New approaches to studying redox metabolism using time-resolved NAD(P)H fluorescence and anisotropy

活细胞需要持续不断的能量输入来维持它们特有的秩序。这是由新陈代谢的化学反应提供的。这些反应中有很大一部分是氧化还原反应，即电子从一个分子转移到另一个分子的氧化还原反应。在分解营养分子以从食物中提取能量的过程中，键被打破，电子被传递给被称为NADH的载体分子。电子的去除称为氧化，电子的加成称为还原。因此，食物的新陈代谢包括糖、蛋白质和脂肪的氧化以及NADH的同时还原。在NADH还原过程中传递给NADH的能量在线粒体内转化为一种可利用的形式，即细胞的能量货币三磷酸腺苷(ATP)。这是通过使用NADH携带的电子将氧气还原为水来实现的。这是人体几乎100%消耗的氧气的命运，释放的能量以ATP的形式储存。这一过程中的缺陷会导致活性氧物种(ROS)的产生。这些都是极具破坏性的分子，因此细胞拥有特定的防御机制来抵御它们的有害影响。这些防御是由另一种称为NADPH的电子载体分子维持的。NADPH支持的抗氧化系统作用于中和NADH调节的能量产生过程中产生的有害ROS。这些反应统称为氧化还原代谢。这两个过程之间的不平衡是包括癌症、糖尿病和神经退行性疾病在内的一系列疾病发展的已知因素。为了在了解这些疾病的发生机制和测试可能的治疗方法方面取得进展，为生物医学研究人员提供高精度的工具来研究细胞和组织中的氧化还原代谢是至关重要的。作为一群擅长使用激光研究分子的动态行为并应用这些方法来研究活组织中代谢过程的科学家，我们建议开发下一代方法来研究氧化还原代谢在健康和疾病中的作用。我们将利用NADH和NADPH的本征荧光来构建我们的新的实验技术。虽然这两个分子发出相同颜色的光，但它们为了发挥不同作用而结合的两组独立的酶会对它们的荧光的其他特性造成不同的影响。我们将首先制备与它们各自的酶结合的NADH和NADPH溶液，以研究它们产生的荧光特性。这些分子附着的不同结合部位可能会对吸收激光脉冲后出现荧光所需的时间造成相反的影响，即所谓的荧光寿命。作用在结合部位的分子上的不同作用力也会导致它们运动自由度的不同，这可以通过测量发射的光相对于吸收的光的偏振变化率来检测，即所谓的时间分辨荧光各向异性。随着NADH和NADPH荧光在结合到它们各自的酶组上时的差异的特征，我们将构建一个显微镜，在其中可以在活组织中检测到这些特性。在细胞的复杂环境中，荧光信号预计来自NADH和NADPH的混合物，两者都与它们的酶结合，并且是自由的。我们将开发从这些信号中提取信息的方法，以便研究它们所涉及的单独氧化还原途径的功能。随着氧化还原代谢被发现在越来越多的过程中发挥作用，这些新的方法将在增强我们对生物学的基本理解方面发挥关键作用。

Living cells require the constant input of energy to maintain their characteristic order. This is provided by the chemical reactions of metabolism. A vast number of these reactions are "redox" (reduction-oxidation) reactions, in which electrons are transferred from one molecule to another. During the breakdown of nutrient molecules to extract energy from food, bonds are broken and electrons are passed to a carrier molecule known as NADH. Removal of electrons is referred to as oxidation, and their addition is known as reduction. The metabolism of food therefore involves the oxidation of sugars, proteins and fats and the simultaneous reduction of NADH.The energy passed to NADH during its reduction is converted into a useable form, the cell's "energy currency" adenosine triphosphate (ATP), inside the mitochondria. This is achieved by using the electrons carried by NADH to reduce oxygen to water. This is the fate of almost 100% of the oxygen consumed by the body, and the energy released is stored as ATP. Defects in this process cause the production of reactive oxygen species (ROS). These are highly damaging molecules, and so the cell possesses specific defence mechanisms against their deleterious effects. These defences are maintained by another electron carrier molecule known as NADPH.The antioxidant systems supported by NADPH act to neutralise the harmful ROS produced during the energy generating processes regulated by NADH. These reactions are collectively known as redox metabolism. Imbalance between the two processes is a known factor in the development of a wide range of diseases, including cancer, diabetes and neurodegenerative disorders. In order to make progress in understanding how these diseases occur and testing potential treatments, it is crucial that biomedical researchers are provided with highly accurate tools to investigate redox metabolism in cells and tissues.As a collection of scientists whose expertise includes the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, we propose to develop the next generation of approaches to studying the role of redox metabolism in health and disease. We will exploit the intrinsic fluorescence of NADH and NADPH to construct our new experimental technique. While the two molecules emit light of the same colour, the two independent sets of enzymes that they bind to in order to perform their distinct roles will cause contrasting effects on other characteristics of their fluorescence.We will first prepare solutions of NADH and NADPH bound to their respective enzymes to investigate the resulting properties of their fluorescence. The distinct binding sites to which these molecules attach may cause contrasting effects on the time taken for fluorescence to emerge following absorption of a laser pulse, the so-called fluorescence lifetime. Different forces acting on the molecules in the binding site will also cause differences in the freedom of their motion, which can be detected by measuring the rate of change of the polarisation of the light emitted with respect to the light absorbed, the so-called time-resolved fluorescence anisotropy.Following characterisation of the differences in NADH and NADPH fluorescence when bound to their separate sets of enzymes, we will construct a microscope in which these properties can be detected inside living tissues. Inside the complex environment of the cell, the fluorescence signals would be expected to arise from a mixture of NADH and NADPH, both bound to their enzymes and free. We will develop approaches to extract information from these signals, allowing the function of the separate redox pathways that they are involved in to be investigated. As redox metabolism is being found to play a role in an ever growing range of processes, these new approaches will play a key role in enhancing our fundamental understanding of biology.