Babcock D F
J Biol Chem. 1983 May 25;258(10):6380-9.
Fluorescein and carboxyfluorescein have found recent application as probes of intracellular pH. The present study examines several parameters required for interpretation of the spectral information derived from fluorescein and carboxyfluorescein generated intracellularly from their permeant diacetate derivatives. Coefficients were determined for the pH dependence of the difference absorbance, of the absorbance ratios, and of the fluorescence emission intensity ratios at selected wavelength pairs for carboxyfluorescein in aqueous buffers. The effect of light scattering on the apparent pH reported by carboxyfluorescein in dilute cell suspensions was assessed. An apparent intracellular acidification associated with increasing internal dye concentration was found to result probably from interactions of the intracellular probe with itself. Working within the experimental limitations imposed by these considerations, protocols utilizing either direct measurement of absorbance or fluorescence or determination of the null spectral response observed upon release of internal carboxyfluorescein all indicate that the cytosolic space of bovine epididymal sperm is maintained at pH 6.5-6.6. The monovalent-cation-specific, carboxylic acid ionophores, nigericin and monensin, were utilized to produce transmembrane proton gradients in cells that were allowed to generate intracellular carboxyfluorescein in a preliminary incubation, then resuspended in media buffered at the same pH as the sperm cytosol but of varying cation composition. By interpolation to the null response, the initial internal Na+ and K+ concentrations in bovine sperm were estimated as 14 +/- 2 and 120 +/- 5 mM, respectively. The ability of initial transmembrane gradients of either protons or of monovalent cations to promote equivalent changes in internal pH following ionophore addition to sperm suspensions supports application of a simple model that predicts steady state cation distributions. With the assumption that the cytosolic proton buffer has a pKa near the determined internal pH, these experiments allow the additional calculation that this buffer is present at a concentration of 190 +/- 20 meq/liter in the bovine sperm.
荧光素和羧基荧光素最近被用作细胞内pH值的探针。本研究考察了从其可渗透的二乙酸酯衍生物在细胞内生成的荧光素和羧基荧光素所获得的光谱信息的解释所需的几个参数。测定了羧基荧光素在水性缓冲液中选定波长对处的吸光度差值、吸光度比值和荧光发射强度比值对pH的依赖性系数。评估了光散射对羧基荧光素在稀细胞悬液中报告的表观pH值的影响。发现随着细胞内染料浓度增加而出现的表观细胞内酸化可能是由于细胞内探针自身相互作用所致。在这些考虑因素所施加的实验限制范围内，利用直接测量吸光度或荧光或测定释放细胞内羧基荧光素时观察到的零光谱响应的方案均表明，牛附睾精子的胞质空间维持在pH 6.5 - 6.6。单价阳离子特异性羧酸离子载体尼日利亚菌素和莫能菌素被用于在细胞中产生跨膜质子梯度，这些细胞在初步孵育中被允许产生细胞内羧基荧光素，然后重悬于与精子胞质溶胶pH相同但阳离子组成不同的培养基中。通过内插到零响应，估计牛精子中初始内部Na⁺和K⁺浓度分别为14±2和120±5 mM。质子或单价阳离子的初始跨膜梯度在离子载体添加到精子悬液后促进内部pH值发生等效变化的能力支持了一个预测稳态阳离子分布的简单模型的应用。假设胞质质子缓冲剂的pKa接近测定的内部pH值，这些实验允许进一步计算出该缓冲剂在牛精子中的浓度为190±20 meq/升。
