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.
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.
Sun
X J
XJ
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Chang
J Y
JY
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
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