In photosynthetic organisms, it is recognized that the intracellular redox ratio of NADPH is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADPH fraction [NADPH/(NADPH + NADP⁺)] quantitatively estimated to date. In the present study, the light response of the NADPH fraction was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light response of NADP(H) observed using PCI extraction was qualitatively consistent with the NAD(P)H fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light–dark transition. Specifically, the fraction of NADPH was 42% in the dark-adapted cell, and saturated at 68% in light conditions.

在光合生物中，人们认识到 NADPH 的细胞内氧化还原比被调节在适当的范围内，以实现多种生理过程的协同作用。然而，尽管它很重要，但迄今为止定量估计的 NADPH 分数 [NADPH/(NADPH + NADP ⁺ )]的值存在很大差异。在本研究中，通过在蓝藻集胞藻中应用苯酚/氯仿/异戊醇 (PCI) 的新型 NADP(H) 提取方法，研究了 NADPH 部分的光响应。sp。PCC 6803. 使用 PCI 提取观察到的 NADP(H) 的光响应在定性上与体内测量的 NAD(P)H 荧光时间过程一致。此外，通过 PCI 提取和基于荧光的方法获得的结果在呼吸链中缺乏氧化 NAD(P)H 能力的突变体中也一致，并表现出独特的 NADPH 光响应。这些观察结果表明，PCI 提取方法允许定量测定 NADP(H) 氧化还原。值得注意的是，PCI 提取方法表明，并非所有的 NADP(H) 都被明暗转变氧化或还原。具体来说，在暗适应细胞中NADPH的比例为42%，在光照条件下饱和度为68%。