pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.
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.
Wakabayashi
Hironao
H
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642, USA.
Zhou
Qian
Q
Nogami
Keiji
K
Ansong
Charles
C
Varfaj
Fatbardha
F
Miles
Stephen
S
Fay
Philip J
PJ
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
D011994:Recombinant Proteins; D003300:Copper; D005169:Factor VIII; D015951:Factor Xa