Reprinted from THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 109, No. 1, April. 1935

ISOLATION OF MUCOITINSULFURIC ACID FROM CANINE GASTRIC JUICE *

BY S. A. KOMAROV

(From the Department of Physiology, McGill University, Montreal, Canada)

(Received for publication, January 10, 1935)

Within the last few years considerable evidence has been accu- mulated in this laboratory justifying the assumption that a muco- protein, which has been designated “dissolved mucin,” is present among the organic constituents of pure canine gastric juice (Bab- kin, 1929, 1931; Webster, 1930, 1931; Webster and Komarov, 1932). The “dissolved mucin” was considered as distinct from the “visible mucus” which covers the surface of the gastric mucosa. It was demonstrated (Webster and Komarov, 1932) that different samples of filtered gastric juice obtained from different animals and under various conditions of stimulation yield, on treatment with acetone as a basis of procedure, a protein material identical in general properties and elementary composition (average per- centage figures: C 53.75, H 6.97, N 13.84, S 1.29). Some of the sulfur (0.25 per cent) was present in the form of organically com- bined sulfates. The substances did not contain any phosphorus, did not reduce Fehling’s or Benedict’s solutions, but after boiling 26 to 3 hours with 2 N H&SO? or N HCl, there was a reduction equivalent to 12.7 per cent glucose. All the general protein color reactions were positive. The consistency of the elementary com- position of the different preparations was thought to prove that this substance is a body sui generis and a product of the normal secretory activity of the gastric glands. The low percentage of nitrogen, the presence of organically combined sulfates, the ab- sence of phosphorus, and the considerable reduction after hydroly- sis were considered to be sufficient evidence to classify this sub- stance as a mucoprotein.

* A preliminary communication was read at the meeting of the Royal Society of Canada, May 24, 1934.

177

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

178 Mucoitinsulfuric Acid

According to the present day theory concerning the structure of mucoproteins, these substances should be regarded as complex proteins containing as a characteristic prosthetic group a conju- gated sulfuric acid of the type of chondroitin- or mucoitinsulfuric acid (Levene, 1925). Therefore only isolation of this prosthetic group from the products of hydrolysis would be direct and indis- putable evidence for classification of the protein in question as a mucoprotein. In the present investigation we believe that we have secured such evidence with regard to the mucoprotein (“dis- solved mucin”) of gastric juice. Three preparations having all the properties characteristic of Subgroup A of the mucoitinsulfuric acids, according to the classification of Levene, were isolated, two of them directly from the products of alkaline hydrolysis of freshly secreted and filtered gastric juice and one from the mucoprotein described by Webster and Komarov (1932).

EXPERIMENTAL

Preparation of Mucoitinsuljuric Acid Directly from Gastric Juice

Gastric juice was obtained by sham feeding from three healthy dogs with gastric fistula and esophagotomy. At 9 a.m. the stomach was usually empty but nevertheless it was washed out twice with warm water. The first 50 or 100 cc. of secretion ob- tained after sham feeding were discarded. Only water-clear, odorless secretion was used. It was collected every 15 minutes, immediately filtered through cotton wool, and placed in the ice box until all the daily secretion (about 500 to 800 cc.) had been collected. This required from 2 to 3 hours. The collected juice was filtered through Whatman filter paper No. 40, immediately neutralized with 5 N NaOH, concentrated under reduced pressure to one-twentieth of the original volume, and preserved for a few days at room temperature under toluene, until sufficient material was accumulated. Two samples of such material obtained from 1.5 and 2.5 liters of gastric juice were treated separately as follows: After removal of toluene by distillation the final concentrate was hydrolyzed for 3 days at room temperature with NaOH, which was added in the proportion of 2 per cent. The product of hy- drolysis was then precipitated by 3 volumes of 96 per cent alcohol. Next day the precipitate was separated on the centrifuge, and

