Assessment of Catalase Inhibition Under e-Beam Irradiation

Abstract

Catalase serves as a crucial component of the antioxidant defense system by catalyzing the decomposition of hydrogen peroxide into water and molecular oxygen. This study investigated the effects of 1 MeV accelerated electron irradiation on catalase activity in model solutions at doses of 100 Gy and 1000 Gy. Enzyme activity was assessed using two complementary methods: spectrophotometric analysis and the oxygen bubble method. The experimental results demonstrated dose-dependent inhibition of catalase activity, indicating that substantial radiation-induced structural modifications may occur in the enzyme molecule as a result of irradiation. To understand the relationship between the irradiation dose and the catalase inhibition, calibration curves plotting the dependencies of hydrogen peroxide decomposition rate and the delayed appearance of oxygen bubbles after adding hydrogen peroxide to catalase saline solution on the catalase concentration showed a 1.5-fold reduction in catalase activity at 100 Gy and a 40-fold decrease at 1000 Gy. Based on these findings, we propose a novel biodosimetry approach utilizing the oxygen bubble formation delay time as an express assessment tool for detecting high radiation doses absorbed by biological objects, for example, food products. The results obtained in the study have important implications for evaluating radiation effects on biological systems, in particular catalase-containing food products, offering potential applications in radiation safety monitoring and food quality control.

Keywords: accelerated electrons; biodosimetry; catalase; catalase activity; hydrogen peroxide; oxygen bubbles; radiation inhibition; radiation processing; spectrophotometry.

Figure 1

Stages of experimental studies: (1) preparation of catalase basic solution; (2) studies with different concentrations of catalase solution alpha (further referred to as α); (3) accelerated electron irradiation of solutions α and studies with irradiated solutions beta (further referred to as β); (4) spectrophotometry measurement of solutions α and β; (5) measurement of oxygen bubble formation in solutions α and β.

Figure 2

Absorption spectra of H₂O₂ (P, curve 1), catalase (Cat_0.03, curve 2), and mix solution (P+Cat_0.03, curve 3) after e-beam irradiation at different doses: (A) D₀ (control), (B) D₁₀₀, and (C) D₁₀₀₀. t_inc = 30 min; λ = 190–400 nm. The red dotted line marks the wavelength λ = 240 nm. The symbol ∆ and the black double headed arrow marks the difference in optical density (absorbances) between P and P+Cat_0.03 at λ = 240 nm.

Figure 3

Absorption spectra of P+Cat_0.03 solutions after accelerated electron irradiation at different doses: (A) D₀ (control), (B) D₁₀₀, and (C) D₁₀₀₀. t_inc = 1–180 min; λ = 190–400 nm. The red dotted line marks the wavelength λ = 240 nm. (D,E) Kinetic curves A(t); symbols represent experimental values (at λ = 240 nm). Fitting curves (Equation (1)) are represented by lines—D₀ (curve 1), D₁₀₀ (curve 2), and D₁₀₀₀ (curve 3). Normalization was performed on the value of optical density at t_inc = 0. (D) The measurements are presented in the range t_inc = 0–18,000 s (5 h); the time of reaching the plateau (t_pl) is indicated by dotted lines (t_pl(3))D₁₀₀₀ for curve 3. (E) The measurements are presented in the range t_inc = 0–1200 s (20 min), and the times of reaching the plateaus are indicated by dotted lines (t_pl(1))D₀ for curve 1, (t_pl(2))D₁₀₀ for curve 2 (E).

Figure 4

Absorption spectra of Cat model solutions after accelerated electron irradiation at different doses (t_inc = 10 min): (A) Cat_0.03 and Cat_0.3 at dose D₀; (B) Cat_0.03 at doses D₀, D₁₀₀, and D₁₀₀₀; (C) Cat_0.3 at doses D₀, D₁₀₀, and D₁₀₀₀. Kinetics of changes in the absorption spectra of Cat_0.3 model solutions at doses D₁₀₀ (D) and D₁₀₀₀ (E). t_inc = 1–300 min; λ = 190–400 nm. The color of the absorption spectra of catalase model solutions from light tint to dark tint corresponds to increasing t_inc. Blue colors—correspond to the dose of 100 Gy, green colors—1000 Gy.

