THE MUCOPROTEINS OF THE SNAILS, HELIX ASPERSA AND HELIX POMATIA.

BY P. A. LEVENE.

(From the Laboratories of The Rockefeller Institute for Medical Research.)

(Received for publication, July 13, 1925.)

The problem of the structure of complex proteins containing carbohydrates in their molecule is still a subject of discussion. The fundamental questions which have been debated during the last decades and which have been the subject of many contro- versies are concerned with the nature of the carbohydrate group present in the molecule of these substances. Since the dis- covery of mucoproteins or mucins, as all of them were termed originally, three mutually antagonistic views were advanced. Chronologically, they were the following: All mucoproteins are physical mixtures of a polysaccharide, resembling dextrin, and proteins. The polysaccharide was termed “animal gum” (Land- wehr’). Through the experimental criticism of Hammarsten,” this view was modified by its author and in its final form it as- sumed a chemical union between the animal gum and the pro- tein. The second view, of more recent date, was that the car- bohydrate complexes of all mucoproteins were conjugated sulfuric acids of the type of mucoitin sulfuric acid. The third theory presented the structure of this group of substances in the form of a peptide or glucosidic linking between simple sugars (amino sugars) and proteins.

There were authorities who believed in the existence of repre- sentatives of each one of the three types of substances. The- oretically, every one of the three theories is possible and, therefore, the final decision will depend only on facts. However, the number of complex proteins containing carbohydrates in their molecules is very great and it will require much time and labor to analyze all of them.

1 Landwehr, H. A., Z. physiol. Chem., 1882, vi, 74. 2 Hammarsten, O., Arch. ges. Physiol., 1885, xxxvi, 412.

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A very critical and, for its time, a classical investigation into the question of mucoproteins was carried out by Hammarsten 40 years ago. Hammarsten came to the conclusion that two types of proteins containing carbohydrates in their molecule were obtainable from Helix pomatia. One type is represented by the substances obtainable from the mucus secreted by these animals and by that extracted from the foot of the animal. These mucoproteins are characterized by their elementary composition which does not differ much from that of simple proteins. In distinction from these mucoproteins, he prepared one from the “protein glands” of these animals. The latter substance pos- sessed a peculiarly low nitrogen content varying between 6 and 8 per cent, and readily yielded on extraction with water or better, on extraction with dilute alkali, a polysaccharide which he named “sinistrin.”

No further work on the mucoproteins of these animals was recorded until very recently. Meanwhile the theory of the animal gum seemed to have been entirely discredited, on the one hand, by the failure of Folin in Hammarsten’s laboratory to prepare nitrogen-free polysaccharides from several mucoproteins and, on the other hand, by the successful isolation of mucoitin and chondroitin sulfuric acids from many mucoproteins which was accomplished in the laboratories of Hofmeister and of the present writer, in cooperation with Lopez-Suarez particularly.

In the year 1920, for the first time after the date of Ham- marsten’s publication, reference was made to the carbohydrate complex obtainable from the proteins of Helix pomatia. Schmied- eberg3 in a very comprehensive publication on mucoproteins made a statement that from the proteins of Helix pomatia a nitrogen- free polysaccharide is obtainable which has the structure of a glucosan-pentosan.

Thus it is seen from this brief review that the work on the mucoproteins of Helix pomatia had a great influence on our conception of the structure of mucoproteins in general. Hence, it seemed expedient to test the old conclusions by more modern methods.

Every new work on this problem, of course, has the benefit of the experience of Hammarsten which led to a differentiation

3 Schmiedeberg, O., Arch. exp. Path. u. Pharmakol., 1920, lxxxvii, 44.

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between the mucoproteins derived from the mucus of these animals and the substances obtained from the foot, on the one hand, and that obtained from the protein glands, on the other. It is unfortunate that the task of dissecting the glands of the animals is too difficult to permit its being carried out on a large scale. The present work was done on a larger scale than that of the older investigators; nevertheless the available material was not sufficiently large to permit as accurate an investigation as is required by modern standards.

