Inhibition profile of a series of phenolic acids on bovine lactoperoxidase enzyme

Figures & data

Figure 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified LPO. Column 1: standard proteins: MBP (maltose-binding protein β-galactosidase, 175 kDa), MBP (maltose binding protein)-paramyosin (fusion of MBP and paramyosin, 80 kDa), maltose-binding protein and chitin binding domain (62 kDa), CBD-Mxe intein-2CBD (fusion of the chitin binding domain and the Mxe intein followed by two chitin-binding domains, 46 kDa). Column 2: purified LPO from bovine milk (80 kDa) (LPO: lactoperoxidase).

Figure 2. Lineweaver–Burk graph in five different substrate (ABTS) concentrations and in three different (A) ferulic acid and (B) quercetin concentrations for the determination of K_i.

Table 1. Chemical structures and K_i values of phenolics compounds of LPO.

	Molecules	Molecules’ name	Ki (µM)	Inhibition type
1		Ellagic acid	14.74 ± 8.03	Noncompetitive
2		Gallic acid	28.29 ± 0.48	Noncompetitive
3		Ferulic acid	4.79 ± 1.87	Noncompetitive
4		Caffeic Acid	2.83 ± 0.55	Noncompetitive
5		Quercetin	5.99 ± 0.41	Noncompetitive
6		P-Coumaric Acid	19.14 ± 9.73	Noncompetitive
7		Syringic Acid	17.76 ± 7.52	Noncompetitive
8		Catechol	7.96 ± 4.44	Noncompetitive
9		Epicatechin	10.04 ± 1.58	Noncompetitive

Table 2. Purification steps of LPO from bovine milk.

Purification steps	Total volume (mL)	Activity (EU/mL)	Total activity (EU)	Protein (mg/mL)	Total protein (mg)	Specific activity (EU/mg)	Recovery (%)	Purification (Fold)
Crude homogenate	196	2.74	537.04	1.75	343.0	1.57	100	1
Ammonium sulphate precipitation	33	13.2	435.6	1.92	63.4	6.88	81.1	4.38
CM-Sephadex C-50 column chromatography	245	1.69	414.05	0.13	31.9	13.00	77.1	8.28
Ammonium sulphate precipitation	31	11.9	368.9	0.69	21.4	17.25	68.7	10.98
Sephadex G-100 column chromatography	179	1.89	338.31	0.08	14.3	23.63	62.9	15.05
Ammonium sulphate precipitation and dialyze	27	5.71	154.17	0.18	4.9	31.46	28.7	20.03