Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide

Abstract

In recent years, the peroxidase enzymes have generated wide interest in several industrial processes, such as wastewater treatments, food processing, pharmaceuticals, and the production of fine chemicals. However, the low stability of the peroxidases in the presence of hydrogen peroxide (H₂O₂) has limited its commercial use. In the present work, the effect of H₂O₂ on the inactivation of horseradish peroxidase (HRP) was evaluated. Three states of HRP (E₀, E₂, and E₃) were identified. While in the absence of H₂O₂, the resting state E₀ was observed, in the presence of low and high concentrations of H₂O₂, E_2, and E₃ were found, respectively. The results showed that HRP catalyzed the H₂O₂ decomposition, forming the species E_x, which was catalytically inactive. Results suggest that this loss of enzymatic activity is an intrinsic characteristic of the studied HRP. A model from a modified version of the Dunford mechanism of peroxidases was developed, which was validated against experimental data and findings reported by the literature.

Figure 1

(a) Soret band, and (b) visible absorption spectra corresponding to the species E₀ (black), E₂ (red), E₃ (green), and E_X (blue). Dotted lines indicate the characteristic absorption bands corresponding to each species.

Figure 2

Change of the UV–Vis spectra of the reaction mixture as a function of time after the repeated additions of 20 µL of H₂O₂ (1 mM) to 3 mL of HRP (500 mg/L of lyophilized enzyme in PB 100 mM, pH 9). In each case, pi represents the number of the pulse of the oxidant.

Figure 3

Enzyme species E₀ (black), E₂ (red), and E₃ (green) as a function of time after the addition of successive pulses (p1 to p6) of H₂O₂ to HRP (500 mg/L) dissolved in phosphate buffer (100 mM, pH 9). Yellow symbols indicate the sum of all the species. Bars represent the standard deviation.

Figure 4

Typical examples of the effect of the initial concentration of H₂O₂ (a), and enzyme (b) on the change of absorbance at 422 nm. Experimental conditions in (a) HRP = 5.2 µM; H₂O₂ (µM) = 26 (black), 48 (red), 67 (green). Experimental conditions in (b) HRP (µM) = 1.7 (black), 3.4 (red), 5.2 (green); H₂O₂ = 26 µM. All assays were performed in PB (100 mM, pH 9) at room temperature. For comparison purposes, results were normalized with respect to the corresponding initial absorbance value (A_i). Continuous lines indicate the proposed model using the coefficients shown in Table 1.

Figure 5

(a) Soret band, and (b) visible absorption spectra corresponding as a function of time. Initial conditions: HRP = 6 µM, H₂O₂ = 550 µM. Dotted lines indicate the characteristic absorption bands corresponding to E₃ (417, 544, and 580 nm), and E_x (670 nm). Arrows indicate the evolution of time.

Figure 6

(a) Total active enzyme concentration (E_A), and (b) peroxidatic activity as a function of time. Enzyme concentrations were obtained from the UV/Vis spectra depicted in Fig. 5. Bars represent the standard deviation. Dotted lines indicate the addition of a new pulse of H₂O₂.

Figure 7

Degradation of E₃ (black) and formation of E_x (red) (a,c,e) and the corresponding H₂O₂ consumption (b,d,f) as a function of time. Bars represent the standard deviation. Continuous lines indicate the proposed model using the coefficients shown in Table 1. Dotted lines represent the time at which the active enzyme concentration was less than 10% of the initial one.

Figure 8

Degradation of HRP as a function of the consumed H₂O₂. Data were obtained from the results shown in Fig. 7. Bars represent the standard deviation.

Figure 9

Effect of the hydrogen peroxide concentration on the fraction of each enzyme species under the absence (black lines) or the presence (red lines) of an external substrate (OII = 1 × 10^–4 M). Calculations were performed using Eqs. (A26) to (A29) along with the coefficients shown in Table 1. For details, see the Supplementary Material, Item 5.