Abstract
SUMMARY A homogeneous substance has been isolated from blood which inhibits bovine testicular hyaluronidase. It has been found to contain amino acids and hexosamine and gives the carbazole color reaction for uranic acid. It contains less than 0.1 per cent sulfur as sulfate, less than 0.2 per cent phosphorus, and less than 5 per cent lipides. No evidence for the presence of heparin was found. The molecular weight was estimated at 100,000, the mobility in Verona1 buffer at pH 8.6, p = 0.1, was -5.4 X 10-S sq. cm. per second per volt, the sedimentation constant was .so,~ = 3.75, and the diffusion constant was DZo = 3.4 f 0.3 x 10-’ sq. cm. per second. BIBLIOGRAPHY 1. Dorfman, A., Ann. New York Acad. SC., 62, 1098 (1950). 2. Glick, D., J. Mt. Sinai Hosp., 17. 207 (1950). 3. Mathews, M. B., and Dorfman, A., Physiol. Rev., in press. 4. Dorfman, A., Ott, M. L., and Whitney, R., J. Biol. Chem., 174, 621 (1948). 5. Glick, D., and Moore, D. H., Arch. Biochem., 19, 173 (1948). 1 The authors are indebted to Dr. Carol Spurling for aid in these studies. by guest, on July 20, 2011www.jbc.orgDownloaded from
Highlights
Dorfman et al [4] showed that Fractions II and III of blood plasma obtained by Method 6 of Cohn demonstrated hyaluronidase inhibitor activity, but Glick and Moore [5] and Moore and Harris [6] found that the activity migrates predominantly with the albumin fraction of serum upon electrophoresis at pH 8.6 in a Verona1 buffer
It was impossible to determine whether any heparin-like activity was present
The mechanism of inhibition of hyaluronidase by this blood substance is obscure, but the demonstration of the presence of glucosamine and possibly uranic acid suggests that the inhibitor may have some chemical similarity to the substrates of this enzyme and may possibly act as a competitive inhibitor
Summary
Dorfman et al [4] showed that Fractions II and III of blood plasma obtained by Method 6 of Cohn demonstrated hyaluronidase inhibitor activity, but Glick and Moore [5] and Moore and Harris [6] found that the activity migrates predominantly with the albumin fraction of serum upon electrophoresis at pH 8.6 in a Verona buffer. Cipitated from plasma in Fractions II and III by Method 6 of Cohn, but subsequent studies indicated that a more efficient procedure could be devised if the first step of Method 10 was utilized Under these conditions 80 to 100 per cent of the activity could be recovered in the combined Fraction I-II-III which presumably contains r-globulins, p-lipoproteins, lipide-poor euglobulins, cold insoluble globulins, isoagglutinins, plasminogen, fibrinogen, and prothrombin. When larger quantities were prepared as described above, purification up to 800 times was achieved
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