Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X.
Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7. 4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.[1]References
- Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X. Persson, E., Selander, M., Linse, S., Drakenberg, T., Ohlin, A.K., Stenflo, J. J. Biol. Chem. (1989) [Pubmed]
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