title: pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.
abstract: Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642, USA.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Charles
authors.initials: C
authors.lastname: Ansong
authors.forename: Fatbardha
authors.initials: F
authors.lastname: Varfaj
authors.forename: Stephen
authors.initials: S
authors.lastname: Miles
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D003300:Copper / Q000737:chemistry*; D019281:Dimerization; D005169:Factor VIII / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015233:Models, Statistical; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D003300:Copper; D005169:Factor VIII; D015951:Factor Xa