Notwithstanding the many shortcomings, the results obtained in the course of the present work are sufficient to warrant several very definite conclusions. First, that the mucoprotein of the mucus belongs to the group derived from mucoitin sulfuric acid. A substance having the properties of the latter was isolated in a state free from protein and perfectly intact in every respect save for a loss of some of its sulfuric acid radicle. It gave on partial hydrolysis a disaccharide (mucosin), and after complete hydroly- sis, there were obtained chitosamine and a volatile fatty acid in a proportion equivalent to one acetyl group for each atom of nitrogen. On distillation with hydrochloric acid, it gave furfural. When mucosin was treated in the same way the yield of furfural was equal to that required by the theory for equal pro- portions of glucuronic acid and of chitosamine. Whether the “uranic” acid was actually glucuronic, or galacturonic, or some other acid of this type was as yet not ascertained.

The present substance, like all other mucoitin sulfuric acids, did not reduce Fehling’s solution directly, but did so after hydroly- sis with mineral acids. It gave a positive test for glucuronic acid with naphthoresorcinol.

Very characteristic for the substance is its solubility. Like all other mucoitin sulfuric acids, it is insoluble in all organic solvents including glacial acetic acid. It is insoluble in water and in alkalies, and is soluble in strong mineral acids. In these solutions the substance was dextrorotatory.

On the basis of solubility the present mucoitin sulfuric acid belongs to the subgroup A, of which the substance derived from “funis mucin” is a representative member.

Thus, the general characteristics of the mucoitin sulfuric acid derived from the mucus of the snails are quite clear and they con-

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firm the conclusions expressed in previous publications that mucoproteins secreted by mucous membranes are derivatives of mucoitin sulfuric acid. The interesting point attached to the mucoitin sulfuric acid obtained from the snails is its insolubility in water. Previously, substances of this type were obtained only from certain forms of connective tissue structures. Since the mucoitin sulfuric acid derived from the snails was prepared by a much simpler process than those which led to the soluble forms of mucoitin sulfuric acid, it is suggestive that the soluble mucoitin sulfuric acid is a product of the insoluble form. This suggestion is only tentative.

Somewhat more complex is the problem of the chemical structure of the polysaccharides obtained from the tissues of the snails. It was stated above that Hammarsten was the first to have pointed out that the carbohydrate obtained from the mucoprotein of the foot differed from that prepared from the body of the snail; the first having the properties of the carbohydrate radicle char- acteristic for other mucoproteins, the second being a distinctive substance. Our observations in certain respects are in harmony with those of Hammarsten. The complex carbohydrate prepared by us from the mucoprotein of the foot of the snails resembled, in the main, the analogous substance prepared from the mucus. The substance prepared from the mucoprotein of the foot, how- ever, contained a small proportion of a different polysaccharide, which is more abundant in the bodies of the snails. As was mentioned above, Hammarsten discovered the polysaccharide and referred to it as “sinistrin.” We believe that the material prepared by Hammarsten was purer than ours as we did not attempt the extirpation of the gland with anatomical accuracy. The polysaccharide prepared by us was sparingly soluble in water but readily soluble in strong mineral acids and also to some extent, in alkalies. In the majority of cases the solution was too opaque to permit a very dependable reading of its optical rotation. Whenever it was possible to take the rotation it was found to be to the right, and on a few occasions, the substance was inactive. The optical behavior alone was sufficient to indicate that the substance was not homogeneous. Furthermore, the substance contained small quantities of nitrogen and sulfur, which indicated that it contained a small proportion of mucoitin sulfuric acid.

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On hydrolysis with dilute mineral acids, the substance gave a maximum yield of about 60 per cent of a monosaccharide, cal- culated on the basis of glucose. Besides, on distillation there was obtained between 20 and 30 per cent of acetic acid. The most interesting point in connection with the polysaccharide was the nature of the hexose. This was found to be that of galactose. The configuration of the hexose was suggested first by the phenyl- osazone prepared from it. This consisted of small platelets, was dextrorotatory, showing, with time, a decline in the numerical value of the rotation. This property is peculiar among hexoses only to phenylgalactosazone and to phenylgulosazone. Besides, the melting point of the osazone was 195”C., (uncorrected) which is characteristic for galactosazone. On oxidation with nitric acid, mucic acid was formed, which was identified by its melting point (m.p. 215”C.), by the absence of optical activity, and by its elementary composition.

Thus, the substance seems to be a polysaccharide composed of galactose, or perhaps of acetyl galactose. It is the first of this kind observed in animal tissues. Galactosans, to the knowledge of the author, have been prepared only from plants in the form of gums. Thus the term animal gum may be well applied to this substance.

