期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1764, 期 6, 页码 1094-1101出版社
ELSEVIER
DOI: 10.1016/j.bbapap.2006.04.004
关键词
factor VIII; copper; factor Xa generation; binding affinity; binding kinetics; pH
资金
- NHLBI NIH HHS [T32 HL07152, R01 HL038199, T32 HL007152, R01 HL076213, HL76213, HL38199] Funding Source: Medline
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 (K-d similar to 4 nM), whereas a K-d similar to 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 mu M 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 similar to 5-fold at pH 7.25. This effect resulted from an similar to 1.5-fold increased association rate constant (k(on)) and an similar to 3-fold reduced dissociation rate constant (k(off)). High affinity (K-d = 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. (c) 2006 Elsevier B.V All rights reserved.
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