The Disappearance of Heparin Activity with Liver Globulins and Determinations of Heparinase^[*]

Summary

Preparations were made of rabbit liver globulin by the method of Jaques for heparinase and their effect on heparin studied. The results confirmed the observations of a progressive loss of anticoagulant activity with globulin in 0.9% saline, of a loss of metachromatic activity after phenol extraction and the reversal of the latter by alkali. The latter observations were due to the solubility in phenol of heparin on combination with protein. With suitable preparations, a decrease in anticoagulant activity without decrease in metachromatic activity was observed, i.e. conversion of heparin to uroheparin. Loss of heparin due to combination with protein and resulting precipitation, solubility in phenol, etc. followed a protein pH-dissociation curve. Loss of heparin anticoagulant activity due to heparinase was maximal at pH 5.4. No loss of heparin occurred at pH values more acid than 5 or more alkaline than 7.

^* Supported by a P.H.S. research grant No. H-6117 from the National Heart Institute, United States Public Health Service.

^** From Department of Physiology and Pharmacology, University of Saskatchewan, Saskatoon, Canada

• References

• 1 Jaques L. B. Heparinase. J. Biol. Chem. 133: 445 1940;
• 2 Jaques L. B, Keeri-Szanto E. Heparin II. Distribution of Enzyme in Various Tissues and Its Action on Natural Heparins and Certain Synthetic Anticoagulants. Can. J. Med. Sc. 30: 353 1952;
• 3 Monkhouse F. C, Jaques L. B. An Improved Method for the Extraction oF Heparin From Blood. J. Lab. and Clin. Med. 36: 782 1950;
• 4 Cho M. H, Jaques L. B. Heparinase III. Preparation and Properties of the Enzyme. Can. J. Biochem. and Physiol. 34: 799 1956;
• 5 Schuytema E. C, Cushing I. B. Attempts to Extract a Mammalian Heparinase, Fed. Proc. 17: 307 1958;
• 6 Loomis T. Failure of Liver to Inactivate Heparin. Proc. Soc. Exptl. Biol, and Med. 101: 444 1959;
• 7 Fischer A. Combination of Heparin with Protein. Biochem. 2. 278: 133 1935;
• 8 Jaques L. B. The Reaction of Heparin with Proteins and Complex Bases. Biochem. J. 37: 189 1943;
• 9 Jaques L. B, Bell H. J. Determination of Heparin. Methods of Biochemical Analysis. 07: 253 1959;
• 10 Jaques L. B, Napke E, Levy S. W. The Metachromatic Activity of Urine Following the Injection of Heparin. Circ. Res. 01: 321 1953;
• 11 Amatuzio D. S, Grande F, Wada S, Hay L. J. A Natural Occurring Lipid That Neutralizes Heparin Activity in Blood. J. Lab. and Clin. Med. 56: 277 1960;
• 12 Jaques L. B, Ballieux R. E, Van Arkel C. The Microelectrophoresis of Heparin. Acta med. Scand. In press 1963
• 13 Jaques L. B, Waters E. T, Charles A. F. A Comparison of the Heparins of Various Mammalian Species. J. Biol. Chem. 144: 229 1942;
• 14 Jaques L. B. Effect of Intravenous Injections of Heparin in the Dog. Am. J. Physiol. 125: 98 1939;
• 15 Jaques L. B, Ricker AnnG. The Relationship between Heparin Dosage and Clotting Time. Blood. 03: 1197 1948;
• 16 Monkhouse F. C. Physiological Factors Concerned with the Removal of Injected Heparin From the Circulating Blood. Am. J. Physiol. 178: 223 1954;