The question then arises whether this polysaccharide is actually a component of a “glucoprotein,” as Hammarsten suggested, or is a substance which occurs in the tissues of the snails in a free state and is adsorbed to the protein during the precipitation. The latter possibility seems to be suggested by the analytical data on the so called “glucoprotein.” Hammarsten and before him, Eichwald and others have found that this protein contained only 8 per cent of nitrogen. No other mucoprotein is known with a nitrogen content as low as the one just mentioned. It was there- fore attempted to extract the polysaccharide directly from the organs by means of hot water. The method of Pfliiger and Nerking was used for separating the polysaccharide from the protein material. Under these conditions, the same polysac- charide was obtained as that from the so called “glucoprotein” of Hammarsten. It was hydrolyzed into galactose and a volatile acid (acetic). This observation was in a way surprising, as Hammarsten reported the isolation of glycogen from the tissues

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of the snail. In this respect the experience of Hammarsten was at variance with that of Landwehr. Since the identification and the isolation of glycogen is a very simple matter, it is possible that the presence or absence of glycogen is conditioned by the nutritional state of the animals. It was fortunate for us that in the tissues of our animals, glycogen was completely absent, and this fact facilitated the isolation of the galactose polysac- charide. It is possible, however, that still another polysaccharide is present in the tissues. This possibility is suggested because frequently the osazone prepared from the products of hydrolysis was optically inactive and melted at ‘202°C. On the other hand, only mucic acid could, with certainty, be isolated from the prod- ucts of nitric acid oxidation of the polysaccharide. It is un- fortunate that the material was not accessible in quantities to permit a more thorough purification of the polysaccharide and of its products of hydrolysis.

EXPERIMENTAL.

PART I.

The work on Helix aspersa Miiller var. maxima Taylor will be reported first in view of the fact that the material was available in larger quantities and therefore permitted more detailed analysis.

Mucoprotein from the Mucus.-This material was collected according to the suggestion of Hammarsten. The shell of the animal was opened and by rubbing the animal with a glass rod it was caused to secrete the mucus. The fresh secretion was transferred into 95 per cent alcohol and kept until a quantity sufficient for work (about 500 gm. of the moist mucoprotein) was collected. In order to facilitate the precipitation of the muco- protein, the alcohol was acidulated with acetic acid. The muco- protein was then separated from the alcohol in part by passing through cheese-cloth, in part by filtration. The precipi- tate was transferred into a fresh portion of alcohol which was refluxed for 2 to 3 hours. The mucoprotein was then freed from the alcohol at first by filtration and subsequently by passing through a hydraulic press. The fairly dry cake was minced in a meat chopper. This material contained a considerable quantity of lime salts and, in order to remove the latter, it was suspended

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in large quantities of hot water containing 1 or 2 per cent of acetic acid and the mixture was turbinated. The water was renewed every 2 hours and the extraction continued until the suspended material on incineration left only a very small mineral residue. The suspended material was finally washed with pure water, again filtered, and pas&d through a hydraulic press.

Mucoitin Sulfuric Acid.

The mucoitin sulfuric acids can be prepared from this material in several ways. Each has its own advantages and the choice of the method should be determined by the purpose for which the material is needed.

First Process.-By this process, the mucoitin sulfuric acid is obtained in the most unaltered state. About 500 gm. of the mucoprotein, still moist, are taken up in 1000 cc. of a 5 per cent solution of sodium hydroxide and the mixture is placed in a shaking machine. After a short interval the mucoprotein is dissolved into a homogeneous viscous fluid. The shaking is continued for 48 hours. At the end of that time the viscous fluid is transferred into twice its volume of 95 per cent alcohol. A precipitate is then formed which is removed by centrifugaliza- tion. It is washed repeatedly with 95 per cent alcohol. The precipitate is then suspended in 800 cc. of 5 per cent aqueous sodium hydroxide and again shaken for 24 hours. At this phase the sodium salt of the mucoitin sulfuric acid appears in the main in the form of a suspension. At the end of the 24 hours the mixture is transferred into twice its volume of 95 per cent alcohol. The precipitate is separated from the fluid by centrifugalization. This material is practically insoluble in water. It has a gelatinous character and still gives a positive biuret test, which undoubtedly is due to the soluble protein which adheres mechanically to the mucoitin sulfuric acid. In order to remove the traces of the adhering soluble protein, the material is suspended in 5 per cent aqueous sodium hydroxide and placed in the shaking machine for an hour. After that time the mucoitin sulfuric acid is sedi- mented by centrifugalization. The alkali is replaced by distilled water and the suspension again is placed in a shaking machine. The latter operation is repeated as long as the mucoitin sulfuric acid gives a positive biuret test. The final product is biuret-