Figure 5

Photographs of oxygen bubbles in P+Cat_0.03 solutions after e-beam irradiation at doses of D₀, D₁₀₀, and D₁₀₀₀. (A) t_inc = 60 min; (B) t_inc = 1–20 min.

Figure 6

Kinetics of changes in oxygen bubble parameters in P+Cat_0.03 solutions after accelerated electron irradiation at doses D₀ (curve 1), D₁₀₀ (curve 2), and D₁₀₀₀ (curve 3). (A) Number of bubbles N(t_inc), (B) average diameter of bubbles d(t_inc), (C) total volume of bubbles V(t_inc). Dots represent experimental values (with error symbols), and curves show fitting curve (Equation (3)). The dotted lines indicate the delay time τ.

Figure 7

Absorption spectra of H₂O₂ (P, curve 1), catalase (Cat_i, curve 2), and mixed solution (P+Cat_i, curve 3) at different concentrations of catalase i. (A) Cat_0.03, (B) Cat_0.003, and (C) Cat_0.0003. t_inc = 30 min; λ = 190–400 nm. The red dotted line marks the wavelength λ = 240 nm. The symbol ∆ and the black double headed arrow marks the difference in optical density between P and P+Cat_i at 240 nm.

Figure 8

Absorption spectra of P+Cat_i solutions at different concentrations of catalase i: (A) Cat_0.03, (B) Cat_0.003, and (C) Cat_0.0003 at t_inc = 1–180 min; λ = 190–400 nm. The red dotted line marks the wavelength λ = 240 nm. (D) Kinetic curves A(t); symbols represent experimental values (at λ = 240 nm). Fitting curves (Equation (1)) are represented by lines—Cat_0.03 (curve 1), Cat_0.003 (curve 2), Cat_0.0005 (curve 3), and Cat_0.0003 (curve 4). Normalization was performed on the value of optical density at t_inc = 0. The measurements are presented in the range t_inc = 0–18,000 s (5 h). The times of reaching the plateau $t_{pl\left(1\right)}$Cat_0.03 = 370 ± 10 s for curve 1, $t_{pl\left(2\right)}$Cat_0.003 = 2600 ± 50 s for curve 2, $t_{pl\left(3\right)}$Cat_0.0005 = 4000 ± 60 s for curve 3, and $t_{pl\left(4\right)}$Cat_0.0003 = 17,000 ± 100 s for curve 4, are indicated by dotted lines.

Figure 9

Photographs of oxygen bubbles in P+Cat_i solutions at different concentrations of Cat_0.0003-Cat_0.3 catalase. (A) t_inc = 60 min; (B) t_inc = 1–20 min. The time—when the bubbles were not identified—is highlighted in red, the time of their inception and development is highlighted in green.

Figure 10

Kinetics of changes in oxygen bubble parameters in P+Cat_i solutions at different concentrations of model catalase solutions: Cat_0.0005 (curve 1), Cat_0.003 (curve 2), Cat_0.03 (curve 3), and Cat_0.3 (curve 4). (A) N(t_inc), (B) d(t_inc), and (C) V(t_inc). Dots represent experimental values, and curves show fitting curve (Equation (3)). The dotted lines indicate the delay time τ.

Figure 11

The experimental data (with error symbols) and calibration linear regressions: (A) υ_A(C_Cat)—red calibration line and (B) 1/τ(C_Cat)—brown calibration line. Areas of study for small concentrations of Cat model solutions are highlighted in circles, with subsequent zooming in.

Figure 12

Dependence of the intact catalase concentration C_Cat as a function of irradiation dose D. Experimental points (squares). Parameters of fitting curve Equation (6) (brown line): C₀ = 0.03 ± 0 units, φ = 0.0041 ± 0.0002 Gy⁻¹, and correlation coefficient 0.98.

Figure 13

Dependence of the delay time τ on the irradiation dose D. Fitting curve parameters Equation (7) (purple line): τ₀ = 1.89 ± 0.36 min, D_max = 1037 ± 13 Gy, K = 0.40 ± 0.13 min·Gy, and correlation coefficient 0.98.