EPR spectra and antioxidant activity of gamma-irradiated papain

The structure and number of paramagnetic centers (PC) stabilized in papain γ-irradiated with a dose of 50 to 2300 kGy were studied using EPR spectroscopy. The radiation yield G≈6 PC/100 eV during radiolysis at 77 K is six times greater than the PC yield in the samples irradiated at 300 K. During radiolysis at 300 K, the maximum concentration of PC is achieved at doses of 200 kGy at a level of 8 × 10¹⁸ PC/g, whereas in papain radiolyzed at 77 K they accumulate up to 2300 kGy and reach 2.2 × 10²⁰ PC/g. During papain radiolysis at 77 K, the cleavage of the peptide bond prevails over the cleavage of bonds in the molecular groups of amino acid residues, including sulfur-containing ones. As a result, $-\text{C}\left(=0\right)-\stackrel{•}{\text{C}}\text{H}-{\text{CH}}_{\text{2}}\text{R}$ radicals are mainly recorded in the EPR spectra. In the multicomponent spectrum of papain irradiated at 300 K, a doublet with splittings of 1.77 mT is distinguished, attributed to the $-\text{C}\left(=0\right)-\stackrel{•}{\text{C}}\text{H}-\text{NH}-$ radical formed by the abstraction of hydrogen from the glycine residue. Peroxide radicals formed during radiation oxidation at 300 K are not retained in the matrix of irradiated papain as stabilized radicals and, most likely, participate in secondary radiation-chemical processes with the formation of oxygen-containing products. A tendency for the antiradical and antioxidant activity of papain to increase with increasing radiation dose is noted as a result of radiation destruction of the peptide bond with the formation of amino acid fragments that are donors of a hydrogen atom.