Horie K, Hagihara H, Wada A, Fukutome H
Anal Biochem. 1984 Feb;137(1):80-7. doi: 10.1016/0003-2697(84)90350-6.
A highly sensitive quantitative method was developed to detect protons released or taken up upon ligand binding. A small change in pH due to proton release or uptake was detected by measuring the difference in the absorbance of a pH indicator upon ligand addition. Owing to the difference detection of protons, the uncertainty of pH due to CO2 dissolution and unknown buffering capacities of sample solutes could be compensated with easy manipulations. Precise calibration of the absolute amount of protons could also be made very easily. The amount of protons measurable by the method is as small as 0.5 nmol that is 10 to 30 times more sensitive than the pH-stat method. We measured the Mg2+ ion-induced proton releases of ADP to confirm the accuracy and reliability of the method and of Escherichia coli ribosomes to show the improvement in sensitivity. The method is useful for protometric studies of biomolecules that are difficult to obtain in large amount.
开发了一种高灵敏度定量方法，用于检测配体结合时释放或摄取的质子。通过测量加入配体后pH指示剂吸光度的差异，检测由于质子释放或摄取导致的pH微小变化。由于对质子的差异检测，因二氧化碳溶解和样品溶质未知缓冲能力导致的pH不确定性可通过简单操作得到补偿。质子绝对量的精确校准也可非常容易地进行。该方法可测量的质子数低至0.5 nmol，比pH计法灵敏10至30倍。我们测量了Mg2+离子诱导的ADP质子释放，以确认该方法的准确性和可靠性，并测量了大肠杆菌核糖体的质子释放，以展示灵敏度的提高。该方法对于难以大量获得的生物分子的质子计量学研究很有用。
