EPR spectra and antioxidant activity of gamma-irradiated papain

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Abstract

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, −C=0−C•H−CH2R 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 −C=0−C•H−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.

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S. V. Tokarev

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

I. I. Faingol’d

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

D. A. Poletaeva

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

A. V. Smolina

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

S. V. Demidov

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

A. V. Akimov

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

U. Yu. Allayarova

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

T. A. Raevskaya

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

S. R. Allayarov

Federal Research Center for Problems of Chemical Physics and Medicinal Chemistry of the RAS

Author for correspondence.
Email: sadush@icp.ac.ru
Russian Federation, Chernogolovka

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2. Fig. 1. Dependence of the change in the concentration of PC on the dose of γ-irradiation at 77 K (a) and on the heating temperature (b) of papain irradiated at 77 K with a dose of 2300 kGy of the sample.

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3. Fig. 2. EPR spectrum of papain γ-irradiated at 77 K. Irradiation dose (kGy): 50 (a), 200 (b), 700 (c), 1700 (d), 2300 (e–i). Registration temperature (K): 77 (a–f), 223 (g), 273 (h), 300 (i). Samples (g–i) were registered during the heating of papain irradiated at 77 K to the indicated temperatures.

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4. Fig. 3. Kinetic curves of the accumulation of paramagnetic centers in papain during radiolysis at 300 K in vacuum (a) and in air (b).

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5. Fig. 4. EPR spectrum of papain γ-irradiated at 300 K in vacuum (solid lines) and in air (dashed lines). Irradiation dose (kGy): 50 (a), 200 (b), 700 (c), 1700 (d), 2300 (d). Registration temperature 300 K.

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6. Fig. 5. EPR spectra of papain γ-irradiated in air at 300 K. Irradiation dose (kGy): 50 (a), 200 (b), 3500 (c). Registration temperature (K): 77 (solid lines), 300 (dashed lines).

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7. Fig. 6. Interception of DPPH radical by irradiated papain, expressed as a percentage. Doses of γ-irradiation (kGy): 0 (a), 250 (b), 500 (c), 1550 (d), 3200 (d).

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8. Fig. 7. Inhibition of malondialdehyde formation as a lipid peroxidation indicator in mouse brain homogenate under the action of irradiated papain. Doses of γ-irradiation (kGy): 0 (a), 250 (b), 500 (c), 1550 (d), 3200 (d).

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