Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation

Abstract

The structural stability of the protease inhibitor antithrombin from bovine plasma was examined as a function of the concentration of guanidinium chloride (GdmCl). A biphasic unfolding curve at pH 7.4, with midpoints for the two phases at 0.8 and 2.8 M GdmCl, was measured by far-ultraviolet circular dichroism. Spectroscopic and hydrodynamic analyses suggest that the intermediate state which exists at 1.5 M GdmCl involves a partial unfolding of the antithrombin molecule that exposes regions of the polypeptide chain through which slow, intermolecular association subsequently takes place. The partially unfolded molecule can be reversed to its fully functional state only before the aggregation occurs. Upon return of the aggregated state to dilute buffer, the partially unfolded antithrombin remains aggregated and does not regain the spectroscopic properties, thrombin-inhibitory activity, or heparin affinity of the native inhibitor. This behavior indicates that the loss of the functional properties of the proteins is caused by the macromolecular association. Comparative experiments gave similar results for the human inhibitor. Analyses of bovine antithrombin in 6 M GdmCl indicated that the second transition reflects the total unfolding of the protein to a disulfide-cross-linked random coil. This transition is spectroscopically reversible; however, on further reversal to dilute buffer, the molecules apparently are trapped in the partially unfolded, aggregated, intermediate state. The results are consistent with the existence of two separate domains in antithrombin which unfold at different concentrations of GdmCl but do not support the contention that the thrombin-binding and heparin-binding regions of the protein are located in different domains [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053].

A Highly Purified Antithrombin III Concentrate Prepared From Human Plasma Fraction IV-1 by Affinity Chromatography

“…This was removed by the second He parin Hyper D column. A similar result was observed by Wickerhauser and Williams [10], Known reasons for lowaffinity interaction of AT-III to heparin are: (a) cleavage of AT-III at the active site [24]; (b) folding into putative latent states by heat or chaotropes [22][23][24]; (c) aggregation fol lowing dénaturation [25]; (d) modification of the N-asparaginyl oligosaccharides from bianterinary to tetra-antennary structures in in vitro culture [26], and (e) point mutations of critical amino acids in the heparin binding region [1,27]. The latter two possibilities are not relevant to this situation.…”

Effect of amidoalkyl group as spacer on aggregation properties of guanidine-type surfactants

“…Also, the guanidine group is known to interact strongly with biological substances having carboxylic and phosphate groups through ion pair formation by means of electrostatic interaction and hydrogen bonding [1]. Many of the studies on guanidine salts in the field of biochemistry have dealt with the effects of the salt on the structure [2] and physiological activity [3,4] of protein molecules. It has also known that insertion of an alkyl group into guanidine salt strengthened the inhibitory effect on physiological activity.…”

Cellular folding pathway of a metastable serpin

“…The cellular folding process of ATIII, including its traversal of the secretory pathway, facilitates its folding to the functional metastable high free energy state. The differing outcomes of unassisted refolding of purified ATIII and its cellular folding underline the profound difference between protein folding reactions in isolation and in cells and invite further exploration of the key players and steps that make cellular folding so successful (8,9). The fact that ATIII and other serpins must adopt metastable states to function and that their misfolding is implicated in several pathologies further raises the importance of understanding their cellular folding pathway.…”