Røjkjaer R, Schousboe I
Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Denmark.
Eur J Biochem. 1997 Jan 15;243(1-2):160-6. doi: 10.1111/j.1432-1033.1997.0160a.x.
The dependency of concentrations of Zn2+ and the negatively charged surfaces, phosphatidylinositol phosphate (PtdInsP), sulfatide and dextran sulfate, on the autoactivation of human factor XII, has been studied. While the autoactivation induced by sulfatide, and low concentrations of dextran sulfate, was unaffected by the presence of Zn2+, that induced by PtdInsP and higher concentrations of dextran sulfate was completely dependent on Zn2+: the excess of Zn2+ needed to induce maximal activity with PtdInsP was 12-fold the concentration of factor XII, while with dextran sulfate it was 40-fold. Determination of the Zn2+-binding properties of factor XII revealed that a total of four zinc ions could bind to each factor XII molecule. The first bound zinc ions (Kd 0.1 microM) induced an increase in the intrinsic tryptophan fluorescence of factor XII, while further titration up to a 40-fold surplus resulted in a quenching of the fluorescence. Binding of the zinc ions that caused the quenching had an average Kd of approximately 1 microM, independent of whether it was determined from the fluorescence changes or by equilibrium filtration. Low concentrations of both sulfatide and PtdInsP induced a fluorescence increase similar to that at low concentrations of Zn2+ but, in contrast to sulfatide, higher concentrations of PtdInsP did not induce a quenching in fluorescence. As the Zn2+-independent activating surface (sulfatide) induced quenching in the fluorescence intensity, while the Zn2+-dependent activating surface (PtdInsP) did not, the quenching, whether it was caused by sulfatide or zinc ions, was assigned to a change in the conformation which resulted in a molecular structure of factor XII that could be autoactivated. Association of factor XII in this conformation on the activating surface was suggested to be responsible for the autoactivation.
研究了锌离子（Zn2+）浓度与带负电荷表面（磷脂酰肌醇磷酸（PtdInsP）、硫苷脂和硫酸葡聚糖）对人凝血因子XII自激活的依赖性。硫苷脂和低浓度硫酸葡聚糖诱导的自激活不受Zn2+存在的影响，而PtdInsP和高浓度硫酸葡聚糖诱导的自激活则完全依赖于Zn2+：用PtdInsP诱导最大活性所需的过量Zn2+是凝血因子XII浓度的12倍，而用硫酸葡聚糖则是40倍。对凝血因子XII的Zn2+结合特性的测定表明，每个凝血因子XII分子总共可以结合四个锌离子。首先结合的锌离子（解离常数Kd为0.1微摩尔）导致凝血因子XII的固有色氨酸荧光增加，而进一步滴定至40倍过量则导致荧光猝灭。导致荧光猝灭的锌离子结合的平均Kd约为1微摩尔，无论通过荧光变化还是平衡过滤测定都是如此。低浓度的硫苷脂和PtdInsP都诱导了与低浓度Zn2+相似的荧光增加，但与硫苷脂不同的是，高浓度的PtdInsP不会诱导荧光猝灭。由于不依赖Zn2+的激活表面（硫苷脂）导致荧光强度猝灭，而依赖Zn2+的激活表面（PtdInsP）则不会，因此，无论猝灭是由硫苷脂还是锌离子引起的，都归因于构象变化，这种变化导致了可自激活的凝血因子XII分子结构。这种构象的凝血因子XII在激活表面上的缔合被认为是自激活的原因。
