Aqueous two-phase system purification for superoxide dismutase induced by menadione from Phanerochaete chrysosporium

Figures & data

Table 1. Effects of molecular weight of PEG and system composition in PEG–K₂HPO₄ on menadione induced SOD partitioning at 25 °C

Sample no.	System	A t (U)	A b (U)	K e	K p	PFt	PFb	R t (%)	R b (%)
1	PEG 1000 10% – K2HPO4 20%	87.12	31.39	2.77	0.25	1.67	0.15	34.47	12.06
2	PEG 1000 15% – K2HPO4 15%	86.99	71.79	1.21	0.57	0.92	0.43	33.42	27.58
3	PEG 2000 10% – K2HPO4 20%	46.11	38.75	1.19	0.27	0.83	0.19	17.72	14.89
4	PEG 2000 15% – K2HPO4 15%	45.38	59.28	0.76	0.55	0.49	0.35	17.43	22.77
5	PEG 3350 10% – K2HPO4 20%	37.45	41.91	0.89	0.25	0.72	0.20	14.39	16.11
6	PEG 3350 15% – K2HPO4 15%	41.31	53.48	0.77	0.48	0.49	0.30	15.87	20.55
7	PEG 6000 10% – K2HPO4 20%	32.82	48.71	0.67	0.14	1.02	0.21	12.61	18.71
8	PEG 6000 15% – K2HPO4 15%	27.54	53.61	0.51	0.18	0.69	0.24	10.58	20.59
9	PEG 8000 10% – K2HPO4 20%	19.83	54.43	0.36	0.09	0.92	0.23	7.62	20.92
10	PEG 8000 15% – K2HPO4 15%	13.12	60.58	0.22	0.12	0.47	0.26	5.04	23.28

About 0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 µg was present in each tube.

Table 2. Effects of molecular weight of PEG and system composition in PEG–K₂HPO₄ on menadione-induced SOD partitioning at 25 °C

Sample no.	System	A t (U)	A b (U)	K e	K p	PFt	PFb	R t (%)	R b (%)
1	PEG 1000 20% - K2HPO4 8%	88.12	133.19	0.66	1.33	0.59	1.19	33.86	51.18
2	PEG 1000 22% - K2HPO4 7%	87.12	134.14	0.65	1.06	0.65	1.06	33.47	51.55
3	PEG 2000 20% - K2HPO4 8%	47.37	139.25	0.34	1.69	0.28	1.44	18.20	53.51
4	PEG 2000 22% - K2HPO4 7%	45.77	145.18	0.32	1.44	0.29	1.36	17.59	55.79
5	PEG 3350%20 - K2HPO4%5	43.39	234.17	0.19	0.68	0.41	1.51	16.67	89.98
6	PEG 3350%22 - K2HPO4%4	44.51	176.75	0.25	0.16	1.22	0.79	17.10	67.92
7	PEG 3350%22 - K2HPO4%3	— No phase formation —
8	PEG 6000%20 - K2HPO4%5	34.03	87.64	0.39	0.13	1.14	0.38	13.08	33.68
9	PEG 6000%22 - K2HPO4%4	27.58	46.50	0.59	0.17	0.72	0.21	10.60	17.87
10	PEG 6000%22 - K2HPO4%3.5	31.09	39.10	0.80	0.29	0.53	0.19	11.95	15.02
11	PEG 6000%20 - K2HPO4%3	— No phase formation —
12	PEG 6000%22 - K2HPO4%3
13	PEG 8000%20 - K2HPO4%5	18.92	46.42	0.41	0.11	0.73	0.19	7.27	17.84
14	PEG 8000%22 - K2HPO4%4	19.49	45.85	0.42	0.14	0.61	0.20	7.48	17.62
15	PEG 8000%22 - K2HPO4%3.5	16.19	44.82	0.36	0.15	0.48	0.19	6.22	17.22
16	PEG 8000%20 - K2HPO4%3	— No phase formation —
17	PEG 8000%22 - K2HPO4%3

0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 µg was present in each tube.

Table 3. Effects of PEG 3350 concentration and system pH on menadione induced SOD partitioning at 25 °C

Sample no.	System	pH	A t (U)	A b (U)	K e	K p	PFt	PFb	R t (%)	R b (%)
1	PEG 3350%20 - K2HPO4%5	9.46	43.39	234.17	0.19	0.68	0.41	1.51	16.67	89.98
2	PEG 3350%20 - K2HPO4%5	7.68	102.23	147.13	0.69	2.38	0.55	1.91	39.26	56.54
3	PEG 3350%22 - K2HPO4%5	9.5	50.44	229.62	0.22	0.80	0.44	1.59	19.38	88.24
4	PEG 3350%22 - K2HPO4%5	7.98	105.52	143.80	0.73	3.04	0.54	2.23	40.55	55.26
5	PEG 3350%24 - K2HPO4%5	9.37	51.70	223.60	0.23	1.12	0.38	1.82	19.87	85.92
6	PEG 3350%24 - K2HPO4%5	7.67	105.74	143.58	0.74	5.15	0.49	3.39	40.63	55.17
7	PEG 3350%26 - K2HPO4%5	9.73	44.34	226.63	0.19	1.88	0.26	2.51	17.04	87.09
8	PEG 3350%26 - K2HPO4%5	7.43	100.93	148.39	0.68	3.48	0.49	2.55	38.79	57.02

0.5 mL crude extract with SOD activity of 260.4 U and total protein amount of 700 µg was present in each tube.

Table 4. Effects of system pH on menadione induced SOD partitioning at 25 °C

Sample no.	System	pH	A t (U)	A b (U)	K e	K p	PFt	PFb	R t (%)	R b (%)
1	PEG 3350%24 - K2HPO4%5	9.00	36.19	132.24	0.27	1.22	0.35	1.58	19.41	71.11
2	PEG 3350%24 - K2HPO4%5	8.50	35.76	113.91	0.31	3.56	0.25	2.79	19.23	61.24
3	PEG 3350%24 - K2HPO4%5	8.00	44.50	97.22	0.46	4.56	0.29	2.91	23.93	52.27
4	PEG 3350%24 - K2HPO4%5	7.75	61.96	85.53	0.72	4.32	0.41	2.45	33.30	45.99
5	PEG 3350%24 - K2HPO4%5	7.50	51.17	108.06	0.47	5.87	0.32	3.99	27.51	58.10
6	PEG 3350%24 - K2HPO4%5	7.25	41.69	132.08	0.32	6.32	0.26	5.19	22.41	71.01
7	PEG 3350%24 - K2HPO4%5	7.00	24.50	146.75	0.17	6.65	0.15	6.04	13.17	78.90
8	PEG 3350%24 - K2HPO4%5	6.75	24.31	112.42	0.22	5.12	0.16	3.69	13.07	60.44
9	PEG 3350%24 - K2HPO4%5	6.50	22.35	89.54	0.25	4.48	0.15	2.64	12.01	48.14

0.5 mL crude extract with SOD activity of 186 U and total protein amount of 500 µg was present in each tube.

Figure 1. (A) Elution profile of SOD from Sephadex G-100 column; (B) Molecular weight calibration curve; standard proteins (–•–) and ATPS purified SOD (–○–).

Figure 2. SDS–PAGE profiles of P. chrysosporium SOD purified by partitioning in ATPS. Lane 1: molecular weight markers (15–260 kDa) (10 μg), lane 2: top phase of the optimized system (25 μg), lane 3: crude SOD extract (25 μg) and lane 4: bottom phase of the optimized system (25 μg).