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free but still contains some calcium. In order to remove the latter, hydrochloric acid is added very cautiously to a suspension of the mucoitin sulfuric acid in water until the mixture reacts acid to Congo red. The suspension is then placed in a shaking machine for about 4 hour. The dilute hydrochloric acid is replaced by distilled water and the shaking with fresh portions of distilled water is repeated until the washings no longer contain chlorine ions. The final product is dried by treatment with alcohol and ether. The average yield from 500 gm. of moist mucoprotein is from 15 to 18 gm. of mucoitin sulfuric acid.

Second Process.-This process is much less laborious and time- consuming but has the disadvantage that it leads to a smaller yield and to a product which contains less sulfur than the sub- stance obtainable by the first process.

The mucoprotein is taken up in twice its weight of 5 per cent solution of sodium hydroxide and placed on a water bath for 3 hours. The clear solution is then cooled and neutralized with acetic acid. A precipitate consisting chiefly of protein material is formed and removed by centrifugalization. From the super- natant liquid the mucoitin sulfuric acid is precipitated by means of basic lead acetate and ammonia. This precipitate is freed from supernatant liquid by centrifugalization. The precipitate is washed several times with water and is then taken up in enough concentrated hydrochloric acid to convert all the lead into its chloride. The lead chloride is removed by centrifugalization and the supernatant liquid is poured into a large excess of glacial acetic acid. The mucoitin sulfuric acid settles out in the form of a flocculent precipitate. This again is freed from the super- natant liquid by centrifugalization. It is taken up in a little water and precipitated by means of alcohol. By this process protein-free mucoitin sulfuric acid can be prepared in about 48 hours. The maximum yield obtainable by this process was 10 gm. out of 500 gm. of moist mucoprotein.

A substance which as regards its sulfur content occupies a position intermediate between the other two is obtained when the lead salt is suspended in water containing barium acetate and a stream of hydrogen sulfide gas is passed through the mixture. The filtrate is then concentrated to a very small volume and is poured into a large excess of glacial acetic acid. The product

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obtained in this way is taken up in water and reprecipitated with alcohol.

Properties of the Mucoitin Sulfuric Acid.-As was already stated by each one of the above methods, a biuret-free substance is obtainable. It is very little soluble in water, in aqueous alka- lies, or in dilute mineral acids. It is soluble in concentrated mineral acids giving a slightly opalescent solution. The substance does not reduce Fehling’s solution but shows a strong reduction after preliminary hydrolysis. The substance gives with naphtho- resorcinol a very strong test for glucuronic acid. The best samples prepared by the first process contained only half the theoretical amount of sulfuric acid; on the other hand, aft.er reflusing for 1 hour over a free flame with a solution of 20 per cent of oxalic acid, a product is obtained which still contains 1 per cent of sulfur.

Composition of the Various Samples of Mucoitin Sulfuric Acid.

Sample I prepared by the first process: 0.1000 gm. substance required for neutralization (Kjeldahl) 1.90 cc.

0.1 N acid. N 2.66. 0.2048 gm. substance: 0.0350 gm. BaSOI. S 2.34.

Sample II prepared by the second process: 0.0924 gm. substance: 0.1370 gm. CO2 and 0.0468 gm. H20. C 40.43,

H 5.66. 0.1626 gm. substance neutralized (Kjeldahl) 3.0 cc. of 0.1 N acid. N

2.58. 0.1635 gm. substance: 0.0136 gm. BaSOI. S 1.14.

Sample III prepared according to the third process: 0.1000 gm. substance neutralized (Kjeldahl) 3.50 cc. of 0.1 N acid.

N490. 0.2590 gm. substance: 0.0312 gm. BaSOI. S 1.65.

C28H18029N2S2. Calculated. C 35.72, H5.18, N 2.99, S 6.82. Found. Sample I. “ 2.66, “ 2.34.

“ II III.

“ 40.43, “ 5.66, “ 2.58, “ 1.14. “ “ 4.90, “ 1.65.

Mucosin.

