Tongshu Yang

Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency.

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibodys binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240-1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251-255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5-10%, 15-20% and 25-30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8 M urea by Co(2+)-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46 x 10(5) M(-1) and 1.03 x 10(5) M(-1) respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394 units/micromol, which approaches the activity of rabbit liver GPX.

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron.

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe‐XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O2 −) and hydroxyl radical (OH), but also alkyl or alkoxyl radicals (R) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe‐XO/HX system. The generated amount of R was dependent on the kind and concentration of saccharides added into the Fe‐XO/HX system and no R were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe‐XO/HX system and saccharide molecules are essential for generating R in the Fe‐XO/HX system. It is expected that the toxicity of R would be greater than of hydrophilic O2 − and OH because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O2 − and OH. The R generated from the saccharide containing Fe‐XO/HX can be effectively scavenged by selenium containing abzyme (Se‐abzyme), indicating Se‐abzyme is a promising antioxidant.

A Selenium-Containing Abzyme, the Activity of Which Surpassed the Level of Native Glutathione Peroxidasea

Abstract: Using two different glutathione derivatives as hapten, we have prepared two abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two abzymes is possibly attributed to the conformation difference of the abzymes.

Protection of myocardial mitochondria against oxidative damage by selenium-containing abzyme m4G3

Selenium-containing abzyme (m4G3) was prepared and its protection of myocardial mitochondria against oxidative damage was studied using the swelling of mitochondria, quantity of lipid peroxidation products, and change in cytochrome-c oxidase activity as a measure of mitochondrial damage. The results showed that m4G3 could inhibit mitochondrial damage caused by the hypoxanthine-xanthine oxidase system in vitro. Electronic spin resonance (ESR) studies demonstrated that m4G3 could decrease the amount of free radicals generated in the damage system.