Abstract
The presence of two unfolding domains in antithrombin III during its denaturation in guanidinium chloride has previously been reported (Villanueva, G. B., and Allen, N. (1983) J. Biol. Chem. 258, 11010-11013). In the present work, we report the results of refolding studies on antithrombin III. Circular dichroism and intrinsic fluorescence studies have demonstrated that the first unfolding domain of low stability (midpoint at 0.7 M guanidinium chloride) is irreversible upon renaturation, whereas the second unfolding domain (midpoint at 2.3 M guanidinium chloride) is reversible. The intermediate form of antithrombin III, termed AT-IIIR, which has lost the structural features of the first domain was investigated. Clotting assays and electrophoretic analyses showed that AT-IIIR had lost 60% of heparin cofactor activity but was still capable of forming sodium dodecyl sulfate-stable complexes with thrombin. Although certain regions of this molecule do not refold to the conformation of native antithrombin III, the tryptophan residues refold to a conformation identical with the native state. This was demonstrated by fluorescence quenching, solvent perturbation, and chemical modification studies. However, the tryptophan-ascribed fluorescence enhancement and absorption difference spectrum which occur when heparin binds to antithrombin III are reduced by 70%. On the basis of these data, the binding of heparin to antithrombin III is interpreted in terms of a two-step mechanism. The primary binding occurs in the region without tryptophan and is followed by a secondary conformational rearrangement which affects the tryptophan environment. The mechanism of the binding of heparin and antithrombin III has been previously studied by kinetic methods, and the data also support a two-step mechanism. The agreement of these two studies employing entirely different approaches to the same problem lends support to the validity of this postulated mechanism.
Highlights
In the present workw,e report the resultosf refolding charide potentiation of inhibition
It is Circular dichroism and intrinsicfluorescence studies have demonstrated that the firstunfolding domain of low stability is irreversible upon renaturation, whereas the second unfolding domain is reversible.Theintermediateform of antithrombin 111,termed AT-IIIR, which hlaosst the strucbelieved that a singlesite in heparin isufficient to accelerate antithrombin III-Factor X. interactions but a second region is required foraugmenting the neutralizatioonf thrombin and Factor IX, by antithrombin 111
In the preceding paper on this study [4], we have demonstrated by circular dichroism, intrinsic fluorescence, and absorption difference spectroscopy the existence of a two-domain structure in antithrombin111during its denaturation in guanidinium chloride
Summary
In the present workw,e report the resultosf refolding charide potentiation of inhibition. It is Circular dichroism and intrinsicfluorescence studies have demonstrated that the firstunfolding domain of low stability (midpoint at 0.7 M guanidinium chloride) is irreversible upon renaturation, whereas the second unfolding domain (midpoinatt 2.3 M guanidinium chloride) is reversible.Theintermediateform of antithrombin 111,termed AT-IIIR, which hlaosst the strucbelieved that a singlesite in heparin isufficient to accelerate antithrombin III-Factor X. interactions but a second region is required foraugmenting the neutralizatioonf thrombin and Factor IX, by antithrombin 111. Certain re- In order to understanhdow antithrombin I11 interacts with gions of this molecule do not refold to theconformation heparin, it is important to know the molecular forces which of nativeantithrombin 111, thetryptophanresidues stabilize the unique three-dimensional structure of this prorefold to a conformation identiwcailth the native state.tein For this purpose, we studied the equilibrium denatura-. Itsstructuralpropertiesare proachestothesame problemlendssupport tothe compared with native antithrombin 111, and a model for the validity of this postulated mechanism
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