It was pointed out in previous publications on mucoitin sul- furic acids that mucosin was much less stable than chondrosin, and that great caution must be exercised not to permit the hydroly- sis to pass beyond the stage of mucosin formation. To avoid the latter danger, it was attempted to hydrolyze the mucoitin

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sulfuric acid by means of oxalic acid. 10 gm. of the substance were taken up in 200 cc. of a 20 per cent solution of oxalic acid and refluxed over a free flame for 1 hour. At the end of that time the heavy floccules of the mucoitin sulfuric acid disappeared leaving only a fine white precipitate consisting, in the main, of calcium oxalate. The solution was practically colorless. It was freed from the sediment by centrifugalization and was poured into 5 volumes of a mixture consisting of equal parts of alcohol and ether.

In this manner a substance was obtained which was more soluble than the original substance. The substance had 50 per cent of its nitrogen in the form of amino nitrogen and still con- tained 1 per cent of sulfur. The yield of the substance was 5 gm. from 10 gm. of the original material. These 5 gm. were then dissolved in 100 cc. of 10 per cent hydrochloric acid, and refluxed for 4 hour on a boiling water bath. The solution was then con- centrated under reduced pressure to 5 cc. and poured into 600 cc. of a mixture of equal parts of alcohol and ether. The material obtained after this operation differed only little from the original. The proportion of amino to total nitrogen had increased to about 60 per cent. The yield from 5 gm. was 4.8 gm. This material was then dissolved in 50 cc. of 20 per cent hydrochloric acid and treated as before; 1.2 gm. of mucosin were obtained. The sub- stance was slightly colored and could not be used for optical measurement. 0.050 gm. of the substance had the reducing power of 0.027 gm. of glucose.

The substance contained 4.69 per cent of total nitrogen and 4.02 per cent of amino nitrogen. 0.400 gm. of the substance gave on distillation 0.065 gm. of phloroglucide of furfural, which corresponds to 50 per cent of glucuronic acid.

Thus the substance had the properties of mucosin but was as yet crude. Unfortunately, further purification was impossible because of the lack of material.

Chitosamine.

5 gm. were taken up in 25 cc. of 20 per cent hydrochloric acid to which 2 gm. of barium chloride and 1 gm. of stannous chloride had been added and refluxed over a free flame. The solution was then freed from barium and from tin and concentrated under

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reduced pressure. Chitosamine hydrochloride crystallized out. The substance turned dark above 2OO”C., but had no melting point. It analyzed as follows:

No. 144. 0.0100 gm. substance: 1.20 cc. nitrogen gas (Van Slyke). CGH130bN.HCl. Calculated. N 6.51.

Found. “ 6.73.

The optical rotation of the substance was

Imtial. Equilibrium.

[al to _ $0.98” x 100 D-

1x1 = $98” D

[(yl 20 = t0.76” X 100 1x1

= +76”

Furjural.

The distillation of furfural was conducted under the usual conditions. The yield of the phloroglucide was much below that required by theory. One of the reasons, however, might have been the fact that only small quantities of material were available for these experiments. On the other hand, from mucosin, the yield of the phloroglucide of furfural agreed with that required by the theory. Thus, 0.5000 gm. of the crude product yielded 0.035 gm. of phloroglucide of furfural, which corresponds to 0.100 gm. or 20 per cent of glucuronic acid, whereas the theory requires 40 per cent.

Estimation of the Volatile Fatty Acids.

The volatile fatty acids were estimated in two ways. In one case, the material was suspended in an excess of barium hydroxide and refluxed over a free flame in an apparatus protected against the absorption of carbon dioxide from the air. The operation was continued for 4 hours at the end of which time it was inter- rupted, and the material allowed to cool. The mixture was then made acid to Congo red by means of sulfuric acid and distilled. The distillate was received in 0.1 N alkali protected against the absorption of carbon dioxide from the air.

Either 1 or 0.500 gm. of the material was used for distillation. Calculated for acetic acid, the yields varied between 10 and 15 per cent. The yield required by theory for free chondroitin sulfuric acid is 12.6 per cent. The small amount of available

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material did not permit the isolation and the identification of the acid.

Polysaccharide from the Mucoproteins of the Foot of the Snails.

