Parui Partha Pratim, Sarakar Yeasmin, Majumder Rini, Das Sanju, Yang Hongxu, Yasuhara Kazuma, Hirota Shun
Department of Chemistry , Jadavpur University , Kolkata 700032 , India . Email:
Division of Materials Science , Nara Institute of Science and Technology , Nara 630-0192 , Japan.
Chem Sci. 2019 Aug 6;10(39):9140-9151. doi: 10.1039/c9sc02993a. eCollection 2019 Oct 21.
The activities of biomolecules are affected by the proton concentrations at biological membranes. Here, we succeeded in evaluating the interface proton concentration (-log[H] defined as pH') of cardiolipin (CL)-enriched membrane models of the inner mitochondrial membrane (IMM) using a spiro-rhodamine-glucose molecule (RHG). According to fluorescence microscopy and H-NMR studies, RHG interacted with the Stern layer of the membrane. The acid/base equilibrium of RHG between its protonated open form (o-RHG) and deprotonated closed spiro-form (c-RHG) at the membrane interface was monitored with UV-vis absorption and fluorescence spectra. The interface pH' of 25% cardiolipin (CL)-containing large unilamellar vesicles (LUVs), which possess similar lipid properties to those of the IMM, was estimated to be ∼3.9, when the bulk pH was similar to the mitochondrial intermembrane space pH (6.8). However, for the membranes containing mono-anionic lipids, the interface pH' was estimated to be ∼5.3 at bulk pH 6.8, indicating that the local negative charges of the lipid headgroups in the lipid membranes are responsible for the deviation of the interface pH' from the bulk pH. The peroxidase activity of cyt increased 5-7 fold upon lowering the pH to 3.9-4.3 or adding CL-containing (10-25% of total lipids) LUVs compared to that at bulk pH 6.8, indicating that the pH' decrease at the IMM interface from the bulk pH enhances the peroxidase activity of cyt . The peroxidase activity of cyt at the membrane interface of tetraoleoyl CL (TOCL)-enriched (50% of total lipids) LUVs was higher than that estimated from the interface pH', while the peroxidase activity was similar to that estimated from the interface pH' for tetramyristoyl CL (TMCL)-enriched LUVs, supporting the hypothesis that when interacting with TOCL (not TMCL), cyt opens the heme crevice to substrates. The present simple methodology allows us to estimate the interface proton concentrations of complex biological membranes.
生物分子的活性受生物膜上质子浓度的影响。在此，我们利用螺罗丹明 - 葡萄糖分子（RHG）成功评估了线粒体内膜（IMM）富含心磷脂（CL）的膜模型的界面质子浓度（定义为pH'的 -log[H]）。根据荧光显微镜和H - NMR研究，RHG与膜的斯特恩层相互作用。通过紫外 - 可见吸收光谱和荧光光谱监测RHG在膜界面处质子化开放形式（o - RHG）和去质子化封闭螺环形式（c - RHG）之间的酸碱平衡。当本体pH与线粒体膜间隙pH（6.8）相似时，含有25%心磷脂（CL）的大单层囊泡（LUVs）（其脂质特性与IMM相似）的界面pH'估计约为3.9。然而，对于含有单阴离子脂质的膜，在本体pH 6.8时界面pH'估计约为5.3，这表明脂质膜中脂质头部基团的局部负电荷是界面pH'与本体pH偏差的原因。与本体pH 6.8相比，将pH降低至3.9 - 4.3或添加含CL（占总脂质的10 - 25%）的LUVs后，细胞色素c的过氧化物酶活性增加了5 - 7倍，表明IMM界面处pH'相对于本体pH的降低增强了细胞色素c的过氧化物酶活性。在富含四油酰基CL（TOCL）（占总脂质的50%）的LUVs的膜界面处，细胞色素c的过氧化物酶活性高于根据界面pH'估计的值，而对于富含四肉豆蔻酰基CL（TMCL）的LUVs，过氧化物酶活性与根据界面pH'估计的值相似，这支持了以下假设：当与TOCL（而非TMCL）相互作用时，细胞色素c会向底物打开血红素裂隙。这种简单的方法使我们能够估计复杂生物膜的界面质子浓度。
