title: Exposed hydrophobic sites in factor VIII and isolated subunits.
abstract: 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.
authors.affiliation: Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: K
authors.initials: K
authors.lastname: Sudhakar
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: 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
chemical_list: D000817:Anilino Naphthalenesulfonates; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D015944:Factor VIIIa; D005169:Factor VIII; D004492:Edetic Acid; D002118:Calcium