June 5 , 1959
CHEMICAL CONSTITUTION OF BUSYCON Mucus [CONTRIBUTION FROM THE
DEPARTMENT O F CHEMISTRY,
2S99
UNIVERSITY OF DELAWARE]
The Structure and Constitution of Mucus Substances. 11. The Chemical Constitution of Busycon Mucus BY VICTORE. SHASHOUA’ AND HAROLD KWART RECEIVED APRIL 8, 1958 The amino acid constitution of the protein portion of Busycon mucus has been elaborated. Ornithine was found to be most unusual among the seventeen amino acids present. Glucosamine and galactosamine have been identified by chromatographic means. The mole ratio of acidic t o basic amino acids was established to be almost three. A relatively large amount of calcium was noted among the several metal ions shown to be present in the purified residue after dialysis and lpophilization. A sulfur constituent has been shown to be entirely in the form of ionic sulfate which is labilized without either acid or base. A structural hypothesis portraying Busycon mucus as a polyhexoseammonium sulfate linked to an acidic protein moiety through calcium ions appears t o correlate all the available data and to be consistent with certain specific tests which have been contrived. T h e possible generality of this type of structure and constitutional similarities between Busycon mucus and other mucus substances are also considered. Synthetic analogs have been prepared in the interest of demonstrating how the structural features account for some of the physical properties of Busycon mucus.
I n a previous paper2 the physical properties of a mucus derived from the large marine snail, Busycon Canaliculatum L., were shown to be due to its polyelectrolyte nature. It was found that this mucus is a high molecular weight polymer (about 3 X lo7) consisting of a polysaccharide (mol. wt. approx. 2.5 X lo6) cornplexed with a protein and that this combination behaved as a swollen, spherical molecule in solution. Our objective in the present undertaking was to determine the chemical structures responsible for this behavior, the mode of linkage of the polysaccharide to the protein, and to compare the mucus derived from this relatively simple source with the various mucus substances which are to be found in a large number of biological fluids. Experimental Preparation of the Samples.-The mucus was extracted by Ronkin’s3 method according t o the procedure outlined in reference 2. The inorganic analyses were carried out on samples extracted with either 0.5 N sodium chloride or 0.5 Jf ammonium acetate. All the preparations were centrifuged in a Sorvall centrifuge t o obtain cell-free extracts before further examination. The analyses on the dry mucus were performed on lyophilized material. KO attempt was made t o remove the last traces of water usually associated with freeze-dried substances. The lyophilized mucus was always obtained as a fluffy white fibrous product. Analyses of the Inorganic Ash.-The ash content was determined by pyrolysis of the dried mucus in silica crucibles a t 900” for 3 hr. in a furnace. The emission spectrum of the ash was analyzed by means of a Bausch and Lomb spectrograph and the elements were identified by their spectral bands and lines according t o published data.’ T h e quantitative analyses for sodium and calcium were performed on solutions of the ash in dilute sulfuric acid (ca. 0.1 N ) using flame photometry. For this a hydrogen-oxygen flame was used in a Beckman D. U. apparatus and spectral lines a t 422 and 589 mp were examined for sodium and calcium, respectively. T h e analytical procedure was similar t o that described by Hawk, Oser and Summersons and the Beckman Flame Photometry manual.‘ In each determination stock solutions of calcium sulfate and sodium sulfate were prepared as controls and compared t o the unknown. The concentration of the standards was adjusted by dilution until two solutions encompassing the unknown were obtained. (1) Taken f r o m the thesis of V. E. Shashoua, submitted in partial fulfillment of the Ph.D. degree a t t h e University of Delaware. ( 2 ) H. Kwart and V. E. Shashoua, THIS JOURNAL, 80, 2230 (1958). (3) R. K. Ronkin, B i d . Bull., 103, 296 (1952). (4) W. R. Brode. “Chemical Spectroscopy,” John Wiley and Sons, Inc., New York, N. Y., 1943. ( 5 ) P. B. Hawk, B. L. Oser and W. H. Summerson. “Practical Physiological Chemistry,” 13th Ed., 1954, p. 853. (6) Beckman Instruction Manual 334-A for Model DU and R Flame Spectrophotometers. Will Corp., Rochester, N. Y.. 1954.
