Purification, characterization, and investigation of in vitro inhibition by metals of paraoxonase from different sheep breeds

Figures & data

Figure 1. SDS-PAGE of merino and kivircik serum paraoxonase. The pooled fractions from ammonium sulfate precipitation and hydrophobic interaction chromatography (sepharose-4B, L-tyrosine, 1-napthylamine) were analyzed by SDS-PAGE (12% and 3%) and revealed by Coomassie Blue staining. Experimental conditions were as described in the method. Lane 3 contained 3 μg of various molecularmass standards: ß-galactosidase (116.0), bovine serum albumin (66.0), ovalbumin (45.0), lactate dehydrogenase (35.0), ∞-lactoglobulin (25.0), lysozyme (19.5).

Table I. Summary of the purification of different sheep serum paraoxonase.

	Volume(ml)	Activity(U/ml)	Totalactivity(U/ml)	Proteinamount(mg/ml)	Totalprotein(mg)	Specificactivity(U/mg)	Overallyield %	Overallpurification(Fold)
Merino
Serum	82	75.3	6174.6	7.6	623.2	9.91	100	−
Ammonium Sulfate Precipitation	32	77.1	2467.2	7.7	246.4	10.01	40.0	1.01
Hydrophobic Interaction Chromatography	4, 5	88.5	398.3	0.019	0.086	4631.4	6.5	462.7
K1v1rc1k
Serum	50	64.4	3220.0	7.5	375.0	8.59	100	−
Ammonium Sulfate Precipitation	18	67.8	1220.4	7.8	140.4	8.69	37.9	1.01
Hydrophobic Interaction Chromatography	3	68.2	204.6	0.017	0.051	4011.8	6.4	461.7

Figure 2. IC50 and Ki graphics of metals on Kivircik paraoxonase enzyme.

Table II. Type of inhibition and IC₅₀ values (mM) of metals on merino and kivircik paraoxonase enzyme.

	Heavy Metal	I50 (mM)	Ki (mM)	Type of Inhibition
Merino
	Cu	0.747	0.397	Competitive
	Hg	1.810	5.445	Noncompetitive
	Ni	3.390	2.820	Competitive
	Cd	2.840	4.015	Uncompetitive
	Co	2.050	4.440	Competitive
	Mn	5.140	4.469	Competitive
Kivircik
	Cu	0.53	0.319	Competitive
	Hg	2.140	0.768	Noncompetitive
	Ni	4.220	1.200	Competitive
	Cd	0.98	0.479	Uncompetitive
	Co	1.810	0.649	Competitive
	Mn	1.980	1.668	Competitive

Figure 3. IC50 and Ki graphics of metals on Merino paraoxonase enzyme.