March 20, 1953
COMMUNICATIONS TO THE EDITOR
1519
Chondroitinsulfuric acid (from cartilage) was essentially homogeneously N-deacetylated with 45% NaOH (25,48 hr.) under nitrogen and in the presence of antioxidants (benzyl alcohol and sodium sulfite) and was sulfated as described above (but a t 8&90°). The product was isolated as the amorphous sodium salt and is under further analytical characterization ; -NH2 (by ninhydrin), absent. The anticoagulant activity was 48 I. U./mg. The same sample of sodium chondroitin sulfate was subjected to the above sulfation procedure without DEPARTMENT OF MICROBIOLOGY preliminary N-deacetylation and the product, isoH. R . WHITELEY~~ SCHOOL OF MEDICINE OF WASHINGTON UNIVERSITY lated in the same manner, showed an anticoagulant SEATTLE 5 , ~VASH. activity of ca. 10 I. U./mg. Sodium heparinate was RECEIVEDJAXUARY 28, 1953 re-sulfated under these conditions with a reduction (10) D. R. Sanadi and J. W. Littlefield, J . Biol. Chew., 193, 883 in its activity from 110 to 55 I. U./mg. and an in(1051). crease in the sulfur content from 12 (initial) to ( I 1) Part o1 this work was dune a t the Hopkins Marine Station, Pacific 14.4%. tirove, California, on an Atomic Energy Commission Postdoctoral Methyl 2-amino-2-deoxy-~-~-glucopyranoside hyI'ellowship. drochloride4 was sulfated as above to produce the amorphous, water-soluble barium salt of the NSULFATED NITROGENOUS POLYSACCHARIDES AND sulfate, tri-O-sulfate; [(Y]*'D $4' (c 3.4, HzO). THEIR ANTICOAGULANT ACTIVITY' Anal. Calcd. for C7HllN017S4Ba2.2H20 : S , 15.63; Sir : Ba, 33.48. Found: S, 15.64; Ba, 32.98. A 3 X N-Deacetylated chitin, previously swollen with M solution of this substance in 0.004 N HC1 a t pyridine, was heterogeneously sulfated a t 100' for 95' lost 1.0 mole of sulfate in 5 2 0 min. with the 1 hour with chlorosulfonic acid and pyridine to concomitant release of the free amino group (ninyield a product, isolated (inorganic salts were re- hydrin). The 0-sulfate was removed relatively moved by dialysis) as the amorphous, water-soluble more slowly and only completely so after 12 hr. sodium salt, containing essentially two N-sulfate Previously reported results2 on the inactivation of and one 0-sulfate groups per anhydrodisaccharide heparin by mild acidity were considered to involve unit; [ a I z 6-2~3' (c 1.5,water). Anal. Calcd. for a negligible sulfate loss. On the basis of our present ClzHle0,(NSOaNa)2(0S03Na):C, 22.93;H, 3.05;N, knowledge of the heparin molecule,6 this sulfate 4.46; S,15.31;Na, 10.98. Found: C, 22.68;H,3.08; loss is about equivalent to the amino group released N, 4.02; S, 15.6; Na, 11.2; -NHz (by ninhydrin), so that a sulfate group shift5is not a required postuabsent; NAc, absent. This preparation exhibited lation. the behavior in the Van Slyke amino acid assay The above results show that the sulfamic acid characteristic of the acid-labile N-sulfate group group is a potent contributor to anticoagulant acpresent in heparin.*n3 Its anticoagulant activity tivity. Experiments are now underway to deterwas 56 International Units (I. U.)/mg. The ani- mine the optimum molecular size for these chemimal (mouse intravenous) toxicity was approxi- cally modified polysaccharides. mately double that of heparin, a finding believed to OF CHEMISTRY M. L. WOLPROM be due to the unsuitably high molecular size of the DEPARTMENT THEOHIOSTATEUNIVERSITY (MISS) T. M. SHEN substance. C. G. SUMMERS COLUMBUS 10, OHIO
amount of ATP and is independent of CoA, thus suggesting that succinyl phosphate may be formed, (d) in experiments with sulfanilamide,l0 a CoAand ATP-dependent decrease in free amide is observed; this decrease is greater if carbon dioxide production is inhibited, suggesting that succinylCoA, as well as succinyl phosphate, may be formed. A detailed treatment of the experimental results and of the postulated mechanism of succinate activation will be presented elsewhere.
(1) Supported by the Bristol Laboratories, Inc., Syracuse, N. Y.,
(R. F. Project 432). (2) M. I,. Wolfrorn and
w.
H. McNeely, THISJ O U R N A L , 67, 748
(1945).
(3) J. E. Juryes, H. BusLruin and V. Mutt, J . Uiul. C h e w . , 183,607 ( l!)50).
RECEIVED FEBRUARY 9, 1953 (4) I. C. Irvine, D. McNicoll and A. Hynd, J . Ckcm. Soc., 99, 230 (1911). ( 5 ) h l . 1,. Wolfroiii, 11. M o n t g o m e r y , J . V. Karabinose and 1'. l
