Diode array detector

Reduction of CYB5 and electron transfer from ferrous CYB5 to the ferric globins was studied under anaerobic conditions. UV-visible spectra and kinetic data were recorded on an SX20 stopped-flow spectrophotometer equipped with a diode-array detector (Applied Photophysics). CYB5 was reduced to the ferrous form with excess sodium dithionite, which was removed using a Sephadex G25 column (PD10, GE Healthcare) equilibrated with 100 mM sodium phosphate buffer. Globins were oxidized with excess potassium ferricyanide (ferricyanide solutions ~1 M were added in a 1:10 ferricyanide:protein ratio (v/v) and incubated for 5–60 seconds at room temperature) and then excess oxidant was removed as described above. The absorbance at 568 nm was used to follow globin reduction, as this wavelength was found to be isosbestic for the transition of CYB5 from the ferrous to the ferric state, but not for that of any of globins tested. The final concentration of all globins was maintained between 5 and 10 μM, while the CYB5 concentration was varied from as high as 60 μM to less than 10 μM. Reactions were followed at 25 °C in 100 mM sodium phosphate, pH 7.4.

The rate of oxygen dissociation from Ngb-H64Q-CCC was determined by ligand replacement. The experiments were carried out at 25 °C in an Applied Photophysics SX-20 stopped-flow spectrophotometer with a diode array detector (Applied Photophysics Ltd.) contained in an anaerobic glove box (Coy Laboratory Products). 100 mM sodium phosphate buffer, pH 7.4 was used for all the solutions. The Ngb samples were reduced by excess sodium dithionite in the glove box and the deoxy-Ngb was run through a gravity size-exclusion column (Econopac 10DG, BioRad) to remove the excess of reductant. The protein was mixed with air-saturated buffer ([O₂] ≈ 260μM) in 1:4 (v/v) or 1.5:1 (v/v) ratios to form quantitatively the Ngb-oxy complex and achieve final oxygen concentrations of ≈ 208 μM (1:4) or ≈ 104 μM (1.5/1). This sample was then mixed different concentrations of CO buffer made by mixing CO-saturated buffer ([CO] ≈ 1 mM) and anaerobic buffer in the stopped-flow instrument. The spectrum in the visible range (350–730 nm) was sampled every 1.24 ms for a reaction time of 1 s. Spectral changes were consistent with a decay of the Ngb-oxy complex to form the Ngb-CO species. Traces over the whole spectral range (300–730nm) where fitted simultaneously for each experiment using the Pro-K software (Applied Photophysics Ltd.) to calculate the observed oxygen dissociation rate.