High Energy Radiation Effects in Single Histones. I. Preparation of Histones and Irradiation of Histone H2B

Summary

Histone H2B from calf thymus was irradiated with 50 or 100 ns pulses of 16 MeV electrons in N₂O-saturated aqueous solution at pH 9 in the presence of NaN₃. All tryrosine moieties in the histone were found to be freely accessible to the attack of ·N₃ radicals (formed by the reaction ·OH + N⁻₃ → OH⁻ + ·N₃). At sufficiently high concentrations of H2B, tyrosyl radicals were formed with G(TyrO·) = 5·4/100 eV and dityrosine groups with G(dityr) = 1·6/100 eV, indicating that about 60 per cent of tyrosyl radicals formed bisphenolic products. There is no polymer effect with respect to G(dityr) as inferred from comparison with other authors' data obtained with low molecular weight compounds.

Kinetic measurements revealed that tyrosyl radicals reacted in two modes, a fast one with a value of τ_1/2 of about several milliseconds and a slow second order process also in the millisecond range. The fast process is assigned to intramolecular reactions of tyrosyl radicals generated in close proximity to each other and the slow process to intermolecular self reactions of isolated tyrosyl radicals distributed statistically in the solution. There is a polymer effect with respect to the rate constant of the slow process: 2k₈ = 4·8 × 10⁷ dm³ mol⁻¹ s⁻¹ (H2B) and 2k₈ = 4 × 10⁸ dm³ mol⁻¹ s⁻¹ (Lys-Tyr-Lys, Prütz et al. (1983)). The five histones contained in calf thymus were isolated chromatographically with the aid of two gels, Bio-Gel P-60 (BioRad) and Sephadex G100 (Pharmacia).

• histone H2B
• ·N ³ radicals
• dityrosine groups