Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study

Figures & data

Table 1.  Purification scheme of GST from rainbow trout erythrocytes.

Purification step	Total volume (mL)	Activity (EU/mL)	Protein (mg/mL)	Total protein (mg)	Total activity (EU)	Specific activity (EU/mg)	Yield (%)	Purification factor
Haemolysate	35	0.073	48.86	1710	2.555	0.0015	100	1
Glutathione-Agarose affinity chromatography	7	0.215	0.013	0.091	1.505	16.54	59	11,026

Table 2.  K_i and IC₅₀ values obtained from regression analysis graphs for rainbow trout erythrocytes GST in the presence of different metal ion concentrations.

Metal ions	IC50 (mM)	Ki (mM)	Inhibition type
Ag^+	0.0348	0.0243 ± 0.009	Non-competitive
Cd^2+	0.1710	0.1117 ± 0.052	Non-competitive
Cr^2+	1.0420	2.506 ± 1.1040	Non-competitive
Mg^2+	46.330	65.73 ± 17.378	Non-competitive

Figure 1.  SDS-PAGE photograph. Lane 1: Rainbow trout erythrocytes GST. Lane 2: Standard proteins: rabbit phosphorylase B (97 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), bovine erythrocyte carbonic anhydrase (29 kDa).

Figure 2.  Standard Rf-Log MW graph of GST using SDS-PAGE. (Standards: Rabbit phosphorylase B (97.4 kDa), bovine albumin (66 kDa), chicken ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa).

Figure 3.  Standard Kav-Log MW graph of GST using gel filtration. (Standards: Cytochrome c (12 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), yeast alcohol dehydrogenase (150 kDa) and β-amylase (200 kDa).

Figure 4.  Activity-pH graph of GST.

Figure 5.  Activity – [K-phosphate] for determination optimal ionic strength of GST.

Figure 6.  The effect of the temperature on GST.

Figure 7.  Stable pH graph of GST in 0.01 M K-phosphate.

Figure 8.  Lineweaver-Burk graph in 5 different GSH concentrations and in constant CDNB concentration.

Figure 9.  Lineweaver-Burk graph in 5 different CDNB concentrations and in constant GSH concentration.

Figure 10.  Activity % vs [metal ions] regression analysis graphs for rainbow trout GST in the presence of 5 different (a) [Ag⁺] (b) [Cd²⁺] (c) [Cr²⁺] (d) [Mg²⁺] concentrations.

Figure 11.  Lineweaver-Burk graph with 5 different substrate (GSH) concentrations and 3 different (a) [Ag⁺] (b) [Cd²⁺] (c) [Cr²⁺] (d) [Mg²⁺] concentrations for determination of Ki.