![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=1735839322&Signature=ylS1zM6uHzaam923E~lVk1oT-2GM5qCQswGsD45TCx6ezksDn-0qPCg2vVCx1oEUupu~bIEQESaq~7r66YdrB89lL9IJsm2E6yWBDydoCd2KWr0iCFqY1gc9LrVJAbgrWqXavfNfvjnPym9~DWj9z~7bufBW7JmUAqasF2OMoAYaeYZBBLP6rNIDNxi2uLA9Gj1DplbhirSN8SC1Inp8a702Xg-tYFBClkFRlceDDrqULjLURmxopJRG-qn~4iJg8go09cFJmNHHIalEOnw3Z1KfIW6dIbopb~OTpp1wwRqIQwvnHPuXhEe7YbtKoIgtAyvCy-GmdtLgekmEQtp1Ww__&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.
