Determination of the overall dead time with a fast electron-transfer reaction.
Ferrocytochrome c (0.48 mM) was reacted with various concentrations of sodium ferrihexacyanide at 27.5°C. Kinetic traces of the reaction show the data after two-component analysis as the fraction of ferrocytochrome c vs time and after calculating the logarithm of this fraction (inset) for the Soret region 352–450 nm (a); and the α-band region 500–600 nm (b). The buffer was 50 mM potassium phosphate (pH 7, ionic strength is 0.1 M). Final ferrihexacyanide concentrations were: 1, 2, 5, 10, 20, 50 and 100 mM (from blue to yellow). The lines through the data points are second-order fits (1 and 2 mM ferrihexacyanide) and first-order exponentials. The fits all cross in a single point (±1 pixel or ±0.3 μs) where the fraction of reduced cytochrome c equals 1. Experimental data are shown with a time offset of 3.8 μs, i.e. the determined dead time. (c) Observed rates (kobserved) as a function of the ferrihexacyanide concentration. Red Δ are from analysis of the Soret band, red o are from the α-band. Blue ● are the observed rate constants after corrections for the ionic strength (see text) and are the average of the analyses performed for the Soret band and α-band; The blue line is a linear fit through the origin and the data points yielding a second-order rate constant of 0.986 ± 0.028 107 M-1s-1.