Purification and Characterization of Glutathione-S-Transferase from Chicken Erythrocyte

Figures & data

Figure 1.  DEAE cellulose chromatography of chicken erythrocyte GST. (…•…) GST activity, (—o—) absorbance at 280 nm.

Figure 2.  Q sepharose chromatography of chicken erythrocyte GST. (…•…) GST activity, (—o—) absorbance at 280 nm.

Figure 3.  Sephadex G-100 gel filtration chromatography of chicken erythrocyte GST. (…•…) GST activity, (—o—) absorbance at 280 nm.

Table 1. Purification steps for chicken erythrocyte GST

	Total Protein (mg)	Specific Activity (U/mg)	Total Activity (U)	Purification Fold	Yield
Crude Extract	590	0.06	37.84	1.0	–
Acetone Precipitation	123	0.21	25.87	3.5	68.3
Ethanol-Chloroform Treatment	28.2	0.66	18.53	11.0	48.9
DEAE-cellulose Chromatography	2.08	3.37	7.02	56.1	18.5
Q-Sepharoz Chromatography	0.434	7.37	3.20	122.8	8.4
SephadexG-100 Gel Filtration	0.048	20.39	0.99	339.8	2.6

Figure 4.  SDS-polyacrylamide gel electrophoresis of chicken erythrocyte GST (a) protein standards; (b) purified chicken erythrocyte GST.

Figure 5.  Optimum pH profile of chicken erythrocyte GST.

Figure 6.  pH stability of chicken erythrocyte GST. pH 7.5(… ♦ …), pH 7 (—○—), pH 8 (…□…), pH 9 (—Δ—), pH 6 (—▴—), pH 5 (— □^___), pH 4 (…•…).

Figure 7.  Heat stability of chicken erythrocyte GST. 10 °C (—•—), 20 °C (—□—), 30 °C (—▴—), 40 °C (—○—), 50 °C (— •—), 60 °C (… •…).

Figure 8.  Effect of substrate (CDNB) concentration on chicken erythrocytes GST (Lineweaver-Burk plot).

Figure 9.  Effect of different diavalent cations on chicken erythrocyte GST activity.

Table 2. Effect of 2-mercaptoethanol and dithiothreitol on chicken erythrocyte GST after l hours incubation

	Remaining Activity (%)
Concentration (mM)	2-mercaptoethanol	Dithiothreitol
5	42	62
10	36	50
20	25	41
30	9	32
40	0	25

Table 3. Effect of some chemicals on chicken erythrocyte GST after 1 hours incubation

Chemicals	Concentration (M) requiredfor 50% inhibitionis text complete?
Mercury (II) acetate	2.8. 10^−7
FeCl3	2.6. 10^−5
TCA	1.3. 10^−6
Thiourea	1.0. 10^−3
EDTA	1.1. 10^−2
Triton X- 100	0.05%