From the foot of the snails the mucoprotein was extracted according to the directions of Hammarsten. The minced organs were extracted with a 0.1 per cent solution of sodium hydroxide, the solution centrifugalized, and the mucoprotein precipitated from the supernatant liquid by means of acetic acid. The precipi- tate thus formed was redissolved and reprecipitated several times. The final product contained 14 per cent of nitrogen. The final precipitate of the mucoprotein was refluxed with 95 per cent alcohol for several hours then taken up in alkali, and further treated exactly according to the second process described above and the product obtained from it was undoubtedly a mixture of mucoitin sulfuric acid with the polysaccharide. The proportion of the former was undoubtedly very large. This is quite natural since the dissecting of the foot from the body was not carried out with anatomical accuracy. The composition of one of the samples obtained by this process was the following.

C 40.39, H 6.19, N 3.58, S 0.48.

The substance gave a negative biuret test and with naphtho- resorcinol a strong test for glucuronic acid.

Polysaccharide from the So Called L‘G’lucoprotein.”

The so called “glucoprotein” was prepared from the bodies of the snails in exactly the same manner as the mucoprotein from the foot. It contained 12 per cent of nitrogen. The carbohy- drate was isolated by the second process employed for the prep- aration of mucoitin sulfuric acid from the mucoprotein of the mucus. The final product obtained in this manner was a slightly grayish white powder. It was never completely soluble in water. The solution was practically always opaque. The substance was completely soluble in hydrochloric acid of specific gravity 1.19 and from this solution it is precipitated by means of alcohol. In hydrochloric acid solution the substance is slightly dextro- rotatory. Some samples appeared entirely inactive. The sub-

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stance always contained small proportions of nitrogen and sulfur, though the biuret test was apparently negative. No perceptible test for glucuronic acid could be obtained with naphthoresorcinol; also with orcinol the test was negative. The yield of furfural on distillation was negligible. The small proportions of nitrogen and sulfur (when present) were undoubtedly due to insignificant admixtures of mucoitin sulfuric acid. The substance gave with iodine a negative test for either starch or dextrin.

Samples were obtained containing as little as 0.3 to 0.6 per cent of nitrogen and contained no sulfur.

The sample which was used for the experiments described below had the following composition. The purer samples were too small for further work.

Sample No. 598. C 42.61, H 6.38, N 0.98, S 0.67.

Hydrolysis of the Polysaccharide.

1 gm. of the substance was hydrolyzed by refluxing over a free flame with 100 cc. of 4 per cent sulfuric acid. The resulting perfectly colorless solution was dextrorotatory ([cx] = +0.32”) and reduced Fehling’s solution equivalent to 0.586 gm. of glucose.

From this solution an osazone was obtained which crystallized from pyridine and alcohol. It crystallized in bright platelets melting at 197’ (uncorrected).

The rotation of the osazone in pyridine and alcohol solution (c = 1 per cent; 1 = 50mm.).

Initial. Equilibrium. a”,” = +0.35” a”,” = +0.16”

The rotation and the direction of the mutarotation of the substance suggested that it was a phenylgalactosazone.

From samples which contained more impurities the osazone was inactive and in such cases, the melting point was somewhat higher; namely, 202°C.

Nitric Acid Oxidation.

3 gm. of the polysaccharide were hydrolyzed by refluxing for 8 hours over a free flame in 150 cc. of 10 per cent nitric acid. The solution was then concentrated to 30 cc., an equal volume of

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nitric acid of specific gravity 1.4 was added, and the solution was allowed to stand overnight and then rapidly oxidized. From this material 0.500 gm. of mucic acid was obtained. The sub- stance was optically inactive, melted at 215”C., and had the following composition.

0.1000 gm. substance : 0.1252 gm. CO2 and 0.0442 gm. HZO. Cd%& Calculated. C 34.22, H 4.80.

Found. “ 34.14, “ 4.95.

Acetyl Estimation.

1 gm. of the material was used for acetyl estimation by the barium hydroxide hydrolysis and subsequent distillation of the hydrolysate acidulated with sulfuric acid as described in an earlier section of this paper. The yield was 0.200 gm. of acetic acid which was identified as the silver salt. For analysis it was re- crystallized from water.

0.050 gm. substance: 0.0324 gm. Ag. C2H302Ag. Calculated. Ag 64.31.

Found. “ 64.80.

Polysaccharide Obtained Directly from the Bodies of the Snails.

