ON THE SO-CALLED SIALIC ACIDS OF BLOOD CELLS AND SERUM

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The Journal
of Biochemistry,
Vol. 43, No. 6, 1956
ON THE SO-CALLED SIALIC ACIDS OF BLOOD CELLS AND SERUM BYTAMIO YAMAKAWA (FromtheDepartment of Chemistry, the Institute for Infectious Diseases, the University of Tokyo, Tokyo) (Receivedfor publication,August 18, 1956) In the previous paper, isolation of a crystalline reducing substance named 'sero-lactaminic acid' from equine serum (1) was reported, which was closely related to sialic acid (2, 3), acetyl-neuraminic acid (4), lactaminic acid (5) or gynaminic acid (6). At about the same time, Dr. Odin (7) isolated probably the same substance from human serum and claimed it as identical with sialic acid previously obtained from sheep submaxillary gland mucin (8) by means of x-ray diffraction pattern. In 1951, Yamakaw a and Suzuki (9) reported the presence and isolation of hemataminic acid (later confirmed as methoxyl neuraminic acid) from hematoside, a glycolipid of equine blood cells, and discussed that it would be present in the lipid probably as N-acetylated form. Two years later, Klenk and Lauenstein (10) reported in their reinvestigation of our work, that hydrolysis of hematoside produced no volatile acid, and denied the presence of acetyl group. More recently, Blix and his co-workers (8) reported bovine submaxillary sialic acid contains an N-acetyl and an O-acetyl group, the latter being very easily split off, whereas the ovine (sheep) substance has only an N-acetyl, and the porcine (hog) acid no acetyl but an N-glycolyl group, instead. Moreover, Svennerholm (11) isolated sialic acid from brain ganglioside, later, Blix and Odin (12) stated it would be the same as sialic acid of sheep submaxillary mucin, which would probably be identical with N-acetyl neuraminic acid. In the course of the studies of influenza virus hemagglutination receptor, Klenk and his collaborators (13, 14), Gottscha1k (15) and B1ix's school (16) became in agreement in regarding the receptor site to be correlated with so-called sialic acid. It was found in this laboratory the mucopolysaccharide fraction (90per cent phenol extract) of delipidized human blood stroma, although not yet satisfactorily puri867
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T. YAMAKAWA
fled, has a marked capacity to inhibit viral hemagglutination and con tains a considerable amount of Bial-chromogen besides hexosamine (17, 18). The structure of hemataminic acid and of sialic acid were suggested by the present author (19) and recently by Gottscha1k (20, 21), but available evidences were not sufficient to exclude other structures. Thereafter, the present author has regarded the formula previously assumed for hemataminic acid as inadequate, because it could not ex plain his recent finding that formalin (as dimedon) was produced after periodate oxidation (unpublished). In a mean while, Klenk and his co-workers (22) have recently pointed out this erroneous assumption and presented a probable struc ture for neuraminic acid. In this report, delipidized human as well as equine stromata, hemato side and human serum were treated with dilute sulfuric acid to release Bial-chromogen (=sialic acids) and the chromogen was purified by adsorption on ion exchanger just in the same way as described in the case of sero-lactaminic acid from equine serum (1). In each case, it was succeeded to obtain crystalline needles. The materials from hematoside as well as delipidized equine stroma gave no volatile acid on hydrolysis and available evidences show the amino group was substituted with glycolyl group as in case of porcine submaxil lary sialic acid (11); on the contrary, the substance from human de lipidized stroma and human serum contained the same amount of acetyl group as equine serum sialic acid (=serolactaminic acid) (1). While the dried sample from human stroma gave no methoxyl value despite after crystallization from methanol-ether, the acid from serum con tained a methoxyl group even after drying over phosphorus pentoxide at elevated temperature in a high vacuum. On the other hand, the serum-sialic acid crystallized from water-acetoneether instead of usual methanol-ether solvent system was devoid of methoxyl and remained methoxyl-free even after retreatment with methanol; although the serum-sialic acid once crystallized from methanol-ether was not con verted to methoxyl-free compound after recrystallization from water acetone and drying under high vacuum at elevated temperature. This methoxyl value of serum sialic acid is now proved to be due to labile methyl ester formation during prolonged treatment with methanol and not to crystal solvent (1), because its aqueous solution is neutral and cannot be titrated electrometrically at low temperature.