Cryoradiolytic reduction of heme proteins: Maximizing dose dependent yield

Abstract

Radiolytic reduction in frozen solutions and crystals is a useful method for generation of trapped intermediates in protein based radical reactions. In this communication we define the conditions which provide the maximum yield of one electron reduced myoglobin at 77 K using (60)Co γ-irradiation in aqueous glycerol glass. The yield reached 50% after 20 kGy, was almost complete at ∼160 kGy total dose, and does not depend on the protein concentration in the range 0.01 - 5 mM.

Figure 1

A Absorption spectra of γ-irradiated met Mb measured at 77 K after photobleaching with white light. (1) Met Mb before irradiation; (2) 10 kGy dose; (3) 20 kGy; (4) 40 kGy; (5) 70 kGy. The samples were prepared from the same stock solution and irradiated at 77 K up to the shown dose. B Absorption spectra of met Mb (ferric) and cryoreduced Mb (ferrous) measured at 77 K and shown in millimolar absorption units. These spectra were used as the spectral basis for the cryoreduction yield measurements. Insert: Spectra of irradiated apo Mb at a dose 70 kGy after photobleaching. Protein concentration were: (1) 0.6 mM, (2) 0.15 mM.

Figure 2

Absorption spectra of γ-irradiated metMb. (1) Cryoreduced met Mb after 90 kGy dose before photobleaching with white light; (2) Same sample after photobleaching; (3) Same sample before irradiation shown for comparison. Concentration of Mb was 4.7 mM, path length 0.05 mm.

Figure 3

Annealing of cryoreduced met Mb (0.04 mM). Spectra were measured after photobleaching at different temperatures: (1) 90 K; (2) 153 K; (3) 188 K.

Figure 4

Calculation of the yield of radiolytic reduction from the experimental absorption spectra of γ-irradiated met Mb. (1) Spectrum of met Mb before irradiation; (2) Spectrum after γ-irradiation at 70 kGy and photobleaching with the white light; (3) Background subtracted as described in Materials and Methods; (4) Full line is the spectrum (2) with subtracted background (3); dotted line is the fit of this spectrum with two basis spectra shown in Fig. 1B. (5) Residuals.

Figure 5

Yield of cryoreduction of met Mb as a function of γ-irradiation dose for different protein concentrations. (x) 0.02 mM; (+) 0.04 mM; (o) 0.15 mM; (*) 4.7 mM.