A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II)

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im₄]²⁺→ [Cu(II)EDTA]²⁻, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t_Q), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV–visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu²⁺ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu²⁺ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]²⁻ and tetraimidazole Cu(II) ([Cu(Im)₄]²⁺) in X-band EPR spectra. The results are implemented in a simple high-precision ‘recipe’ for determining t_Q. These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.