In each case an effort was made to be as similar in concentration t o the test sample as possible. Fractionation of the Mucus.-The following is a typical fractionation procedure, adapted from the method of Bacila and Ronkin? for the gross fractionation of this mucus into its component polysaccharide and protein fragments. A 100-ml. solution of the mucus (about 0.2 g. in 0.5 il’ KaC1 at pH 7 ) was stirred with 100 ml. of chloroform for 16 hr. a t room temperature. The resultant milky product was then centrifuged (10 min. a t 2600 r.p.m.) to give a clear aqueous layer above a clear chloroform layer with the denatured protein as a precipitate a t the interface. Kext, the aqueous layer was isolated and the fractionation continued with a fresh quantity of chloroform; the process was repeated until there was no further precipitate a t the interface (generally four such treatments were sufficient). The combined protein fractions were then filtered, washed with distilled water to remove any adhering carbohydrate fraction and then dialyzed for three days against a continuous flow of distilled water t o remove salts. The product was next lyophilized to give a white fibrous protein (approx. 40 mg.). The carbohydrate fraction remained in the aqueous layer. It was purified by dialysis for three days and lyophilized to give a white fibrous product (approx. 30 mg.). The carbohydrate fraction gave a positive Molisch and a negative biuret test, while the protein fraction gave the opposite results. The biuret and hlolisch tests were carried out according t o the methods described in Hawk, Oser and Summerson.6 A study of the conditions for mucus fractionation by the chloroform technique showed that an electrolyte was necessary in order to cause the separation of the polysaccharide-protein complex; a dialyzed sample gave no precipitate even on prolonged stirring m4th chloroform. Furthermore, the type of salt in the aqueous phase did not appear t o be critical; for example, SHaC1, KHdAc, (NH4)2so4 were as effective as NaC1. It seemed very clear that the protein was denatured by the chloroform only in the presence of an electrolyte. Hydrolysis of the Mucus.-The mucus was hydrolyzed into its component amino acids and carbohydrates with 6 N HCl a t 120’. The method consisted of heating 20-40 mg. of mucus in a sealed tube with 20 ml. of the acid for 48 hr. When the polysaccharide-protein complex was hydrolyzed, a black precipitate was obtained together with a brown solution of the amino acids, whereas the isolated proteiii gave clear yellow solutions and no precipitate. The products of hydrolysis were isolated and purified according t o the method described by Block, Durrum and Zweig.6 The amino acids and carbohydrates were then either dissolved in a 10% solution of 2-propanol in water for paper chromatographic studies or in a citrate buffer for ion-exchange chromatographic analysis. Table I summarizes the results of a study of a number of different hydrolytic conditions. I t was found that the use (7) M. Bacila and R. R. Ronkin, Btol. B u l l , 103, 296 (1952); see also E. A. Kabat, “Blood Group Substances,” Academic Press, New York, N. Y., 1958, p. 130. (8) R. J. Block, E. L. Durrum and A. Zweig. “Paper Chromatography and Paper Electrophoresis,” Academic Press, Inc.. NQW York. N. Y.. 1955.
VICTORE.SHASHOUA AND HAROLD KW.\RT
2!100
( i f sulfuric acid as the reagent for hydrolysis gave poor re.;ults in spite of the fact t h a t the mucus completely dissolved in the acid. Alkaline hydrolysis with barium hydroxide also failed to give good results. The isolated carbohydrate fraction was hydrolyzed with 1 A' HC1 for four hours on a boiling water-bath and examined by paper and ion ruchange chromatography for its cninpoIlents.
1 I ~ T D R O L T OF S I Sb1vcr.s AT 120' '1.4JJ1.14;
Solvrnt
Hydrolysis time (hr.)
Results
24 \-dine 1 JV HnSOA 6 S HrSOi 48 Alaninr l(lLj& Ba(OI-I)r 48 Aspartic acid, glucose, alanine 6 S HCl 24 17 amino acidsband glucosamine c, AT HC1 48 17 amino acids' and glucosamine '4 s HC1" 24 17 amino acids5 '' This analysis was performed on the isolated mucoprotein fraction. See Table I X for details. Paper Chromatographic Analysis.-The qualitative cornposition of the hydrolyzates of the mucus and the isolated mucoprotein fraction was determined by descending paper chromatography. The techniques employed were essentially those described in Block, Durrum and Zweig.8 Three separate solvent systems were used to analyze and compare the unknown mixtures with standards on Whatman #I papers. These mere the phenol-water (4/1) mixture of Consden, Gordon and MartinQ using sodium cyanide to evolve hydrogen cyanide for stabilization of the phenol; the lutidine-collidine-water mixture with 2YG added diethylamine of Dentlo and the recently described solvent of Connell and Haines," n-propanol-phosphate buffer [80/ 20 (0.03 M p H 6.7)]. This latter solvent has the advantage t h a t traces of salts d o not hinder the motion of the amino ,icids. I n the use of the propanol solvent, the papers were first thoroughly soaked with the buffer and dried before placement of the amino acids and the unknown mixtures. -111 the constituents of the solvents were obtained from Eastman Organic Chemicals Co., with the exception of the phenol which was obtained from Mallinkrodt Chemical c o. Table I1 shows the RF values for the standard amino acids using the 1-propanol-phosphate buffer together with the specific colors of the spots obtained immediately after spraying with ninhydrin reagent (0.17,in acetone) and drying (1-2 min. at 80'). These colors present a n additional means of identification of the amino acids. The RF values for the other two solvents used in this work have been published e l ~ e w h e r e . ~ Ion-exchange Chromatography.-The quantitative anal!& of the protein was carried out by ion-exchange chromatography using the techniques of Moore and Stein1* as modified by Hamilton and Xnderson.13 A 10-mg. sample was used for each analysis on a column (I00crn. X 1 cm.) o f Dowcx 50 X 8 resin. T h e acidic and the neutral amino :Icids were determined in a separate experiment from thc basic amino acids using a buffer sequence of 0.1 11t p H :3 citrate a t 10' followed by 0.1 A l pH 4.5 citrate as shown in Fig. 2. The basic amino acids were eluted with a series of three buffers: 0.1 iZf citrate p H 3.83, 0.1 A t phosphate containing 0.01LjC Triton X-155 and l . 5 7 i benzyl alcohol a t "5" , 0.1 d.f citrate p 3.41 (see Fig. 1). The tyrosine content of the phenylalanine-tyrosine mixture was estimated h y the method of Thomas.14 The proline and ornithine were analyzed by the method of Chinard.I6 Degradation of Chitosan.-Chitosan (essentially a poly2-aminoglucose from hydrolyzed crab shells)16 was degraded into a water-soluble fraction for use in studies on synthetic (9) K. Consden, A. H.Gordon and A. J. P. Martin, Biochem. J . , 38, 22-I (1944) (10) C. E . Dent, ibid., 43, ItiO (lYt8). i l l ) G. I
