Purification And Determination Of Paraoxonase In Rabbit Serum

ObjectivePON is a kind of phospholipase generated by organism itself. Current research also found that it can hydrolyze organophosphate, aromatic ester, carbamate and unsaturated fatty acid. PON widely distributes among many kinds of animals, while the PON in rabbit serum has the highest activity. This experiment is designed to derive and purify the PON in rabbit serum and establish the method to test its activity in order to make basis to furtherly explore its characteristics of molecular biology and mechanism of hydrolyzing organophosphorus compound, and to supply clew to treat organophosophorus poisoning.MethodEstablish the method to test the activity of PON. Paraoxon can be hydrolyzed by PON. Making use of that the product p-nitrophenol has absorption peak at 405nm, we can test the activity of PON by observing the change of absorbance continuously under such wave length.Choose 10 New Zealand rabbits to acquire 100 mixed serum by centrifuge the blood acquired from them. By the method of salting out precipitation to get the crude enzyme protein.Use the method of liquid chromatography to derive the paraoxonase in rabbit serum and purify it. Firstly derive the protein in serum by affinity chromatography.Cibacron Blue Agarose has specific affinity to lipoprotein in serum, choose it as affinity choramtography medium to absorb the paraoxonase in the serum. After the equilibrating, scrubbing and eluting, get the crude liquid of paraoxonase. Choose the parts having higher activity for dialysis and concentration。Use the method of ion exchange chromatography to separate the protein of different isoelectric point and molecular weight. According to IP of PON is 5.1, choose the DEAE-Sepharose CL-6B as the ion exchange medium under the condition of PH=8.0. For PON is hydrophibic protein, surfactant can increase its solubility and affinity to resine during the ion exchange chromatography. Make use of its molecular seive mechanism with sodium chloride of gradient concentration to separate the protein of different IP and molecular weight.During the purification, choose EDTA of suitable density to retain the density of Ca²⁺ so as to stablize the activity of PON.Use the method of Commassie Brillinat Blue to draw standard curve so as to test the density of partly purified protein.Use the method of SDS-PAG electrophoresis to determine the puritiry of partly purified paraoxonase.ResultThe result of the determination of the 100 ml mixed rabbit serum is that its total activity is 317.64 U and the specific activity of paraoxonase is 0.058 U/mg, while the specific activity is elevated to 12.15U/mg after purification, which means the purification has elevated the PON activity in rabbit serum about 210 fold . However the total activity descends to 32.13 U with the yield near 10% or so. On the SDS-PAGE electrophoresis, only one band appears near the 43 kd, which shows that the purification is of good quality.ConclusionChromatography method can be used to derive and purify the paraoxonase in rabbit serum. Choosing suitable gel can greatly improved the efficacy of purification.