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. Komarov 179

washed twice with 80 per cent alcohol, and twice with acetone. Acetone was removed in vacua and the precipitate was dissolved in 20 cc. of water; the filtered solution was poured into 200 cc. of glacial acetic acid. A characteristic flocculent precipitate, which appeared instantaneously, was centrifuged, washed twice with 95 per cent acetic acid, and then with 96 per cent alcohol until practically all the acetic acid was removed. At this stage the precipitate, on being stirred in alcohol, tends to form a fine stable suspension. The addition of a few cc. of concentrated solution of NaCl in 80 per cent alcohol caused a good flocculation. The precipitate was then washed twice with 80 per cent alcohol con- taining traces of NaCl, then again with 96 per cent aclohol, with absolute alcohol, and with ether, and finally dried in vacua. This product was not entirely soluble in water. It was extracted twice with 20 cc. of water; the combined extracts were concentrated to 20 cc. and precipitated again with 200 cc. of glacial acetic acid, the precipitate being carefully washed with alcohol and ether. All the residues, except the filtrates from glacial acetic acid precipita- tion, were combined, neutralized, concentrated, and then hydro- lyzed again with 2 per cent NaOH for 2 days, the whole procedure being then repeated once more. A further, smaller yield of a product apparently ident#ical with the first yield was obtained. Both were combined, dried in a vacuum desiccator over sulfuric acid in the presence of soda-lime, and then analyzed. From the first sample of 1.5 liters of gastric juice, 66.8 mg. of a substance (Preparation I), which on analysis was found to be a fairly pure sodium salt of mucoitinsulfuric acid, was obtained. This corre- sponds to a yield of 4.16 mg. per cent of free acid. From another sample of 2.5 liters of gastric juice 65 mg. of a substance (Prepara- tion II) containing 5.6 per cent ash were obtained. Apparently it was a mixture of mucoitin and mucoitinsulfuric acid. The yield of free acid thus was about 2.5 mg. per cent. Difference in the yield and in the composition of the two preparations obviously is due to the fact that during such a long and complicated procedure some uncontrollable slight variations were unavoidable. Varia- tions in the room temperature at the stage of alkaline hydrolysis and in the amount of NaCl added at the stage when washing with alcohol was carried out, should be particularly mentioned in this connection.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

180 Mucoitinsulfuric Acid

Preparation of Mucoitinsulfuric Acid from Mucoprotein of Gastric Juice

Mucoprotein used in this work was isolated from 15.4 liters of gastric juice exactly as in Preparation III, described by Webster and Komarov (1932). The substance had the same general properties and practically the same elementary composition: C 52.68, H 7.00, N 14.02, S (as organically combined sulfates) 0.37 per cent. 9 gm. of this substance, corresponding to 6.8 liters of gastric juice, were dissolved in 80 cc. of 2 per cent NaOH and treated further in the same way as the concentrates of gastric juice described in the previous section. In precipitating the prod- uct of alkaline hydrolysis with alcohol it was necessary to add 2 gm. of sodium chloride in order to cause a good precipitation. The final product was converted into a barium salt by dissolving in 20 cc. of water and adding saturated barium hydroxide solution up to the point at which the reaction just began to turn slightly alka- line to litmus. After centrifuging, the solution was filtered into an equal volume of absolute alcohol. The precipitate was washed on the centrifuge with alcohol in increasing concentrations and finally with absolute alcohol and anhydrous ether, and then dried in a vacuum desiccator over sulfuric acid in the presence of soda- lime. The total yield was 365 mg. When dry the substance was a heavy, snow-white powder. It is described below as Prepara- tion III.

