Inhibitory effects of selected pesticides on peroxidases purified by affinity chromatography

Figures & data

Table 1. Purification steps of rPOD and tPOD with a sepharose-4B-tyrosine-4-aminobenzohydrazide affinity column chromatography.

Purification steps		Total volume (mL)	Enzyme activity (EU/mL min)	Protein (mg/mL)	Total enzyme activity (EU/mL⋅min)	Total protein (mg)	Specific activity (EU/mg)	Yield (%)	Purification fold
rPOD	Crude homogenate	5	22.4	0.783	112	3.915	28.61	100	1.0
	Sepharose-4B-l-tyrosine-4-aminobenzohydrazide affinity chromatography	5	1.546	0.0015	7.73	0.0075	1030.67	6.9	36.02
tPOD	Crude homogenate	5	31.46	0.6116	157.3	3.058	51.351	100	1.0
	Sepharose-4B-l-tyrosine-4-aminobenzohydrazide affinity chromatography	5	4.056	0.0003	20.28	0.0015	13520	12.89	263.29

rPOD: peroxidase purified from black radish (Raphanus sativus L.); tPOD: peroxidase purified from turnip (Brassica rapa L.).

Table 2. IC₅₀ value, K_i constant, and inhibition type of pesticides for rPOD

Compounds	IC50 (mM)	r^2	Ki (mM)	Inhibition type
2,4-d-acid dimethylamine	2.68	0.9874	5.33 ± 1.43	Noncompetitive
Fenoxaprop-p-ethyl	7.70 × 10^−2	0.9482	7.12 × 10^−2 ± 1.83 × 10^−2	Uncompetitive
Glyphosate, isopropylamine	7.70	0.9151	6.76 ± 1.59	Uncompetitive
Haloxyfop-p-methyl	23.0	0.9180	220.0 ± 23.0	Competitive
Cypermethrin	3.85 × 10^−2	0.9412	2.14 × 10^−2 ± 0.08 × 10^−2	Competitive
λ-Cyhalothrin	1.82 × 10^−2	0.9591	3.80 × 10^−2 ± 0.72 × 10^−2	Competitive
Dichlorvos	16.0	0.9882	11.7x±4.4	Noncompetitive

rPOD: peroxidase purified from black radish (Raphanus sativus L.).

Table 3. IC₅₀ values, K_i constants, and inhibition types of pesticides for peroxidase purified from turnip (Brassica rapa L.) (tPOD).

Compounds	IC50 (mM)	r^2	Ki (mM)	Inhibition type
2,4-d-acid dimethylamine	7.07	0.9558	6.98 ± 2.97	Competitive
Fenoxaprop-p-ethyl	4.07 × 10^−2	0.9044	2.87 × 10^−2 ± 0.41 × 10^−2	Competitive
Glyphosate, isopropylamine	0.147	0.8990	1.230 ± 0.240	Uncompetitive
Haloxyfop-p-methyl	17.30	0.9475	0137 ± 0.024	Competitive
Cypermethrin	63.0 × 10^−2	0.9718	1.89 × 10^−2 ± 0.26 × 10^−2	Competitive
λ-Cyhalothrin	3.01 × 10^−2	0.9320	1.23 × 10^−2 ± 0.21 × 10^−2	Noncompetitive
Dichlorvos	1.48 × 10^−2	0.9364	4.76 × 10^−2 ± 1.20 × 10^−2	Competitive

Figure 1. (A) Activity (%)–[λ-cyhalothrin] plot for the tPOD in five different λ-cyhalothrin concentrations; (B) Lineweaver–Burk graph of λ-cyhalothrin for the tPOD using three different λ-cyhalothrin concentrations for the determination of K_i and inhibition type; (C) activity (%)–[λ-cyhalothrin] plot for the rPOD in five different λ-cyhalothrin concentrations; (D) Lineweaver–Burk graph of λ-cyhalothrin for the rPOD using three different λ-cyhalothrin concentrations for the determination of K_i and inhibition type.

Figure 2. Chemical structures of the used pesticides.