[COXTRIBUTION FROM THE NUTRITION AND PHYSIOLOGY SECTION, RESEARCH DI\'ISION, LEDERLE LABORATORTES]
AMERICAN CYAXAMIn COMPAUY,
Isolation and Purification of Mactins, Heparin-like Anticoagulants from Mollusca1 BY S. L. BURSON, JR., M. J. FAHRENBACH, L. H. FROMMHAGEN, B. A. RICCARDI, R. A. BROWN,J. A. BROCKMAN, H. V. LEWRYAND E. L. R. STOKSTAD RECEIVED MAY14, 1956 Anticoagulants, designated niactin A and inactin B, were isolated from the rnollusca SPissuZn (Muctra) sdidissiri~cz:md Cyprina (Arctica) islandica, respectively. In vitro anticoagulant activities of the purified neutral sodium salts u-ere 130-150 U.S.P. units/ing. for mactiii A and 150-180 U.S.P. units/mg. for mactin B, as compared with 120-145 U.S.P. uiiits/iiig. for the most active samples of heparin neutral sodium salts available. Both mactiiis were sulfated polys:~cclinridcs :mci contained glucosamine and glucuronic acid. Mactin A, mactin B and heparin neutral sodium salts appeared t o be siiiii1:ir except for their molecular weights, which were 24,800, 28,700 and 14,200, respectively, and their optical rotations, ivliich were 4-71, + G l and +47', respectively.
Discussion servations have been made by Ricketts and WalThe presence of a heparin-like substance in the ton1' with dextran sulfate. Accordingly, columns of IRA-40012and IR-1BOL2 common surf clam, Spissula (Muctra) solidissima, has been demonstrated by Thomas.2 The isolation were utilized for partial deionization of crude macand partial purification of such anticoagulants have tin solutions. Addition of lead acetate to the dilute been r e p ~ r t e d . ~I?n~the present study the isola- aqueous effluents allowed the resultant insoluble tion and purification procedures of Frommhagen, mactin lead salts to be isolated by filtration. This et u Z . , ~ were extended and improved to yield highly treatment serves as a convenient means for isoh purified anticoagulants, designated mactin A and tion of the anticoagulants from the very dilute cfmactin B, from the two species of clams, Spissula fluent and as an effective method for the separation (Mactra) solidissima and Cyprina ('4rctica) is- of the anticoagulants from many impurities which are not normally removed in the conventional solZundica, respectively. For the preparation of crude extracts of mactin vent precipitation procedures. However, complete A and mactin B procedures were employed which removal of lead-precipitable ions other than the macwere similar to those already reported for the isoh- tins was found to he undesirable, since the subsetion of crude heparin from mammalian t i s s ~ e s . j - ~quent treatment of such thoroughly deionized ef Ground fresh clam meat was autolyzed a t 43' for fluents invariably led to colored products which 24 to 72 hours, extracted with alkali and ammoniuni could not be conveniently decolorized. Optimum sulfate, and the solid isolated from this extraction results in the deionization step were obtained when by acidification to pH 1.5 to 2.0 was then digested the capacity of the resin was exceeded by approxiwith pancreatin or trypsin. Crude products iso- mately 30%, thereby obtaining a solution contamilated from these digestions by precipitation with nated with a controlled quantity of extraneous ions. such solvents as acetone or alcohol had in vitro an- Preparation of the acid sodium salt of either mactin ticoagulant activities which ranged between 15 and was accomplished by suspension of the correspond20 U.S.P. units/mg.l0 Over-all yield of anticoagu- ing lead salt in 20% sodium chloride solution a t 60°, lant activity isolated in these crude products was followed by the addition of sodium sulfide to p H 0 30,000 to 3G,OOO U.S.P. unitsikg. of ground whole to 10, clarification of the warm mixture and addiclam meat in the case of mactin A, and 20,000 to tion of two volumes of glacial acetic acid to the fil26,000 U.S.P. uriitskg. of ground whole clam meat trate a t room temperature. The amorphous white in the case of mnctin R . These values are appreci- acid sodium salt was isolated by filtration with ably greater than those reported for the isolation of filter aid, then redissolved in water and reprecipicrude heparin from mammalian tissuesg and also tated with glacial acetic acid in order to remove for the isolation of sitnilrtr products from Spissubn traces of salt. Conversion to the neutral sodium salt was effected by addition of sodium hydroxide to ( A l f ( i c f r asolidissinza. ) a suspension of the acid sodium salt in anhydrous 3A In spite of their strong negative charges, neither mactiri A nor rnactin B could be absorbed on anion- alcohol-ether (1 : 1) employing brom thymol blue as indicator. An alternate procedure for the prepexchange resins. Presumably this is because of the aration of the neutral sodium salt consisted of the large molecular size of these substances. Similar ob- passage of a solution of the corresponding acid sodium salt through a monobed column containing ( I ) l'xiented a t t h e 12!lth Iteetirig of the American Chemical SI,ciety, n ; ~ l l n +T, e x a s . A p i i l 1 1 , 19.ifi. IRA-IZ0012and 1R-120,l2followed by titration of the (21 I,. J . Thomas, J r . , Riiil. Bvli., 101, 230 (19,51). effluent to the equivalence point ( p H 7.S5) with so( 3 ) I,. 11. I'rommhagen, h f . J . Fahrenbach, J. A. Brockman, Jr.* dium hydroxide and finally isolation of the neutral and I?. I,. R . Stokstad, PYOC. Soc. E.xfiU. Bid A J e d , 82, 280 (1953). sodium salt by freeze drying. (4) I.. J . Thomns, J r . , B i d Bill[ , 106, 129 (1454). ( 5 ) D. A . Scott and A . F. Charles, J . B i d . CIzem., 102, 42.5 In vitro anticoagulant activities of various prep( 1 !133). arations of the acid and neutral sodium salts of mac(0) A . 1'. Charles and D. A . ScrAt, Bioihem. J . , 30, I!J?? tin A ranged between 130 and 1.50 U.S.P. units/mg., Cl93C'I). whereas those of the corresponding sodium salts of ( 7 ) A . IC. Charles anrl A . R . Todd, i b i d . , 34, 112 (1910). (X) I.. I3 J:iqiicq, 13. T. Waters and A . P. Chnrles, .I. B i d . C ' h t , i i i , niactin B were between 150 and 180 U.S.P. units/ 144, ?2!l (1812). ( 0 ) \I. €1. Kiiizengn anrl I,. B. Spaulding, i h i d , 148, 011 (1813). (10) Heparin sodiiim, U. S. Phnrninropein X I V , 271 (19.50).
(11) C K Ricketts and K. N U'dlt