Properties of Xubstances Isolated

All three preparations showed identical general properties. As free acid and sodium salt (Preparations I and II) the substance is very easily soluble in distilled water, in aqueous alkalies, and in dilute mineral acids, giving slightly opalescent solutions. It is soluble in dilute acetic acid, but gives a very characteristic floccu- lent precipitate in 89 per cent or stronger acetic acid. It is in- soluble in 80 per cent or stronger ethyl alcohol in the presence of small amounts of electrolytes; insoluble in acetone and ether. As barium salt (Preparation III) the substance is easily soluble in water and in weak solutions of hydrochloric acid, giving a per- fectly clear solution; insoluble in 50 per cent or stronger ethyl alco- hol; insoluble in acetone and ether. As free acid (Preparations I and II) or as Ba salt (Preparation III) the substance does not re-

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. Komarov 181

duce Fehling’s solution, but shows a strong reduction after pre- liminary hydrolysis with dilute mineral acids. With naphtho- resorcinol and with cy-naphthol it gives positive tests for glucuronic acid (Tollens’ and Goldschmiedt’s tests). It does not give any of the general protein color reactions (negative biuret, xanthoproteic, Millon, Hopkins, lead acetate test for unoxidized sulfur) but gives a strongly positive Molisch test. It contains all the sulfur (nearly the theoretical amount) in the form of organically combined sul- fates. Among the products of alkaline hydrolysis volatile acids were found to be present; the amounts, calculated on the assump- tion that the volatile acid is acetic acid, are very close to the theo- retical. The substance did not contain any phosphorus detectable by the procedure of Fiske and Subbarow (1925) after moist diges- tion with HzS04 + HNOS.

Composition of Substances Isolated

Preparation I-15.4 mg. of substance gave on incineration 3.20 mg. of ash, which was entirely soluble in water, giving a neutral solution. BaClz caused a characteristic BaSOa precipitate; AgN03 in the presence of nitric acid gave only a very slight opal- escence.

5.08 mg. of substance required 0.50 cc. of 0.02 N HzS04 for neu- tralization of NH, (Kjeldahl). A duplicate determination gave identical results.

9.072 mg. of substance gave 3.1 mg. of BaSOa after preliminary hydrolysis with N HCl in a sealed tube at 100” for 6 hours.

0.4064 mg. of substance reduced respectively 1.26, 1.26, and 1.28 cc. of 0.005 N KsFe(CN)E after preliminary hydrolysis with N HCl in a sealed tube at 100” for 6 hours, the Hagedorn-Jensen (1923) method being used.

Calculated. Ash 20.8, N 2.75, S 4.69; reducing power equivalent to 55.4% glucose

Theory for C28Hd10*0N2S2NaI. Ash 27.6, N 2.72, S 6.23

The composition of this substance evidently corresponds well with that of sodium salt of mucoitinsulfuric acid, according to Levene’s theory of its structure.

Preparation 11-20.6 mg. of substance gave on incineration 1.15 mg. of ash. The ash was readily soluble in water, giving a clear

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

182 Mucoitinsulfuric Acid

neutral solution, which yielded a characteristic BaSOa precipitate on addition of Bach in the presence of dilute HCl and also a char- acteristic AgCl precipitate on addition of AgN03 in the presence of dilute HNO,.

5.328 mg. of substance required 0.61 cc. of 0.02 N H&SO4 for neutralization of NH3 (micro-Kjeldahl). A duplicate determina- tion gave identical results.

0.444 mg. of substance reduced respectively 1.38, 1.38, and 1.40 cc. of 0.005 N K3Fe(CN)6 after preliminary hydrolysis with N HCl in a sealed tube at 100” for 6 hours.

7.9 mg. of substance hydrolyzed as in the above analysis gave a heavy precipitate with BaClz; quantitative analysis was lost.

Calculated. Ash 5.6, N 3.12, reduction equivalent to 56.7 glucose I‘ for ash-free substance. N 3.40, reduction equivalent to

60.0 glucose Theory for C28H48029N&. N 2.98

,‘ “ C:~HUOBNZ. “ 3.59

Thus this preparation is apparently a mixture of mucoitin and mucoitinsulfuric acid.

Preparation III-52.7 mg. of substance gave on incineration 17.6 mg. of ash, all of which was insoluble in dilute hydrochloric acid.

