some additional mechanism must be present to bring about its rapid hydrolysis.

Urease by itself appears to have no hydrolytic action on potas- sium cyanate. The behavior of the free acid in presence of the enzyme is not to be elucidated by a simple experiment, since the continuous formation of urea from the action of the isocyanate on ammonia, and the continuous decomposition of urea owing to the action of urease introduce a set of variables not easily con- trolled.

That soy urease preparations are not without some action on cyanates is shown by the inhibiting effect on the color test.

In his recent experiments, Sumner has employed a preparation of insoluble jack bean urease, which could be filtered off from the substrate before applying the tests for cyanate. The possibility arises here that in removing the urease any cyanate present may have been removed along with the enzyme.

It would be instructive to know if the results of Sumner would be altered were he to add the silver nitrate reagent directly to the system without any previous separation of the enzyme, and, having washed the precipitate free from ammonia, to decompose the precipitate directly without any previous separation from carbonate and carbamate, a process which may lead to the loss of a small but significant amount of cyanate.

BIBLIOGRAPHY.

1. Fearon, W. R., Biochewz. J., 1923, xvii, 84, 800. 2. Fleming, R., Analyst, 1924, xlix, 275. 3. Fosse, R., cj. Werner, E. A., The chemistry of urea, London, 1923.

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4. Gottlieb, E., Biochem. J., 1926, 1. xx, 5. Kay, H. D., Biochem. J., 1923, xvii, 277, and private communication. 6. Mack, E., and Villars, D. S., J. Am. Chem. Sot., 1923, xiv, 505. 7. Marston, H. R., Biochem. J., 1923, xvii, 851. 8. Pertusi, C., and Gastaldi, E., Chem.-Ztg., 1913, xxxvii, 609. 9. Spacu, G., Z. anal. Chem., 1925, lxvii, 31.

10. Sumner, J. B., Graham, V. A., and Noback, C. V., PTOC. Sot. Ezp. Biol. and Med., 1924, xxi, 551.

11. Sumner, J. B., J. Biol. Chem., 1926, Ixviii, 101. 12. Wailer, A. D., Proc. Roy. Sot. London, Series B, 1910, Ixxxii, 574.

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William Robert FearonCOLOR TEST FOR CYANIC ACID

IN THE UREA-UREASE SYSTEM: A THE SIGNIFICANCE OF CYANIC ACID

1926, 70:785-792.J. Biol. Chem. 

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