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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
- Source :
- The Journal of biological chemistry. 264(19)
- Publication Year :
- 1989
-
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.
- Subjects :
- Binding Sites
Alkylation
Molecular Structure
Heparin
Antithrombin III
Thrombin
Heparin, Low-Molecular-Weight
Structure-Activity Relationship
p-Dimethylaminoazobenzene
Spectrometry, Fluorescence
Humans
Indicators and Reagents
Cysteine
Disulfides
Dithioerythritol
Oxidation-Reduction
Chromatography, High Pressure Liquid
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 264
- Issue :
- 19
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.pmid..........e0eebe0ea4d275352b1f5a2867683b9c