Radiolysis of myoglobin concentrated gels by protons: specific changes in secondary structure and production of carbon monoxide

Abstract

While particle therapy has been used for decades for cancer treatment, there is still a lack of information on the molecular mechanisms of biomolecules radiolysis by accelerated ions. Here, we examine the effects of accelerated protons on highly concentrated native myoglobin, by means of Fourier transform infrared and UV-Visible spectroscopies. Upon irradiation, the secondary structure of the protein is drastically modified, from mostly alpha helices conformation to mostly beta elements at highest fluence. These changes are accompanied by significant production of carbon monoxide, which was shown to come from heme degradation under irradiation. The radiolytic yields of formation of denatured protein, carbon monoxide, and of heme degradation were determined, and found very close to each other: G_{+denatured Mb} ≈ G_+CO ≈ G_-heme = 1.6 × 10^-8 ± 0.1 × 10^-8 mol/J = 0.16 ± 0.01 species/100 eV. The denaturation of the protein to a beta structure and the production of carbon monoxide under ion irradiation are phenomena that may play an important role in the biological effects of ionizing radiation.

Figure 1

Evolution of the secondary structure of myoglobin under irradiation by 2 MeV protons, as determined by infrared spectroscopy. Light-color circles, 30% metMb solution in D₂O. Deep-color circles, protein gel, [metMb]₀ = 1.1 × 10^–2 M in D₂O, Gel thickness 26 µm. Error bars were determined with a duplicate of irradiation experiments.

Figure 2

Evolution of the concentrations of carbon monoxide bound to iron complexes with ion fluence. The quantities were determined from infrared measurements of the bands at 1943 cm⁻¹ and 1964 cm⁻¹. Insert: evolution of IR spectra with increasing irradiation doses, as shown by the arrows. The isosbestic point is observed for ion fluences above 10 ions/cm⁻². Error bars were determined with a duplicate of irradiation experiments.

Figure 3

UV–Visible spectra of myoglobin gel recorded for significant ion fluences. The arrows show the evolution of the Soret band with increasing ion fluence up to 2.7 × 10¹³ ions cm⁻². Soret band of unirradiated protein is saturated due its strong absorption explaining this plateau shape at an absorbance of 2. [metMb]₀ = 1.1 × 10^–2 M, in D₂O. Gel thickness 26 µm.

Figure 4

Concentrations of metMb, deoxyMb, MbCO and total heme determined by MCA of the UV–Visible spectra, as a function of ion fluence. Met-Myoglobin, black dots; deoxy myoglobin, green dots; carboxymyoglobin, blue dots; total heme concentration, red diamonds. [metMb]₀ = 1.1 × 10^–2 M, in D₂O. Gel thickness 26 µm.

Figure 5

Comparison between the evolutions of quantities of carboxymyoglobin MbCO, determined by UV–Visible spectrometry with that of the species absorbing at 1943 cm⁻¹ in infrared, and of total CO measured and global heme degradation, under irradiation. +: total CO concentration, blue triangle: concentration of the species absorbing at 1943 cm⁻¹, pink circle: MbCO concentration, green diamond: global concentration of degraded heme, Yellow open diamonds: Global quantities of degraded heme after subtraction of MbCO degraded.

Figure 6

Proposed description of the formation of heme species identified in myoglobin radiolysis.

Figure 7

Evolution of the concentrations of native myoglobin (blue dots), denatured protein (orange dots), of degraded heme (green diamonds) and total CO (black crosses), with the energy deposited in the sample per liter. Error bars on native and denatured protein concentrations were determined with a duplicate of irradiation experiments on two gels.

Figure 8

Secondary structures determined by various methods for myoglobin, HSA, BSA and β-lactoglobulin. The crystallographic data used for comparison were determined from pdb structures 1WLA, 1AO6, 3V03 and 1BEB respectively.