Purification and characterization of a phenoloxidase in the hemocytes of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

Figures & data

Table 1. Purification of PO from the hemolymph of E. kuehniella.

Purification steps	U	Protein (mg)	U/mg protein	Recovery (%)	Purification fold
Crude sample	0.41 ± 0.054	2.21	0.070 ± 0.021	100	1
Amonium sulfate 70%	0.163 ± 0.032	1.35	0.120 ± 0.017	39.75	1.71
Sepharyl G-100	0.099 ± 0.047	0.70	0.142 ± 0.025	24.14	2.02
SEAE-Cellulose fast flow	0.084 ± 0.070	0.072	1.17 ± 0.097	20.48	16.71

Figure 1. Optimal pH and temperature of the purified PO from the hemolymph of E. kuehniella. Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Figure 2. Double reciprocal plot to show the kinetic parameters of the purified PO from E. kuehniella by using L-DOPA, pyrocatechol and hydroquinone as substrates. (1/Vmax = intercept on the 1/V0 ordinate, −1/Km = intercept on the negative side of the 1/[S] abscissa).

Table 2. Kinetic parameters of the purified PO from E. kuehniella.

Substrate	Vmax* (U/mg protein)	Km* (mM)	R^2
L-DOPA	1.92 ± 0.12a	8.32 ± 2.36a	0.97
Pyrocatechol	0.420 ± 0.34c	10.27 ± 4.55a	0.80
Hydroquinone	1.19 ± 0.25b	9.77 ± 3.13a	0.96

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Table 3. Effects of mono- and divalent cations on the purified PO activities of E. kkuehniella.

Cations	Concentrations (mM)	Relative activity of PO (%) ± SE*
Na^+	Control (0)	100 ± 5.72b
Na^+	0.5	58 ± 6.78a
Na^+	3	78 ± 4.12a
Na^+	5	54 ± 14.54a
K^+	Control (0)	100 ± 12.98a
K^+	0.5	92.98 ± 5.70ab
K^+	3	76.97 ± 5.62c
K^+	5	78.94 ± 4.31bc
Mn^+	Control (0)	100 ± 6.25a
Mn^+	0.5	30.17 ± 15.17b
Mn^+	3	10.94 ± 1.93b
Mn^+	5	0 ± 0c
Zn^2+	Control (0)	100 ± 4.36a
Zn^2+	0.5	91.08 ± 7.36ab
Zn^2+	3	79.06 ± 2.01ab
Zn^2+	5	63.06 ± 26.57b
Mg^2+	Control (0)	100 ± 27.30a
Mg^2+	0.5	72.04 ± 6.81ab
Mg^2+	3	33.33 ± 19.24c
Mg^2+	5	52.59 ± 8.21b
Ca^2+	Control (0)	100 ± 21.85b
Ca^2+	0.5	322 ± 17.20a
Ca^2+	3	284 ± 8.14a
Ca^2+	5	296 ± 21.37a
Cu^2+	Control (0)	100 ± 12.28c
Cu^2+	0.5	224 ± 16.75b
Cu^2+	3	303 ± 8.11ab
Cu^2+	5	317 ± 15.64a

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Table 4. Effects of chelating agents on the purified PO activities of E. kkuehniella.

Compounds	Concentrations (mM)	Relative activity of PO (%) ± SE*
EDTA	Control (0)	100 ± 6.49a
	2	71.91 ± 7.00ab
	4	53.55 ± 7.01b
	6	57.30 ± 9.77b
	8	63.25 ± 3.64b
	10	25.09 ± 5.09c
DTC	Control (0)	100 ± 22.55a
	2	30.27 ± 15.16b
	4	9.07 ± 3.62c
	6	10.92 ± 6.64c
	8	8.96 ± 0.60c
	10	3.27 ± 0.94c
EGTA	Control (0)	100 ± 27.35a
	2	28.98 ± 3.62b
	4	45.65 ± 5.47b
	6	77.53 ± 6.91ab
	8	39.85 ± 20.17b
	10	69.56 ± 6.52ab
TTHA	Control (0)	100 ± 14.06b
	2	102.15 ± 5.68a
	4	105.5 ± 24.35a
	6	117.84 ± 20.25a
	8	112.2 ± 30.65a
	10	110 ± 15.83a

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Table 5. Inhibition parameters of hexaflumuron and pyriproxyfen on the purified PO of E. kuehniella.

IGR	IC50 (µg/ml)	Slope ± SE	CL (upper and lower limits)	X^2	df
Hexaflumuron	96.41*	1.51 ± 0.107	60.73–161.53	12.73	4
Pyriproxyfen	38.59	1.59 ± 0.136	15.51–86.83	29.12	3

*Asterisk shows statistical differences between two used IGRs by t-test (p ≤ 0.05).

Table 6. Kinetic parameters of the purified PO from E. kuehniella in control and treated conditions of hexaflumuron and pyriproxyfen.

Treatments	Vmax (U/mg protein)	Km (mM)	R^2
Control*	1.92 ± 0.12a	8.32 ± 2.36a	0.97
Hexaflumuron treated	2 ± 0.54a	10.92 ± 1.35b	0.94
Pyriproxyfen treated	1.78 ± 0.39a	15.66 ± 0.79c	0.96

*Control means without any IGR. Used substrate for the experiment was L-DOPA. Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).