Anticoagulation factor Ⅱ(ACFⅡ) from the venom of Agkistrodon acutus is a non-enzymatic anticoagulant and forms a 1:1 complex with activated coagulation factor X (FXa), to thereby prolong clotting time. It has been discovered by HPLC that the binding of ACFⅡ with FXa is dependent on the concentration of Ca(Ⅱ) and the maximal binding of ACFⅡ to FXa occurs at concentration of Ca(Ⅱ) of about 1× 10^-3mol/L. The binding of Ca(Ⅱ) to ACFⅡ is analyzed by equilibrium dialysis and two Ca(Ⅱ)-binding sites with different affinities are identified. At pH 8.0, the apparent association constants k1 and k2 values for these two sites are (1.1±0.3)×10^5L/mol and (1.7±0.4)× 10^4L/mol, respectively. It is evident from the observation of Ca(Ⅱ) induced changes in the intrinsic fluorescence of ACFⅡ that ACFⅡ undergoes a conformational change upon binding of Ca(Ⅱ). The occupation of both Ca(Ⅱ) -binding sites in ACFⅡ requires a concentration of Ca(Ⅱ) of about 1×10^-3mol/L, which is equal to the effective concentration of Ca(Ⅱ) required for the maximal binding of ACFⅡ to FXa and for the maximal Ca(Ⅱ)-induced enhancement of emission fluorescence of ACFⅡ. It can be deduced, from these results, that the occupation of both Ca(Ⅱ)-binding sites in ACFⅡ with Ca(Ⅱ) and subsequent conformational rearrangement might be essential for the binding of ACFⅡ with FXa.

Key words: CONJUGATED PROTEIN, CALCIUM ION