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An enzymatic method for precise oxygen affinity measurements over nanomolar-to-millimolar concentration regime.
JBIC Journal of Biological Inorganic Chemistry ( IF 2.7 ) Pub Date : 2020-01-02 , DOI: 10.1007/s00775-019-01750-6
Ria Sanyal 1 , Ambika Bhagi-Damodaran 1
Affiliation  

Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source. Using this method, we measure thermodynamic and kinetic oxygen affinities (Kd and KM) of different classes of heme and non-heme metalloproteins involved in oxygen transport, sensing, and catalysis. The method enables oxygen affinity measurements over a wide concentration range from 10 nM to 5 mM which is unattainable by simply diluting oxygen-saturated buffers. In turn, we were able to precisely measure oxygen affinities of a model set of eight different metalloproteins with affinities ranging from 48 ± 3 nM to 1.18 ± 0.03 mM. Overall, the Cld method is easy and inexpensive to set up, requires significantly lower quantities of protein, enables precise oxygen affinity measurements, and is applicable for proteins exhibiting nanomolar-to-millimolar affinity values.

Graphic abstract



中文翻译:

一种酶法,用于在纳摩尔到毫摩尔浓度范围内精确测量氧亲和力。

氧亲和力是金属蛋白的一个重要特性,有助于阐明它们的反应特性和机制。迄今为止,氧亲和值的测定要么使用需要昂贵仪器的闪光光解和极谱技术,要么使用在低纳摩尔和高毫摩尔氧浓度下错误的氧滴定方法。在这里,我们描述了一种廉价、易于设置和一锅法的氧亲和力测量方法,该方法使用亚氯酸盐歧化酶 (Cld) 作为精确的原位氧源。使用这种方法,我们测量热力学和动力学氧亲和力(K dK M) 涉及氧运输、传感和催化的不同类别的血红素和非血红素金属蛋白。该方法能够在从 10 nM 到 5 mM 的宽浓度范围内进行氧亲和力测量,这是通过简单地稀释氧饱和缓冲液无法实现的。反过来,我们能够精确测量由八种不同金属蛋白组成的模型组的氧亲和力,亲和力范围从 48 ± 3 nM 到 1.18 ± 0.03 mM。总体而言,Cld 方法易于设置且成本低廉,需要的蛋白质数量显着减少,能够进行精确的氧亲和力测量,并且适用于表现出纳摩尔至毫摩尔亲和力值的蛋白质。

图形摘要

更新日期:2020-01-02
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