RESUMO
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+â [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.