Exposed hydrophobic sites in factor VIII and isolated subunits.
Hydrophobic sites on the surface of factor VIII, factor VIIIa, and their derived subunits were evaluated using the fluorescent, apolar probe, bisanilinonapthalsulfonic acid (bis-ANS). Two hydrophobic sites, with indicated affinities for the probe, were identified on factor VIII (Kd = 0.2 and 1.22 microM), the isolated heavy chain (HC; Kd = 0.21 and 1.44 microM), and light chain (LC; Kd = 0.04 and 0.22 microM). Comparison of these values and fluorescence emission maxima parameters suggested that the higher affinity site on each isolated subunit contributes to the divalent metal ion-dependent, intersubunit interaction while the two lower affinity sites are retained on the surface of the factor VIII heterodimer. A single bis-ANS site was identified on both the A1/A3-C1-C2 dimer (Kd = 0.19 microM) and A2 subunit (Kd = 0.11 microM), whereas two sites, equivalent to the sites of factor VIII, were observed on factor VIIIa. These results suggested the absence of interactive hydrophobic sites between A2 subunit and dimer, a major conformational change around the hydrophobic site in A2 upon dissociation, and the lack of accessible hydrophobic regions on the B domain of HC. Ca2+ reduced the emission intensity of bis-ANS bound to the isolated LC, HC, and A1 subunit, but not the A2 subunit. Reconstitution of factor VIII activity from isolated HC and LC was inhibited by >90% in the presence of 20 microM bis-ANS, whereas this concentration of probe had no effect on the reconstitution of FVIIIa from the A1/A3-C1-C2 dimer and A2 subunit. These results indicate that intersubunit hydrophobic interactions are important for the metal ion-dependent association between A1 and A3 domains, but are not required for the metal ion-independent interaction between A2 subunit and the A1/A3-C1-C2 dimer in factor VIIIa.
Sudhakar
K
K
Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Fay
P J
PJ
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002942:Circular Dichroism; D004492:Edetic Acid / Q000494:pharmacology; D004735:Energy Transfer; D005169:Factor VIII / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D015944:Factor VIIIa; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017186:Titrimetry
D000817:Anilino Naphthalenesulfonates; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D015944:Factor VIIIa; D005169:Factor VIII; D004492:Edetic Acid; D002118:Calcium