When absorption was measured against wavelength at various pH values, the changes at wavelength 408 nm and 470 nm yielded a sigmoidal line with a midpoint at 5.1. This indicated that the absorbance at these two wavelengths depends upon the ionization of the chromophoric portion of TNP-ATP and is unaffected by ionization of ATP. Although this ionization constant of 5.1 is not in physiological range, it has been shown that the absorbance of TNP-ATP is sensitive enough to detect changes due to slight shifts in neutral pH. Spectroscopic superposition indicated TNP-ATP’s isosbestic point to be 339 nm.