| It has been well established that a variety of human diseases are generated by oxidative stress of reactive oxygen species(ROS).ROS are produced naturally during oxygen metabolism,and include superoxide(·O2-),hydrogen peroxide(H2O2),hydroxyl radical(·OH),and singlet oxygen (1O2)[1].Oxidative stress caused by ROS can increase lipid peroxidation,protein oxidation,and damage of DNA.They can also affect cell viability by damaging membranes and suppressing antioxidant enzyme activity,thereby accelerating cell senescence and apoptosis[2].ROS are mainly controlled by antioxidant defense system,involving lipid-soluble antioxidants(such as ct-tocopherol and carotenoids),water-soluble reductants(such as glutathione and ascorbate) and antioxidant enzymes such as superoxide dismutase(SOD)[3],catalase(CAT)[4],and glutathione peroxidase(GPX)[5].These enzymes contribute dominantly to the cellular antioxidant defense against oxidative stress.Among the three enzymes,GPX(EC 1.11.1.9) is a well-known selenoenzyme.Although the structure of GPX and the biologic effect of selenium have been understood fully,the effect mechanism of GPX needs to be studied further.The GPX mimics can be used to do this.On the other hand,GPX has strong antioxidant ability and it is important for treat and prevent Keshan disease,angiocardiopathy,inflammation and cancer.Due to the limitations associated with native GPX,such as instability,limited availability,big molecular weight and immunogenicity,many scientists have made a great deal of efforts to study the GPX mimics.Due to the lack of binding site,early GPX mimics usually had rather low activities.Wilson et al.[13]introduced a quaternary ammonium salt close to the diselenide bridge of a GPX mimic to improve its ability to bind GSH by electrostatic attraction,and as a result,the activity was increased.Therefore,in the initial study it was known that the key to imitation of GPX is to generate affinity for GSH.Towards to this purpose,great deals of GPX mimics had been produced such as selenium-containing cyclodextrin[14-16],bioimprinted protein[17],catalytic antibodies [18],semisynthetic enzyme[19]and so on,and some of the mimics also possessed good GPX activities.However,early designs of GPX mimics were more experiential and dependent on a long-term laboratorial accumulation,thus short of the sufficient support in theory,which is more scientific and compellent.In all the factors of theory,enzyme kinetics is considered to be the microcosmic description of the enzymic biochemical behavior,thus,in order to design an excellent mimic which is close to the natural enzyme at the macroscopical level,it should first start with the kinetics,from microcosmic to macroscopical,and in this way it is possible to achieve the ultimate purpose. Therefore,the kinetics of natural GPX should be first studied and some of the rules are established using natural enzymes as the standard,which could guide the design of GPX mimic in theory and then a GPX mimic is designed and synthesized according to the dynamics conclusions.Here we report the small-molecule water-soluble salicylaldehyde schiff base-like GPX mimic with a double-manganese core and its kinetic and enzymic properties and it is hoped that the question will be solved with our proposed method. The reaction catalyzed by natural GPX includes double substrates and double products and can be simplified as follows. A+B→P+QIn steady-state kinetics of natural GPX,when[A]is variable,[B]is fixed,[P]is a constant at different concentrations and Q = 0,at that time P is not the inhibitor of A;whereas when[A]is variable,[B]is fixed,[Q]is a constant at different concentrations and P = 0,at that time Q is a competitive inhibitor of A.Therefore,from the point of view of mimic,simply increased affinity of GSH as mentioned above(such as B is saturated) could cause the competitive inhibition of H202(such as A) by GSSG which is unfavorable to the catalytic process.Because the enzyme reaction is carried out in the water solution,so the small quantity of water,which is produced during the enzyme reaction,can not produce the evident inhibition of binding between GSH and enzyme when the affinity of another substrate H202 is enhanced.Thus,in order to improve the reaction rate,the state should be ideal for the GPX imitation that the affinity of the GSH should be slightly lower(of course,not too low),and the affinity of H202 must be high,and the kinetics data of natural enzyme are also be matched.According to the parameters given from the literature,the two values of the natural enzyme Km(GSH) = 3.0×10-3 M and Km(H2O2) = 1.0×10-5 M,the latter is smaller than the former at about two orders of magnitude,the conclusion obtained from the theory is confirmed by the data of natural enzyme.In pre-steady state kinetics of natural GPX,the discriminant of the final kinetic equation is decided byαand theαis mostly decided by ks which is binding constant between natural enzyme and H2O2.Evidently,for