The Studies On Modified Hyaluronic Acid As Novel Glutathione Peroxidase(GPX) Mimic

GPX plays an important role in antioxidant defense system of enzymatic action. It can not only get rid of ROOH, but also take place of CAT to exclude H2O2 in the organism of lower CAT containing or lower H2O2 producing. Thus GPX has fundamental function in physiology of aberrance and caducity avoidance, and synthetical prostaglandin participation. Without it the body will result in Colonic Cancer, physical caducity, hemal disease and so on for excess of free radicals attack. So it is very significant to explore the catalytic mechanism of enzyme and study prevention from disease. However, due to the shortcomings of native GPX (solution instability, poor availability, short half-lives and proteolytic digestion), mimics with high GPX efficiency were prepared by many biochemists. One of the well-known GPX mimics is Ebselen (2-phenyl-1, 2-benziososelenazol-3(2H)-one). Although this interesting molecule is undergoing phase III clinical trial in Japan as antioxidant, it has some drawbacks such as its low GPX activity(0.99U/μmol) and its insolubility in water. Based on the structure-function correlation of native GPX, we produced two new kinds of HA-derived GPX mimics out of hyaluronidase, SeHA and TeHA to imitate the functional action of native GPX,and researched their enzymologic character and biological effects. The biological effects of the GPX mimic have been verified in the level of the cells and sub-cells in terms of protection effect to red cell and mitochondrion.1. Selenide hyaluronic acid (SeHA) to mimic GPXHyaluronic acid is a high molecular mass mucopolysacchride, which consists of N-acetyl-d-glucosamine (GLcNAc) and d-glucuronic acid, and exists at high concentrations in the tissues of human, animal and in extracellular matrix. HA is a ubiquitous matrix and constructs fundamental matrix with others, which plays an important role in the process of growth and accommodation. This spatial structure of HA has a hydrophobic cavity, which is about 8 CH units, and may serve as a substrate-binding site. Basing on the framework of hyaluronic acid (HA) and chemical modification method, enzymes catalytic group of GPX was incorporated into activated hyaluronic acid, and a novel selenide hyaluronic acid (SeHA) was synthesized to be functioned as a mimic of glutathione peroxidase (GPX). The structure of SeHA was characterized by means of IR and NMR with the conclusion that the target SeHA was located at -CH₂OH of the N-acetyl-D-glucosamine. The GPX activities of SeHA was determined to be 103.9 U/μmol, after the new compound was prepared and purified, SeHA was applied for DTNB method to identify the selenium content of 152 SeH. The GPX activities of SeHA, reducing H₂O₂, tert-butyl hydroperoxide (t-BuOOH) and cumenyl hydroperoxide (CuOOH) by glutathione, are 103.9U/μmol, 32.2 U/μmol and 152.7U/μmol respectively,which illustrated CuOOH is the optimal substance of the mimic. In the steady-state kinetic studies of the SeHA-catalyzed reactions with three substances, a ping-pong mechanism was observed from the data of the double reciprocal plots.2. Telluride hyaluronic acid to mimic GPXTo mimic the enzyme behavior of GPX, based on the cooperation effect of the groups in the catalytic active center, scientists have synthesized a great deal of small molecule organoselenium compound GPX mimics through chemical/biological methods and principle, including the well-known Ebselen, Spector, Tomado and Mugesh catalyst. Moreover, the further researches find that the organotellurium compounds also exhibit excellent GPX activities. In the GPX's Catalytic cycle, the easy redox of selenium is one of the important reasons of the high catalytic effect for reducing hydroperoxides. Tellurium and selenium are in the same family, so the redox character of them is similar, and the tellurol can decompose hydroperoxides more easily than selenol. In order to have a platform to study the GPX activity of tellurium, basing on the framework of HA and through the method of chemical modification, we incorporated the catalytic group–TeH into HA and synthesized the novel telluric hyraluronic acid compound TeHA to imitate the functional action of native GPX. IR, 13C NMR were used to characterize the mimic. The GPX activity of the mimic was 163.6 U/μmol. Detailed kinetic studies of telluride hyaluronic acid (TeHA) were undertaken. Saturation kinetics was observed for both H2O2 and GSH. Double reciprocal plots of the initial velocity versus the concentration of substrates were a family of parallel lines, which is consistent with a Ping-Pong mechanism. The kinetic equation was similar to native GPX.Since GPX plays a pivotal role in protection cells against oxidative damage, selenium compounds with GPX activity attract great attentions of scientists. Not only can the mimic of GPX illustrate the mechanism of enzymatic catalysis, but also high active GPX mimic is mostly capable of being forehand compound of the efficient medications to prevent and treat disease caused by ROS in future. We constructed two damaged model systems to evaluate the biological effects of the GPX mimics in the level of the cells and sub-cells. (1) Ferrous sulfate/ ascorbate-induced mitochondria damaged model systemWe constructed the ferrous sulfate/ascorbate-induced mitochondria damaged model system, demonstrated the damaged mitochondria have great changes in morphology, structure and function. The extent of swelling, MDA content and CCO activity of mitochondria were chosen as standards to be used to determine the extent of injury and protection of mitochondria. Mimics reduced the swelling of mitochondria during damage and decrease the maximal level of MDA accumulation in its rapid phase.(2)H₂O₂ induced erythrocytes hemolysis and Hydroper -Oxidation model systemH₂O₂ induced erythrocytes hemolysis and Hydroper-Oxidation model system was used to observe mimics against the lipid peroxidation of damaged erythrocytes. The results showed that mimics inhibited hemolysis and MDA production of erythrocytes in a dose dependent manner and prohibited the formation of methemoglobin so that protected the erythrocytes against oxidative damage. These results demonstrate that, as two kinds of new GPX mimics, SeHA and TeHA will play very important role in the ROS-mediated diseases therapy.