Isolation and Characterization of Heparin from Human Lung

Dean D. Metcalfe; Robert A. Lewis; Jeremiah E. Silbert; Robert D. Rosenberg; Stephen I. Wasserman; K. Frank Austen

doi:10.1172/JCI109613

Isolation and Characterization of Heparin from Human Lung

Dean D. Metcalfe, Robert A. Lewis, Jeremiah E. Silbert, Robert D. Rosenberg, Stephen I. Wasserman, and K. Frank Austen

Published December 1, 1979 - More info

View PDF

Abstract

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from ³⁵S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The ³⁵S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The ³⁵S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [³⁵S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 μg/10⁶ cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.