The initial stages of the isolation of this polysaccharide were those recommended .by Pfliiger and Nerking for the isolation of glycogen. The minced material was allowed to stand on the boil- ing water bath for 3 hours with 2 volumes of hot water. The mix- ture was thenmade to contain 3 per cent of pot,assium hydroxide and 8 per cent of potassium iodide and to the mixture alcohol was added as long as it continued to produce precipitation. The precipitate was collected by centrifugalizat,ion and was washed several times with a 50 per cent solution of alcohol containing 3 per cent of alkali and 8 per cent of potassium iodide.

Further purification was very tedious. The crude material was dissolved in hydrochloric acid of specific gravity 1.19, care being taken to avoid warming the solution. The solution (cooled in an ice and salt mixture) was centrifugalized and the super- natant liquid was poured into alcohol. The precipitate was then centrifugalized and washed with alcohol until all the free mineral acid was removed. The precipitate was then suspended

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in water and reprecipitated by alcohol. In this manner the still adhering traces of hydrochloric acid were removed completely. The product obtained in this manner was a mixture of the new polysaccharide and mucoitin sulfuric acid.

For the separation of the two, advantage is taken of the differ- ences in their solubilities. The mucoitin sulfuric acid is the more insoluble product, and the polysaccharide is more soluble. The product just described was taken up in water and centrifu- galized. From the supernatant liquid, the polysaccharide was precipitated by means of alcohol. The insoluble fractions could be purified by taking up in a little hydrochloric acid so that only part went into solution. The insoluble part contained 3.15 per cent of nitrogen and 1.5 per cent of sulfur.

The polysaccharide was again dissolved in water and centrifugal- ized, and the supernatant liquid was precipitated with alcohol.

The final product had the same properties as the polysaccharide obtained from the so called “glucoprotein.” It also contained a small admixture of mucoitin sulfuric acid which could undoubtedly be entirely removed if sufficient material were available.

Sample I. 0.0925 gm. substance required (Kjeldahl) 0.65 cc. 0.1 N alkali. 0.2041 gm. substance: 0.0100 gm. BaSOl (Carius). 0.1052 “ “ * 0.1644 gm. COZ and 0.0600 gm. H?O.

Found. C 42.61, H 6.38, N 0.98, S 0.67.

Purer samples were obtainable when the hot aqueous extracts of the bodies were removed by centrifugalization and only the residues were treated as above. The product obtained in this manner contained practically no sulfur and only minimal propor- tions of nitrogen, as is illustrated by the following two samples.

No. 572. 0.1000 gm. substance required for neutralization (Kje!dahl) 0.20 cc. 0.1 N alkali. N 0.28.

No. 580. 0.1000 gm. substance required for neutralization (Kjeldahl) 0.30 cc. 0.1 N alkali. N 0.42.

1 gni. of the substance containing 0.9 per cent of nitrogen was hydrolyzed with 100 cc. of a 4 per cent solution of sulfuric acid. The solution was dextrorotatory (CX = +0.60” in a 100 mm. tube) and contained 0.6287 gm. of reducing sugar, calculated on the basis of glucose. (The volume of the solution was 87 CC.)

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The osazone prepared from it was optically inactive and had a melting point of 202°C.

No. 614. 0.0626 gm. substance: 8.70 cc. of nitrogen gas at 28°C. and 756.2 mm.

C18H~t0,N,. Calculated. N 15.63. Found. “ 15.69.

Oxidation with Nitric Acid.

3 gm. of the substance were hydrolyzed by refluxing over a free flame for 8 hours with 10 per cent nitric acid and the opera- tion was repeated as in the above described experiment. The yield of crude mucic acid was 0.565 gm. For analysis it was twice re- crystallized out of water. It was optically inactive, had a melting point of 215”C., and had the following composition.

0.1040 gm. substance: 0.1298 gm. CO* and 0.0458 gm. H20. CsH,oOs. Calculated. C 34.22, H 4.80.

Found. “ 34.03, “ 4.91.

It, is possible that the mother liquor from mucic acid contained saccharic acid. The mother liquor from mucic acid was rendered alkaline by means of potassium hydroxide and the solution was then acidulated with acetic acid. On addition of a little alcohol and subsequent scratching the walls of the beaker with a glass rod a crystalline deposit began to form.