20.6 mg. of substance gave on incineration 6.9 mg. of ash, all of which was insoluble in dilute hydrochloric acid. Calculated, ash 33.4, 33.5; S 4.59, 4.61; base 19.65, 19.7 per cent.

For neutralization of NHs (Kjeldahl)

4.9 mg. substance required 0.44 cc. 0.02 N H2SOd 4.9 ‘I ‘I I‘ 0.43 “ 0.02“ “

5.675“ “ “ 0.49 “ 0.02“ “

5.675 “ I1 “ 0.49 “ 0102“ (I Calculated. N 2.46

The carbon and hydrogen were determined by semimicrocom- bustion, this being kindly undertaken by Mr. S. C. Overbaugh, of the Laboratory of Organic Chemistry, McGill University. The filling of the combustion tube was as required for the combustion of substances containing N, S, and Cl. Reported, C 30.6, H 4.25, ash 33.2 per cent.

70.0 mg. of substance were dissolved in 25 cc. of water. The solution was perfectly clear.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. Komarov 183

10 cc., containing 28.0 mg. of substance, were heated with 2 cc. of 5 N HCI in a sealed tube for 10 hours at 100’. In about 1 hour the precipitate of BaS04 began to appear. 9.0 mg. of BaSOa were recovered without addition of BaClz to the product of hydrolysis. Addition of BaClz to the filtrate did not cause any precipitation. Calculated, S 4.41 (in the form of organically combined sulfates).

10 cc. of the solution, containing 28.0 mg. of substance, were heated for 10 hours at 100’ in a sealed tube with 5 cc. of hot satu- rated Ba(OH)2 solution. The contents of the tube were perfectly clear. Soon after the commencement of heating a yellow precipi- tate appeared. The product of hydrolysis was filtered directly into Pregl’s (1930) micro-Kjeldahl apparatus, washed twice wit,h COZ- free water, and 12 cc. of 50 per cent HzS04 then added. Distilla- tion with steam was carried out for 30 minutes, the distillate being passed into an ice-cooled receiver, provided witha guard containing soda-lime. 4.25 cc. of 0.02 N NaOH were required to neutralize volatile acids. The blank was 1.55 cc. of 0.02 N NaOH. The distillate was carefully neutralized with sulfuric acid (slightly acid to litmus, but basic to Congo red) and treated with 2 cc. of 0.02 N

AgS04 until there was no more precipitate; the solution was fil- tered when hot and the precipitate was washed three times with 5 cc. of hot COz-free water. The total volume of ‘the filtrate was about 40 cc. It was again distilled for 30 minutes with 10 cc. of 50 per cent sulfuric acid (freshly opened bottle of Merck’s reagent). 2.84 cc. of 0.02 N NaOH were required to neutralize the volatile acids to phenolphthalein. The blank was 0.60 cc. of 0.02 N NaOH. Calculated, volatile acid (as acetic acid) 9.61 per cent.

0.98 mg. of substance reduced 2.50 cc. of 0.005 N K3Fe(CN)e (Hagedorn-Jensen) after preliminary hydrolysis with N HCl in a sealed tube at 100” for 6 hours. A duplicate determination gave identical results. Calculated, reducing power equivalent to.48.3 glucose.

Found. C 30.6, H 4.25, N 2.46, S 4.60, base 19.7, volatile acids equivalent to 9.61 acetic acid

Theory. C 27.8, H 3.48, N 2.32, S 5.30, base 22.7, acetic acid 9.91

Data obtained on analysis of our Preparation III show that this substance has a composition very close to that required by the theory for barium salt of mucoitinsulfuric acid, as established by Levene.

