Naslov
Use of surface plasmon resonance for studies of protein–protein and protein–phospholipid membrane interactions

Autori
Saenko, E ; Sarafanov, A ; Greco, N ; Shima, M ; Loster, K ; Schwinn, H ; Josic, D

Izvornik
Journal of Chromatography A (0021-9673) 852 (1999), 1; 59-71

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Von Willebrand facto ; Factor VIII ; Membranes ; Surface plasmon resonance detection ; LC

Sažetak
The surface plasmon resonance phenomenon is used for real time measurements of protein–protein and protein–membrane interactions. In the present study two surface plasmon resonance-based binding assays permitting study of the interaction of coagulation factor VIII (fVIII) with von Willebrand factor (vWf) and phospholipid have been developed. These interactions of fVIII are required for maintenance of fVIII concentration in circulation and for the assembly of the functional factor Xase complex, respectively. With these binding assays, the role of the light chain (LCh) in fVIII binding to vWf and to immobilized phospholipid monolayers and intact vesicles containing 25% phosphatidylserine (PS) and 4% PS was examined. The finding that Kd of LCh binding to vWf (3.8 nM) is 9.5 times higher than that of fVIII (0.4 nM), indicates that the heavy chain (HCh) is required for the maximal affinity of fVIII for vWf. In contrast, affinities of LCh for 25/75 PS/phosphatidylcholine (PC) monolayers and 4/76/20 PSPC-phosphatidylethanolamine (PE) vesicles are similar to that of fVIII, indicating that LCh is solely responsible for these interactions. It was also examined how removal of the acidic region affects the binding affinity of the remaining part of LCh for vWf and phospholipid. It was demonstrated that the loss of the LCh acidic region upon thrombin cleavage leads to an 11 and 160-fold increase in the dissociation rate constant (koff value) and a 165 and 1500-fold increase in the Kd value of the binding of fVIII fragment A3–C1–C2 to vWf compared to that of LCh and fVIII, respectively. In contrast, the binding affinity of A3–C1–C2 for PS-containing membranes was 8–11-fold higher than that of LCh. Possible conformational change(s) in C2 domain upon removal of the acidic region were studied using anti-fVIII monoclonal antibody NMC-VIII/5 with an epitope within the C2 domain of LCh as a probe. The determined lower binding affinity of A3–C1–C2 for NMC-VIII/5 immobilized to a sensor chip than that of LCh, indicates that these conformational changes do occur.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

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