DETERMINATION OF ATP AND ADP IN BLOOD-PLATELETS - MODIFICATION OF FIREFLY LUCIFERASE ASSAY FOR PLASMA
DETERMINATION OF ATP AND ADP IN BLOOD-PLATELETS - MODIFICATION OF FIREFLY LUCIFERASE ASSAY FOR PLASMA
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DOI：
10.1016/0003-2697(72)90323-5
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发表时间：
1972-01-01
影响因子：
2.9
通讯作者：
DAY, HJ
中科院分区：
文献类型：
--
作者：
HOLMSEN, H;STORM, E;DAY, HJ
本文改进了乙醇提取-萤火虫发光法测定血浆中血小板ATP-ADP的方法。EDTA必须包含在乙醇中以适当地灭活血小板腺苷酸激酶。EDTA的存在抑制了ADP向ATP的转化（在发光测量之前），推测是通过H2 EDTA 2-和所用的磷酸激酶系统的Mg 2+之间的相互作用引起的pH值下降。加入Tris马来酸盐缓冲液后，ADP-ATP转化率达100%。50%EDTA乙醇溶解最佳量的血小板ATP（99%）和ADP（80-90%）;较少的EDTA乙醇不能抑制ATP-ADP降解血小板酶，而增加EDTA乙醇浓度使核苷酸的溶解减少。ATP和ADP可以从EDTA乙醇不溶性材料中提取0.6 M HClO 4。ADP和ATP加入到这种EDTA-乙醇和HClO 4提取物的血小板丰富的血浆和悬浮液洗涤血小板给予发光反应每摩尔核苷酸等于ATP-ADP标准。核苷酸测定的最佳条件，以及杜邦发光生物计的有益用途。
A method for ATP-ADP determination in plasma by ethanol extraction and firefly luminescence has been modified for platelets. EDTA had to be included in the ethanol for proper inactivation of platelet adenylate kinase. Presence of EDTA inhibited conversion of ADP to ATP (prior to luminescence measurement), presumably through a drop in pH caused by the interaction between H2EDTA2−and the Mg2+of the pyruvate-kinase system employed. Introduction of Tris maleate buffer gave 100% ADP-ATP conversion. Optimal amounts of platelet ATP (99%) and ADP (80–90%) were solubilized by 50% EDTA ethanol; less EDTA ethanol failed to inactivate ATP-ADP degrading platelet enzymes whereas increasing EDTA ethanol concentrations gave decreasing solubilization of the nucleotides. ATP and ADP could be extracted from the EDTA ethanol-insoluble material by 0.6 M HClO4. ADP and ATP added to such EDTA-ethanol and HClO4extracts of both platelet-rich plasma and suspensions of washed platelets gave luminescence responses per mole nucleotide equaling that of ATP-ADP standards. Optimal conditions for nucleotide determination are described as well as the beneficial use of the DuPont luminescence biometer.