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

184 Mucoitinsulfuric Acid

DISCUSSION

The method used for isolation of mucoitinsulfuric acid from gas- tric juice was adopted as a result of another study which was carried out on commercial gastric mucin (also with a view to isola- tion of mucoitinsulfuric acid), and which will form the subject of a special communication. By the use of different methods it was possible to obtain from commercial gastric mucin preparations of mucoitinsulfuric acid having all the characteristics of that particu- lar group of mucoitinsulfuric acids which was classified by Levene as Subgroup A. The elementary composition and general proper- ties were practically identical, but the yield varied from 4.2 to 26.0 per cent, depending on the method used. Since the source of commercial gastric mucin as certified by the manufacturers was hog gastric mucosa and the gastric mucin as manufactured is only a by-product in the preparation of commercial pepsin, it was thought that possibly the dissolved mucin of gastric juice is also a derivative of this group of mucoitinsulfuric acids. The distinc- tive peculiarity of this group of conjugated sulfuric acids, as em- phasized by Levene, lies in the solubilities of free acids and their salts. As free acids and as barium salts they are very soluble in water and are precipitated from aqueous solutions only by a large excess of glacial acetic acid, and when thus precipitated the sub- stances come down in the form of very light floccules, in contra- distinction to the substances of the second subgroup. These, under the same conditions, form a heavy gelatinous mass, and only a slight excess of acetic acid is required to produce such a precipitation. Another important distinction of the substances belonging to the second subgroup is that their barium salts are practically insoluble in water. The method outlined in the ex- perimental part of this study is based entirely on these character- istic features.

Our preparations obtained from gastric juice have identical general properties. Unfortunately only one of the preparations was obtained in sufficient quantity for a more complete analysis. However, quantitative estimation of nitrogen and of reducing power after preliminary hydrolysis could be carried out in all cases. These data, when supplemented by a negative biuret test and a negative test for phosphorus as well as by a positive naph-

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. Komarov

thoresorcinol test and a positive test for organically combined sul- fates, are entirely sufficient. for identification. We wish to em- phasize here the importance of quantitative determination of reducing power after preliminary hydrolysis for identification of mucoitinsulfuric acid. In our experience seven different prepara- tions of mucoitinsulfuric acid, identification of which was sup- ported by a complete elementary analysis, had reducing power equivalent on the average to 60.0 per cent. glucose content, with observed variations of from 58.0 to 61.6 per cent; hydrolysis was carried out in sealed tubes with N HCl at 100” for 6 hours, and

TABLE I Analytical Data for Preparations of Mucoitinsulfuric Acid from Gastric

Juice

Nitrogen and sulfur are expressed in per cent; reducing power in per cent of glucose content; yield in mg. per cent of gastric juice used for isolation. Preparations I, II, and III were analyzed as Na salt, free acid, and Ba salt respectively. All the values have been calculated for the free acid.

Preparation No ........... I II III Theory

N .................... 2 95 3 40 s .................... 5.04

Reducing power .... ._, 59.4 60 0 Yield ................. 4 1 25

3.06 5.73 5 40

60.0 4.32

2 98 6.81

determination of sugar by the method of Hagedorn-Jensen, pre- liminary precipitation with zinc sulfate being omitted.

In spite of the fact that our preparations were analyzed as different compounds of mucoitinsulfuric acid-sodium salts as free acid and as barium salt--the results of the analysis show that in all these cases we are dealing with the same substance. Table I represents analytical data calculated for free mucoitinsulfuric acid. The figures for nitrogen, sulfur, and reducing power are identical or within the limits of experimental error.

The striking feature of the more completely studied Preparation III, isolated from the mucoprotein of gastric juice, is its surpris- ingly high degree of purity, which approximates to the best, known preparations of chondroitinsulfuric acid. Sulfur was determined

