![Fig. 2. Binding of protein variants to immobilized phospholipid evaluated using BIAcore. / The various protein C variants were infused at a flow rate of 30 μL/min over 2 different phopholipid membranes, one being 100% PC, the other being PS/PC, 20:80, wt/wt. The signals (resonance units [RU]) obtained from PC membranes were subtracted from the PS/PC-derived signals to obtain the specific binding to PS/PC membranes. (A) The 5 different protein C variants were injected at a concentration of 0.25 μM (arrows at left). After approximately 90 seconds, the protein solution was replaced with buffer (arrows at right) and the dissociation was followed. (B) WT protein C (upper panel) and QGNSEDY protein C (lower panel) were infused at the 3 indicated concentrations, and association and dissociation followed over time. (C) Increasing concentrations of WT protein C or QGNSEDY protein C were infused until a stable level was obtained, when association and dissociation is at equilibrium. The RU obtained at equilibrium was plotted against the protein C concentration, and the curves were used to calculate the Kd's. (D) Membrane binding was determined with light scatter intensity. M2 is the molecular weight of the protein-membrane complex, and M1 is that of the vesicles alone. The concentration of phospholipid was 5 μg/mL, and the protein C concentrations were varied from 0 to 1 μM, yielding protein/phospholipid (P/PL) ratios up to 16. QGNSEDY, (▴); SEDY, (▵); QGN, (■); WT protein C, (●). The means of 2 to 3 different experiments are shown with error bars representing ± SD.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/6/10.1182_blood-2002-06-1691/4/h80633972002.jpeg?Expires=1722654694&Signature=U5EZ5syBPRssofmEko2bu9k4bZ3cBpr3vAcVzy2ammPKDkTo2OB~NczUhc-E7MXbc84Q4MPmY6oeD-EcGqIg59PJY3djsa0WkmcxIOEbjkHv4NrbpQeHVFoQyWJaKrkuXKGOzRjCa9Cng4lripY2O5Ga60eUeqc12Zx0nrQ2PekPMjZh0YO~SxyVARDE1v~DYTE2Jxj3ti3PrFAZNp5Z9wYvOGWzPr13R9i6~IsXWp-VycrGovcz2M3Xf-k68bDwjdH4Edo01LcEZIpToQZJ8Uotxn4rE~0yDQb6gSUkiuGn8qcJxD5wkTsMi9GHCYNcDDoLUeYaeTa1Euqz034mPA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Binding of protein variants to immobilized phospholipid evaluated using BIAcore.
The various protein C variants were infused at a flow rate of 30 μL/min over 2 different phopholipid membranes, one being 100% PC, the other being PS/PC, 20:80, wt/wt. The signals (resonance units [RU]) obtained from PC membranes were subtracted from the PS/PC-derived signals to obtain the specific binding to PS/PC membranes. (A) The 5 different protein C variants were injected at a concentration of 0.25 μM (arrows at left). After approximately 90 seconds, the protein solution was replaced with buffer (arrows at right) and the dissociation was followed. (B) WT protein C (upper panel) and QGNSEDY protein C (lower panel) were infused at the 3 indicated concentrations, and association and dissociation followed over time. (C) Increasing concentrations of WT protein C or QGNSEDY protein C were infused until a stable level was obtained, when association and dissociation is at equilibrium. The RU obtained at equilibrium was plotted against the protein C concentration, and the curves were used to calculate the Kd's. (D) Membrane binding was determined with light scatter intensity. M2 is the molecular weight of the protein-membrane complex, and M1 is that of the vesicles alone. The concentration of phospholipid was 5 μg/mL, and the protein C concentrations were varied from 0 to 1 μM, yielding protein/phospholipid (P/PL) ratios up to 16. QGNSEDY, (▴); SEDY, (▵); QGN, (■); WT protein C, (●). The means of 2 to 3 different experiments are shown with error bars representing ± SD.
