title: Heparin binding domain of human antithrombin III inferred from the sequential reduction of its three disulfide linkages. An efficient method for structural analysis of partially reduced proteins.
abstract: Human antithrombin III (AT-III) was partially reduced under mild conditions in the absence or presence of low molecular weight heparin. Quantitation of reduced disulfide bonds was facilitated by the application of a water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-iodoacetamido-2'-sulfonic acid (S-DABIA). The study shows that the three disulfide linkages of AT-III can be sequentially reduced, with Cys8-Cys128 being the most sensitive, followed by Cys21-Cys95, while Cys247-Cys430 is the most resistant to the mild reduction conditions. The rate of reduction of Cys8-Cys128 and Cys21-Cys95 was significantly decreased in the presence of heparin. The reduction of Cys8-Cys128 was also found to correlate quantitatively with the loss of heparin-accelerated antithrombin activity, heparin binding affinity, and heparin-induced fluorescence enhancement. These results suggest that Cys8-Cys128 is required for the integrity of the heparin binding domain of AT-III and support previous findings that lysyl residues surrounding Cys128 (Lys107, Lys114, Lys125, and Lys136) constitute an important part of the heparin binding site in AT-III.
authors.affiliation: Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
authors.forename: X J
authors.initials: XJ
authors.lastname: Sun
authors.forename: J Y
authors.initials: JY
authors.lastname: Chang
mesh_terms: D000478:Alkylation; D000990:Antithrombin III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D004226:Dithioerythritol; D006493:Heparin / Q000378:metabolism*; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D004220:Disulfides; D006495:Heparin, Low-Molecular-Weight; D007202:Indicators and Reagents; D004226:Dithioerythritol; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D000990:Antithrombin III; D006493:Heparin; D004124:p-Dimethylaminoazobenzene; D013917:Thrombin; D003545:Cysteine