Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy
Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy
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DOI：
10.1016/j.ab.2004.02.043
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发表时间：
2004-06-01
影响因子：
2.9
通讯作者：
Smith, C
中科院分区：
文献类型：
--
作者：
Tait, JF;Gibson, DF;Smith, C
我们开发了一种方法，用于测量膜联蛋白V的磷脂囊泡和细胞在非常低的膜占有率水平的结合亲和力。膜联蛋白V-117突变体的标记与荧光素碘乙酰胺在其单一的N-末端半胱氨酸残基;结合到磷脂囊泡含有磷脂酰丝氨酸（PS）和2%罗丹明-磷脂酰乙醇胺测定由于共振能量转移的荧光猝灭;结合到细胞与暴露的PS洗脱结合蛋白后，通过荧光测定法测定。平衡常数计算为钙滴定曲线的中点、希尔系数和膜结合位点的浓度的函数。在非常低的蛋白质与膜的比率下的钙滴定揭示了对于囊泡和细胞的约8的希尔系数，远高于先前测量的，但是随着蛋白质-膜比率增加到最大膜占有率的3%以上，希尔系数的值逐渐降低到约2的极限值。高希尔系数也观察到在不同的离子强度和膜PS含量在20至50%的范围内变化的测量。该方法允许准确测定膜联蛋白V-膜结合的亲和力和协同性，并将用于评价用于诊断和治疗应用的修饰的膜联蛋白V衍生物。(C)2004年爱思唯尔公司All rights reserved.
We developed a method for measuring the binding affinity of annexin V for phospholipid vesicles and cells at very low levels of membrane occupancy. The annexin V-117 mutant was labeled with fluorescein iodoacetamide on its single N-terminal cysteine residue; binding to phospholipid vesicles containing phosphatidylserine (PS) and 2% rhodamine-phosphatidylethanolamine was measured by fluorescence quenching due to resonance energy transfer; binding to cells with exposed PS was measured by fluorometry after elution of bound protein. The equilibrium constant was calculated as a function of the midpoint of the calcium titration curve, the Hill coefficient, and the concentration of membrane binding sites. Calcium titrations at very low ratios of protein to membrane revealed Hill coefficients of approximately 8 for both vesicles and cells, far higher than previously measured, but as the protein-membrane ratio was increased above 3% of maximum membrane occupancy, the value of the Hill coefficient progressively decreased to a limiting value of about 2. High Hill coefficients were also observed for measurements performed at different ionic strengths and with membrane PS content varied over the range from 20 to 50%. This method allows the accurate determination of the affinity and cooperativity of annexin V-membrane binding and will be useful for the evaluation of modified annexin V derivatives intended for diagnostic and therapeutic applications. (C) 2004 Elsevier Inc. All rights reserved.