Purification of bovine serum paraoxonase and its immobilization on Eupergit C 250 L by covalent attachment

Figures & data

Figure 1. Schematic illustration of the covalent immobilization of bovine serum PON1 on a Eupergit® C 250 L support.

Table 1. Summary of the purification of bovine serum paraoxonase.

Steps	Volume (ml)	Activity (U/ml)	Total activity (U)	Protein amount (mg/ml)	Total protein (mg)	Specific activity (U/mg)	Overall yield (%)	Overall purification (fold)
Bovine serum	45	28.25	1271.25	287.15	12921.75	0.098	100	–
Ammonium sulfate fractionation	53	20.88	1106.64	152.24	8068.72	0.137	87.05	1.40
Hydrophobic interaction  chromatography	12	82.28	987.36	1.78	21.36	46.22	77.67	337.3

Units: 1 nmol 4-nitrophenol formed per minute. Purification (fold): specific activity, n purification step/specific activity in serum. Yield: activity of fractions combined for the next purification step/total activity in serum × 100. Yields figures do not include all of the activity actually recovered. Usually, three tubes were pooled for hydrophobic interaction chromatography.

Figure 2. SDS-PAGE of bovine serum PON1. The pooled fractions from ammonium sulfate precipitation and hydrophobic interaction. Experimental conditions were as described in the Methods section. Lane 1 contained 3 μg of various molecular-mass standards: Bovine serum albumin (66.7 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (35 kDa), REase βsp98I (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa).

Table 2. Summary of immobilization of PON1 on the Eupergit C 250 L support.

Support	Enzyme loading (mg g^−1 support)	Enzyme coupling yield (%)	Immobilized paraoxonase activity (U g^−1support)	Specific activity of immobilized enzyme (U mg^−1enzyme)	Specific activity of free enzyme (U mg^−1 enzyme)	Activity coupling yield (%)
Eupergit® C 250 L	7.46	74.6	58.9	0.78	46.22	1.69

Figure 3. Effect of pH on the activity of native and immobilized bovine serum PON1.

Figure 4. Thermal stability of native and immobilized bovine serum PON1 at 25 °C.

Figure 5. Thermal stability of native and immobilized bovine serum PON1 at 45 °C.

Figure 6. Thermal stability of native and immobilized bovine serum PON1 at 65 °C.

Table 3. Influence of immobilization process on kinetic constants.

Derivative	Km (mM)	Vmax (U ml/min)
Native enzyme	6.26	169.65
Immobilized enzyme	2.47	149.44