Detailed circular dichroism(CD) and Fourier transform infrared(FTIR) studies have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme C(HRP) inhibited by CN -(HRP CN). The results suggest...Detailed circular dichroism(CD) and Fourier transform infrared(FTIR) studies have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme C(HRP) inhibited by CN -(HRP CN). The results suggest that HRP CN is quite different from native HRP with different spin states of Fe of heme and different coordinated states. Cyanide becomes the sixth ligand of Fe(Ⅲ) of heme and the hydrogen binding network is destroyed partly at the same time, which cause the drastic decrease of thermal stability of HRP. The FTIR and Soret CD spectra analysis demonstrate that during the heating process there is an intermediate state(I) which has both partly destroyed secondary and tertiary structures of native HRP, then it is the appearance of protein aggregation state(A) after fully unfolding. The unfolding pathway thus can be shown as follows: IIUA.展开更多
为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根...为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根过氧化物酶标记,对标记前和标记后纯化产物进行活性测定,同时对标记后产物进行Western-blot鉴定,以及将其应用到验证已知的阳性噬菌体H11、H12、H14、H16、H18中。结果表明:兔抗M13KE多克隆抗体效价高于1∶32 000,标记后活性高于1∶4 000,标记抗体与已知阳性噬菌体的ELISA鉴定结果与前期测序结果一致。说明此酶标抗体有一定的应用价值。展开更多
Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and elect...Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.展开更多
文摘Detailed circular dichroism(CD) and Fourier transform infrared(FTIR) studies have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme C(HRP) inhibited by CN -(HRP CN). The results suggest that HRP CN is quite different from native HRP with different spin states of Fe of heme and different coordinated states. Cyanide becomes the sixth ligand of Fe(Ⅲ) of heme and the hydrogen binding network is destroyed partly at the same time, which cause the drastic decrease of thermal stability of HRP. The FTIR and Soret CD spectra analysis demonstrate that during the heating process there is an intermediate state(I) which has both partly destroyed secondary and tertiary structures of native HRP, then it is the appearance of protein aggregation state(A) after fully unfolding. The unfolding pathway thus can be shown as follows: IIUA.
文摘为了制备用于噬菌体展示肽库的筛选及鉴定等环节的辣根过氧化物酶(HRP)标记的兔抗丝状噬菌体(M13)衣壳蛋白抗体,试验制备了兔源的M13KE多克隆抗体,将多克隆抗体经饱和硫酸铵法和Protein G Resin层析柱纯化后运用过碘酸钠法对其进行辣根过氧化物酶标记,对标记前和标记后纯化产物进行活性测定,同时对标记后产物进行Western-blot鉴定,以及将其应用到验证已知的阳性噬菌体H11、H12、H14、H16、H18中。结果表明:兔抗M13KE多克隆抗体效价高于1∶32 000,标记后活性高于1∶4 000,标记抗体与已知阳性噬菌体的ELISA鉴定结果与前期测序结果一致。说明此酶标抗体有一定的应用价值。
基金the National Natural Science Foundation of China(Grant No.20675064)Natural Science Foundation of Chongqing City(Grant Nos.CSTC-2004BB4149 and 2005BB4100)the High Technology Project Foundation ofSouthwest University(XSGX02),China
文摘Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.
