Palmgren M G
Department of Plant Biochemistry, University of Lund, Sweden.
Anal Biochem. 1991 Feb 1;192(2):316-21. doi: 10.1016/0003-2697(91)90542-2.
Acridine orange is an optical probe commonly used to monitor pH gradients across membranes. In the present study, the changes observed in the visible absorption spectrum of acridine orange during intravesicular acidification of oat root plasma membrane vesicles are shown to be identical with those obtained by increasing the free dye concentration, adding anions, or lowering the temperature, but different from those obtained on addition of biological membranes. It is therefore suggested that the absorbance changes observed during the formation of the pH gradient are simply due to accumulation of free dye inside the vesicles and subsequent dimerization, and not the result of dye-membrane interactions. The proportion of monomeric acridine orange that could undergo dimerization decreased with decreasing temperature. Furthermore, in a membrane-free system different anions induced the formation of dimer-excimer complexes to different degrees. During the formation of the pH gradient permeant anions present in the reaction medium follow the movement of protons into the vesicles, and the intravesicular accumulation of anions thereby amplifies acridine orange quenching, the degree of amplification being dependent on the anion species. Therefore, the use of acridine orange, and probably all metachromatic dyes, as probes for monitoring pH gradients is limited, since these probes neither reflect quantitatively the amount of H+ pumped nor the effect of anions and temperature on transmembrane H+ transport.
吖啶橙是一种常用的光学探针，用于监测跨膜pH梯度。在本研究中，燕麦根质膜囊泡内酸化过程中吖啶橙可见吸收光谱的变化，与通过增加游离染料浓度、添加阴离子或降低温度所获得的变化相同，但与添加生物膜后获得的变化不同。因此，有人认为在pH梯度形成过程中观察到的吸光度变化仅仅是由于游离染料在囊泡内积累并随后二聚化，而不是染料与膜相互作用的结果。可发生二聚化的单体吖啶橙比例随温度降低而减少。此外，在无膜体系中，不同阴离子诱导二聚体-准分子复合物形成的程度不同。在pH梯度形成过程中，反应介质中存在的渗透性阴离子跟随质子进入囊泡，阴离子在囊泡内的积累从而放大了吖啶橙的淬灭，放大程度取决于阴离子种类。因此，使用吖啶橙，可能还有所有的异染染料作为监测pH梯度的探针是有限的，因为这些探针既不能定量反映泵出的H+量，也不能反映阴离子和温度对跨膜H+转运的影响。
