Fig 1.
Binding of C6PS and C6PE to PZ measured by intrinsic fluorescence.
To detect binding, intrinsic tryptophan fluorescence intensities of 150 nM PZ in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl2, 0.6% PEG were measured at 25° C as a function of added C6PS (●) or C6PE (○). Solid lines show fits of the data to a simple single-site binding model, which predicted apparent Kd values for binding of C6PS and C6PE as 47.7 ± 10.7 μM and 47.6 ±8.2 μM, respectively. Fluorescence titrations were also performed under two more conditions: in presence of 5 mM EDTA with C6PS (■) and C6PE (□); and in presence of 1 mM Ca2+ with C6PS (▲) and C6PE (Δ).
Fig 2.
Binding of different six-carbon chain soluble lipids to PZ.
Intrinsic tryptophan fluorescence intensities of 150 nM PZ in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl2, 0.6% PEG were measured as a function of added A. C6PA (○), B. C6PC (●), C. C6PG (Δ) and D. C6(D)PS (▼) to obtain binding constants. The apparent Kd values for binding are 165 ± 25 μM, 129 ± 32, 131± 39, for PA, PC and PG respectively. (D)-PS shows extremely weak binding with Kd~900 μM.
Table 1.
Summary of the results obtained from equilibrium dialysis experiment.*
Table 2.
Linkage between sites for soluble lipids on human PZ.*
Fig 3.
Binding of human PZ to phospholipid vesicles.
Fluorescence measurements were performed in 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM CaCl2, 0.6% PEG by adding increasing concentrations of PZ to 1 μM-labeled DOPC: DOPS: DOPE: Dansyl-PE vesicles of varying composition: A. 69:1:27.5:2.5 (●) 96.5:1: 0: 2.5 (○), 97.5: 0:0:2.5 (Δ); B. 60:10: 27.5: 2.5 (●) and 87.5:10: 0: 2.5 (○).Details of the experimental procedure are described in Methods.
Table 3.
Contribution of PS and PE toward binding of PZ to membrane.*
Fig 4.
Binding of PZ to DEGR-Xa [(5-(Dimethylamino)-1-naphthalenesulfonyl]-Glutamylycylarginyl)-Xa] on phospholipid membranes.
DEGR-Xa (30 nM) fluorescence emission intensities were measured with increasing concentrations of PZ in the presence of 50 μM membrane composed of DOPS: DOPC 30:70 (●), DOPE: DOPC 30:70 (○), 100% DOPC (▼) and in the absence of any membrane (□). Dissociation constants in the presence of PC: PS and PC: PE were 8.6 ± 2.2 nM and 20.6 ± 3 nM, respectively. There was no association of fXa and PZ observed in presence of 100% PC or in absence of any membrane.
Fig 5.
FXa-PZ interaction on phospholipid membranes as measured by AT-mediated inhibition of fXa activity.
The amidolytic activity of 5 nM fXa in a buffer containing 50 mM Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM Ca2+ and 0.6% PEG was measured in the presence of AT, with or without PZ in the absence and presence of membranes (DOPC: DOPS 70: 30; DOPC: DOPE 70: 30, 100% DOPC) using substrate S2765.
Fig 6.
Cartoon diagram describing the major findings of the study.
The diagram shows that PZ does not bind to 100% PC membrane and does not form a complex with fXa (A); PZ binds to PS or PE containing membranes with comparable affinity and form a stable PZ-fXa complex (B & C); PS and PE synergistically act to enhance the binding affinity of PZ towards membrane (D).