Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study

Figures & data

Table I.  Purification of GR from human erythrocyte.

Purification step	Total volume (mL)	Activity (U/mL)	Total activity (U)	Protein (mg/mL)	Specific activity (U/mg)	Yield (%)	Purification factor
Hemolysate	180	0.32	57.80	32	0.01	100	1
Ammonnium sulfate precipitation (30–70%)	50	0.75	37.60	35	0.02	65	2
2′, 5′-ADP Sepharose 4B affinity chromatography	24	1.35	32.40	0.39	3.45	53.60	345
Sephadex G-200 gel filtration chromatography	20	0.84	16.80	0.03	28	29	2800

Figure 1 SDS-PAGE bands of GR (Lane 1: Hemolysate; Lane 2: Ammonium sulfate precipitation; Lane 3–4: Gel filtration chromatography; Lane 5: Standards: E.Coli βgalactosidase (116,000), rabbit phosphorylase B (97,400), bovine albumin (66,000), chicken ovalbumin (45,000), and bovine carbonic anhydrase (29,000); Lane 6: 2′, 5′-ADP Sepharose 4B affinity chromatography.

Table II.  K_i and IC₅₀ values obtained from regression analysis graphs for GR in the presence of different drugs.

Drugs	I50 (mM)	Ki (mM)	Inhibition type
Imipenem	0.030	0.060 ± 0.01	Competitive
Rifamycin	0.146	0.275 ± 0.10	Competitive
Sulfanylacetamide	0.590	0.850 ± 0.05	Competitive
Ceftazidime	2.476	3.590 ± 0.51	Competitive
Chloramphenicol	2.360	3.850 ± 0.40	Competitive
Seftriaxon	2.880	3.710 ± 0.60	Competitive
Vancomycin	4.830	15.110 ± 2.50	Non-competitive
Cefuroxime	15.430	23.500 ± 2.94	Competitive
Ornidazole	19.632	28.490 ± 6.50	Competitive

Figure 2 Activity % vs [Imipenem] regression analysis graphs for human erythrocytes GR in the presence of 5 different imipenem concentrations.

Figure 3 Lineweaver-Burk graph for 5 different substrate (GSSG) concentrations and 3 different imipenem concentrations for determination of K_i.