A highly sensitive photometric method for proton release or uptake: difference protometry.

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.