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

Mucoitinsulfuric Acid

three times; twice in the ash and once after hydrolysis in a sealed tube with N HCl. The first two determinations gave identical results: S 4.60 per cent; the third gave S 4.41 per cent. Volatile acids after hydrolysis with Ba(OH)2 also gave almost the theoreti- cal value for acetic acid. Unfortunately the nature of the amino sugar could not be exactly determined, owing to the lack of ma- terial. However, a few mg. of crystalline material were isolated as hydrochloride from the products of acid hydrolysis in the sam- ples used for the determination of organically combined sulfates. This substance reduced Fehling’s solution, did not melt at 200”, and turned black at 220”, but it was contaminated with some in- organic material. These data might be considered as suggestive that the sugar is probably chitosamine. Another consideration in favor of such a conclusion is that so far chondrosamine has been isolated only from chondromucoids, which occur in the connective tissue of various organs (aorta, tendons, cartilage, sclera) and have never been found in animal fluids, mucous membranes, and their secretions or in any other tissues, except connective tissue (Levene, 1925). Therefore we believe that the substance de- scribed in this communication must be termed mucoitinsulfuric acid in accordance with the classification of Levene, although the direct proof-identification of amino sugar-is at present not available. The importance of identification of amino sugar in this preparation cannot be overestimated. Its indisputable identification as chitosamine would considerably substantiate Levene’s theory of the structure of mucoitinsulfuric acid and of mucoproteins in general. However, even as the question now stands, our data cannot be reconciled with Schmiedeberg’s (1920) view that only chondromucoids contain a carbohydrate complex of the structure of a sulfuric ester of a complex carbohydrate, and that true mucins do not contain sulfuric acid in their carbohydrate compounds. On the other hand, our data are entirely concordant with the postulates of Levene’s theory.

However important the exact nature of the substance described in this paper may be in relation to the theory of mucoproteins, for the main purpose of this investigation it is immaterial, since in any case-whether it is chondroitinsulfuric acid or mucoitinsulfuric acid-the protein containing this carbohydrate complex in a quantity allowing of its isolation in an analytically pure state in

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. Komarov 187

the proportion of 4 per cent of the original material must unques- tionably be regarded as a mucoprotein. In this connection it is very interesting to compare the yield of the substance, when iso- lated directly from the gastric juice and from the mucoprotein obtained from the juice. The yield in the latter case was 4.32 mg. per cent, as compared with 4.1 mg. and 2.5 mg. per cent obtained directly from the gastric juice. This indicates clearly that mucoi- tinsulfuric acid is present in the gastric juice only as an integral part of mucoprotein-in the form of “dissolved mucin”-and not as free mucoitinsulfuric acid which might be liberated from the surface mucus as the result of peptic digestion. Peptic digestion of the surface mucus, however, is very improbable for the reason that the gastric juice used in the investigation was filtered within 15 to 30 minutes after being secreted.

SUMMARY

1. Two preparations, identified as sodium salt of mupoitinsul- furic acid and free mucoitinsulfuric acid respectively, were iso- lated from the products of alkaline hydrolysis of two samples of freshly collected and immediately filtered canine gastric juice.

2. Barium salt of mucoitinsulfuric acid was isolated from the mucoprotein, “dissolved mucin,” of gastric juice.

The writer wishes to express his thanks to Dr. B. P. Babkin who directed this work.

BIBLIOGRAPHY

Babkin, B. P., Am. J. Surg., ‘7, 498 (1929); Canad. Med. Assn. J., 28, 134 (1931).

Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925). Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 136, 46 (1923); 137, 92

(1923).

Levene, P. A., Hexosamines and mucoproteins, London and New York (1925).

Pregl, F., Die quantitative organische Mikroanalyse, Berlin, 3rd edition

(1930). Schmiedeberg, O., Arch. ezp. Path. u. Pharmakol., 87, 1 (1920). Webster, D. R., Tr. Roy. Sot. Canada, sect. 5,24, 199 (1930); sect. 5,26,213

(1931).

Webster, D.‘R., and Komarov, S. A., J. Biol. Chem., 96, 133 (1932).

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

S. A. KomarovACID FROM CANINE GASTRIC JUICEISOLATION OF MUCOITINSULFURIC

1935, 109:177-187.J. Biol. Chem. 

  http://www.jbc.org/content/109/1/177.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/109/1/177.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from