Tait Jonathan F, Smith Christina, Levashova Zoia, Patel Bhavesh, Blankenberg Francis G, Vanderheyden Jean-Luc
Department of Laboratory Medicine, University of Washington, Seattle, 98195-7110, USA.
J Nucl Med. 2006 Sep;47(9):1546-53.
Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy.
Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate.
In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups.
The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.
标记的膜联蛋白V广泛用于体外和体内检测细胞死亡。几乎所有研究都是使用通过胺导向双功能试剂衍生化的膜联蛋白V进行的；人们认为这些分子与未修饰的蛋白质相比保留了完整的生物活性。我们现在通过在低膜占有率条件下通过定量钙滴定测量膜联蛋白V对体外细胞的亲和力来表明这一假设是不正确的。
用4种不同的胺导向试剂修饰膜联蛋白V：肼基烟酸、巯基乙酰三甘氨酸和生物素的N-羟基琥珀酰亚胺酯；以及异硫氰酸荧光素。
在所有情况下，即使在非常低的平均化学计量比下，衍生化也会降低膜结合亲和力。基于泊松分布的统计模型准确地预测了观察到的衍生化异质性作为平均衍生化化学计量比的函数。该模型还表明，多重衍生化形式最有可能具有受损的生物活性，对结合和成像信号的贡献不成比例。体外结合试验正确地预测了所有测试蛋白质在小鼠肝脏凋亡模型中的体内摄取情况。在N端单个特定位点标记的膜联蛋白V-128蛋白，其凋亡特异性肝脏摄取量是通过氨基随机衍生化的所有形式膜联蛋白V的两倍。
膜联蛋白V的膜结合活性对胺导向的化学修饰比以前认识到的要敏感得多。通过位点特异性方法标记的新膜联蛋白V分子将大大提高体内检测细胞死亡的灵敏度。