The acid potassium salt was then converted into the silver salt. Two fractions were obtained which were analyzed without re- crystallization. One (the major portion) contained 54.0 per cent of silver, the other, 62.0 per cent of silver. The silver salts of saccharic and anhydrosaccharic acids contain 50.95 and 53.2 per cent of silver respectively. Silver oxalate contains 71.0 per cent. Since the mother liquor from the mucic acid did contain some oxalic acid, it is not possible for the present to interpret the significance of these silver salts. If saccharic acid were present, it may be derived from chitosamine, since the polysaccharide still contained a small impurity of mucoitin sulfuric acid.

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P. A. Levene 699

PART II.

Helix pomatia.

This snail was avaiiable in smaller quantities. However, practically all that was observed on the Helix aspersa was noted also on Helix pomatia. The mucoprotein from the mucus con- tained a mucoitin sulfuric acid, the so called “glucoprotein.” However, in this case, no attempt was made to isolate the poly- saccharide directly from the bodies of the snails.

The procedures for isolation and analysis were exactly the same as described in the first part of the paper and will therefore be omitted in this place. Only the analytical results will be reported.

Mucoitin Sulfuric Acid Obtained from Mucoprotein of the Mucks.

The substance was obtained by the two procedures described in the first part.

One of the samples obtained by the first process was converted into barium salt and analyzed as follows:

0.2WO gm. substance neutralized (Kjeldahl) 3.65 cc. 0.1 N acid. N 2.32.

0.2086 gm. substance: 0.0762 gm. BaSO,. S 5.37. 0.2054 “ “ : 0.0294 “ “ Ba 9.01.

C28H,h029N2SZBa2. Calculated. N 2.32, S 5.30, Ba 22.70. Found. “ 2.32, “ 5.37, “ 9.01.

The composition of the products varied from one experiment to the other.

Chitosamine.

The chitosamine obtained from this material had the following composition.

0.020 gm. substance: (Van Slyke) 2.35 cc. nitrogen gas at 25°C. and 769.9 mm.

C,&OsN-HCl. Calculated. N 6.51. Found. “ 6.61.

The optical rotation of the substance in 2.5 per cent hydrochloric acid was

Initial. Equilibrium.

[al; = $0.96” X 100 1x1

= +96” ial~~ _ +0.76’ X 100

D- 1x1

= +76”

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700 Mucoproteins of Snails

The mucoitin sulfuric acid gave on distillation 8 per cent of the phloroglucide of furfural, which corresponds to about 24 per cent of glucuronic acid. The acetyl value corresponded to 15 per cent. The acetic acid was not isolated.

Polysaccharide from the So Called “Glucoprotein.”

The polysaccharide was optically inactive and had the following composition. The purest samples contained 0.3 and 0.4 per cent of nitrogen. The larger sample had the following composition.

C 36.88, H 5.09, N 1.2, S 0.8, and 8 per cent ash.

1 gm. on hydrolysis gave in 84 cc. a dextrorotation of LYE = +0.46’ and reducing power equivalent to 0.560 gm. of glucose. The phenylosazone prepared from it had the appearance of a galactose. It had in pyridine-alcohol solution (c = 1 per cent, I = 50 mm.), an initial rotation of cr, = +O.lO” and an equilib- rium rotation of LYE = +O.lO”.

0.0748 gm. substance: 10.20 cc. nitrogen gas at 26.O”C. and 7.53 mm. &H220aN,. Calculated. N 15.63.

Found. “ 15.45.

1 gm. of the substance was oxidized with nitric acid and gave about 0.200 gm. of mucic acid. The substance was optically inactive and had a melting point of 215°C.

CONCLUSIONS.

1. The carbohydrate radicle isolated from the mucoproteins of the mucus of Helix aspersa and Helix pomatia belongs to the group of mucoitin sulfuric acid. On partial hydrolysis the polysaccharides yield the disaccharide mucosin. From the product of complete hydrolysis there were isolated sulfuric acid, chitosamine, and a volatile fatty acid. On distillation with hydrochloric acid, the mucosin yielded furfural in a quantity required by the theory of the structure of this disaccharide.

2. From the bodies of the snails a substance is obtainable which may be regarded as animal gum and which consists largely of a polygalactose, perhaps of an acetylated polygalactose.

3. From the bodies of the snails by mere extractionwith water and by further treatment by the process of Pfliiger and Nerking, an identical polysaccharide can be isolated.

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P. A. LevenePOMATIA

HELIX ASPERSA AND HELIX THE MUCOPROTEINS OF THE SNAILS,

1925, 65:683-700.J. Biol. Chem. 

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