Catalytic and regulatory functions of N-bromosuccinimide-modified bovine thrombin.
At pH 4.1, bovine thrombin reacts rapidly with N-bromo-succinimide to yield modified enzyme containing oxidized tryptophan residue. Both fibrinogen clotting activity and esterase activity are reduced considerably when three moles of tryptophan residues per mole of thrombin are oxidized, but the Michaelis constants for synthetic substrates are not appreciably altered. Reaction of NBS also results in a decrease in the affinity of thrombin for heparin. The dissociation constant for heparin-thrombin complex is increased by 2.6-fold due to the modification of one tryptophan residue. However, the magnitude of the increase in the dissociation constant remains the same for modified enzymes containing approximately two or three oxidized tryptophan residues. The rate constant for the inactivation of thrombin by antithrombin III is increased by 2.5-fold due to the modification of a single tryptophan residue. This increase in rate constant is not further amplified when more than one tryptophan residue is oxidized. In contrast, in the presence of heparin the rate of inactivation of modified and unmodified thrombins by antithrombin III are not significantly different. Thus, the heparin-sensitized inactivation of thrombin by antithrombin III is affected by the modification of one tryptophan residue. Spectrophotometric titrations of the phenolic hydroxyl groups suggest that the structural environments of tyrosyl groups for both unmodified and modified thrombin containing one oxidized tryptophan residue, are similar. The temperature for half loss of catalytic activity of control and NBS-modified thrombin, containing one oxidized tryptophan, are 52 and 51.5 degrees C respectively. It appears that the one tryptophan residue of thrombin is situated at or close to the binding site of heparin.
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology*; D002417:Cattle; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000494:pharmacology*; D013917:Thrombin / Q000378:metabolism*; D014364:Tryptophan / Q000502:physiology
D013388:Succinimides; D014364:Tryptophan; D000990:Antithrombin III; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin; D001974:Bromosuccinimide