Development of a heme sensor using fluorescently labeled heme oxygenase-1
Development of a heme sensor using fluorescently labeled heme oxygenase-1
复制标题
DOI：
10.1016/j.ab.2012.10.002
复制
发表时间：
2013-02-01
影响因子：
2.9
通讯作者：
Sakamoto, Hiroshi
中科院分区：
文献类型：
--
作者：
Koga, Shinya;Yoshihara, Shun;Sakamoto, Hiroshi
游离血红素是血红素的蛋白质非结合形式，对细胞具有毒性和调节作用。为了检测低浓度的游离血红素，我们开发了一种血红素传感器，使用荧光标记的血红素加氧酶-1（HO-1），一种催化血红素氧化降解的酶，对血红素具有高亲和力。血红素传感器的响应是基于血红素与酶结合时发生的荧光猝灭。三种荧光标记的HO-1中的每一种都表现出1：1的结合化学计量和与野生型HO-1的血红素复合物类似的吸收光谱。用氯化血红素滴定标记的蛋白质导致以氯化血红素浓度依赖的方式荧光猝灭，推测是由于从荧光团到与HO-1结合的血红素的能量转移。该传感器对血红素具有很强的亲和力，解离常数在低纳摩尔范围内，对血红素具有很高的选择性。基于传感器对血红素的线性响应，我们进行了荧光微孔板测定。该传感器能够选择性地检测游离血红素，但对与天然血红蛋白结合的血红素没有反应。该方法将是一个有用的工具，用于测定游离血红素在生物样品中含有蛋白结合血红素。(C)2012 Elsevier Inc. All rights reserved.
Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-Is exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme. (C) 2012 Elsevier Inc. All rights reserved.