| Although many evidences show P450 play important roles in insects, knowledge regarding the structure and function of individual P450 in insects is limiting. Isolation and purification of individual P450 have been vastly improving our understanding of several important aspects of P450 in insect such as P450 diversity, insecticide resistance, tolerance to plant toxins and metabolism of endogenous compounds, which have been the focus and nodus of P450 research .Helicoverpa armigera is an economically important pest around the world. It has caused damage to many different crops. In order to know more about the structures and functions of individual P450 in H. armigera, attempts were given to develop purification methods suitable for P450s in this pest. The main results are as followed.The effects of naphathalene (NA) , pentamethylbenzene (PMB) , quercetin, rutin and tannic acid on P450 content of microsomes in H.armigera were compared. NA and PMB had significant induction effects on microsomal P450s, and increased the total P450 of midgut microsome up to 2.60 and 2.34 fold, respectively, and that of fatbody microsome up to 2.51 and 3.15 fold, respectively. Quercetin, rutin and tannic acid had no induction effects on P450 of fatbody but decreased the total P450 of midgut, and increased the susceptibility of the cotton bollworm to deltamethrin.CHAPS efficiently solubilized the P450 of microsomes in midgut and fatbody, while lubrol PX , emulgen 911 and sodium cholate were less efficient. 0.5% and 0.5% or 0.8% CHAPS were suitable for solubilization P450 of microsomes in midgut and fatbody, respectively. Four detergents above mentioned didn't denature P450 of microsomes in midgut and fatbody at concentration of 0.5%.PEG precipitation was used in prefractionation of microsomes P450. At concentration of 2% PEG8000, 30% microsomal protein containing 10% of total P450 was precipitated. At concentration of 5% PEG8000, 55% microsomal protein containing 20% of total P450 was precipitated. At concentration of 8% PEG8000, 78% microsomal protein containing 30% of total P450 was precipitated. At concentration of 11% PEG8000, 85% microsomal protein containing 70% of total P450 was precipitated. 8%PEG was found to be suitable for prefractionation of microsomes P450. After precipitation by PEG, purity of P450 from midgut microsome was increased up to 2.1 fold, and recovery was 72%. Most proteins with molecular weights of 14.4-40kDa and 66-97kDa were precipitated by PEG8000.The optimal conditions of HPHIC for purifying P450 of microsomes were established, i.e.(1)detecting wavelength is 405 nm,(2)flow rate is 0.5ml per min,(3)50min linear gradient from 100% HICA2 to 100% HIC B. Separated by HPHIC, P450s of microsomes were eluted during 39'0''-44'0''(fraction 7) and 44'01''-49'0'' (fraction 8). Compared with microsomes, purity of P450 increased upto 3.04 fold, recovery of P450 was 55.3%.The optimal conditions of HPIEC for purifying P450 of microsomes were established, i.e. (1)detecting wavelength is 405 nm,(2)flow rate is 0.5ml per min,(3)40min linear gradient from 100%IEC A2 to 100%IEC B2. Separated by HPIEC, P450s in fraction 7 and fraction 8 are all eluted during 5'0''-7'0''.Compared with microsomes, purity of P450 increased up to 6.3 fold, recovery of P450 was 17.3%.SDS-PAGE (stained with silver) showed that,separated by HPIEC, fraction 7 and 8 both have two protein brands with molecular weights of 59±1.0, 48±1.9kDa and 55±0.2, 46±1.9kDa, respectively.
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