Properties of thrombin- and elastase-modified human antithrombin III.
Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)
Gettins
P
P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Harten
B
B
D000818:Animals; D000990:Antithrombin III / Q000031:analogs & derivatives / Q000378:metabolism*; D006639:Histidine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009504:Neutrophils / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000378:metabolism*; D011487:Protein Conformation; D013552:Swine; D013917:Thrombin / Q000378:metabolism*
D006639:Histidine; D000990:Antithrombin III; D010196:Pancreatic Elastase; D013917:Thrombin