natural GPX,the intensity of the affinity of H2O2 is the most important factor to its kinetic behavior.That is consistent with the steady-state kinetic conclusion.According to these kinetic conclusions,we synthesized the salicylaldehyde schiff base with a double-manganese core which had the best ability of binding H2O2 among the small-molecule compounds as the "female parent" of the GPX mimic.Then,the selenium-containing catalytic center was introduced into the mimic to obtain trifunctional enzyme with GPX,superoxide dismutase(SOD) and catalase(CAT) activities.2.Synthesis and Characterization of Mn(Ⅲ)2(L-Se-SO3Na)L-Se-SO3Na was a dimmer and a loop structure was formed depending on the diselenium bridge(-Se-Se-).The structure of L-Se-SO3Na and Mn(Ⅲ)2(L-Se-SO3Na) was analyzed by means of MS,NMR and IR.The selenium content of Mn(Ⅲ)2(L-Se-SO3Na) was measured by using 5,5-dithiobis(2- nitrobenzoic acid) method and found to be 3.87±0.18 M of selenium per mole of molecule.This indicates that 1 tool of molecule contains 4 mol of selenium.3.The GPX/SOD/CAT-Iike Activity and the GPX-like Steady State KineticsThe GPX activity of Mn(Ⅲ)2(L-Se-SO3Na) for the reduction of H2O2 by GSH was found to be 5.56 U/μmol,which is 5.6 times that of ebselen.The SOD activity of Mn(Ⅲ)2(L-Se-SO3Na) was 138.9 U/μmol.The CAT activity of Mn(Ⅲ)2(L-Se-SO3Na) was 75.02 U/μmol. Steady state kinetics was observed for both H2O2 and GSH.The initial velocities for the reduction of H2O2 by GSH were determined as a function of substrate concentration at 37℃and pH 7.0,varying one substrate concentration while another was fixed.Double reciprocal plots of the initial velocity versus the concentration of substrates yielded a family of parallel lines,which indicated that a ping-pong mechanism was involved.This result demonstrated that the catalytic mechanism of Mn(Ⅲ)2(L-Se-SO3Na) was same as that of native GPX.The apparent kinetic parameters were calculated from the double-reciprocal plots.The apparent second-order rate constants kcat/KmH2O2 and kcat/KmGSH were 1.14×107 M-1min-1 and 1.00×106 M-1min-1, respectively,which were significantly higher than those of other small-molecule GPX mimics.It is true that the ability of Mn(Ⅲ)2(L-Se-SO3Na) to bind H2O2 is really larger than its ability to bind GSH,and the ratio between the values of kcat/KmH2O2 and kcat/KmGSH is quite similar to that of nature enzyme,demonstrating that as a small-molecule mimic,Mn(Ⅲ)2(L-Se-SO3Na) imitated natural GPX quite better in kinetics behavior.4.The antioxidant ability of the mimic(1) Effect of the GPX mimic on swelling of damaged mitochondriaThe mitochondria swelling was greatly increased by ferrous sulfate/ascorbate- induced mitochondria damage,but the swelling was considerably decreased by the addition of Mn(III)2(L-Se-SO3Na).The absorbance at 520 nm for the control group was basically constant, whereas the absorbance for the damage group was considerably decreased with time,indicating that the mitochondria swelling was considerably increased.But,the swelling for the protection group,which contained a certain concentration of Mn(Ⅲ)2(L-Se-SO3Na),was apparently inhibited, and the swelling of mitochondria was decreased with an increase of Mn(Ⅲ)2(L-Se-SO3Na) concentration.The abilities of Mn(Ⅲ)2(L-Se-SO3Na) and 2-SeCD to inhibit the swelling of mitochondria were different as evidenced and the Mn(Ⅲ)2(L-Se-SO3Na) was better than 2-SeCD in the protective effect.(2) Inhibition of lipid peroxidation of mitochondria by Mn(Ⅲ)2(L-Se-SO3Na)The malondialdehyde(MDA) amount accumulated during damage of mitochondria was considerably reduced in the presence of Mn(Ⅲ)2(L-Se-SO3Na),and the decrease of MDA amount was increased with the increase of the concentration of Mn(Ⅲ)2(L-Se-SO3Na).In order to gauge the abilities of Mn(Ⅲ)2(L-Se-SO3Na) and 2-SeCD to inhibit MDA accumulation,their antioxidant activities were undertaken under the identical condition.As evidenced,the ability of Mn(Ⅲ) 2(L-Se-SO3Na) to decrease the MDA accumulation was also better than that of 2-SeCD.Among the experiments above,although the GPX activity of Mn(Ⅲ)2(L-Se-SO3Na) was just 75%of that of 2-SeCD,the protective effect of Mn(Ⅲ)2(L-Se-SO3Na) was better than 2-SeCD, that might be related to its trifunctional with GPX,SOD and CAT activities and this is the first time to unify the three mainly antioxidant enzyme activities of organisms in one small-molecule mimic.Actually,there exists a synergism among GPX,SOD and CAT.This balance may be more important for protection against oxidative stress than the level of any single antioxidant enzyme. Therefore it can be boldly predicted that one mimic with several enzyme activities(functions) would possibly become one of the potential mainstream direction.Moreover,manganese enzymes were widely considered to be less toxicity towards living organism,thus,Mn(Ⅲ)2(L-Se-SO3Na) should have a potential perspective for pharmaceutical applications.
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