Analysis Of Antioxidant Capacity Of Cytochrome C From Different Species

The main aim of this work is to compare the regulation and modification formitochondrial reactive oxygen species (ROS) by cytochrome c from different species.We believe that different cytochrome c has different effects to eliminate ROS inmitochondrion and try to establish the relationship between the cytochrome c’smolecular structure and it antioxidant efficiency. The detail information about thisrelationship should be useful for human disease treatment Longevity Research.Cytochrome c is a small and stable hemoprotein. It plays a very important rolenot only on energy metabolism but also on cell apoptosis, radical regulation and so on[1]. In early papers[2,3], our lab suggested an antioxidant pathway named “alternativeelectron-leak pathway mediated by cytochrome c” to explain how cytochrome caffects on the ROS levels in cell. This pathway can independently eliminate ROS nearthe generation sites on respiratory chain and give its contribution on redox balance incell. Preliminary work shows that there is a possible relationship between the sourceof cytochrome c (species) and its antioxidant ability.Through the depletion-reconstitution of cytochrome c we determined theantioxidant capacity of different concentrations of cytochrome c. Cytochrome cdepleted heart muscle preparation (c-dHMP) was used as detection system.Lucigenin-derived chemiluminescence was used for superoxide anion assay andluminal plus HRP derived chemiluminescence for H2O2. According the results, twoconcentrations were picked as final reactive concentration:2μM (high concentration)and0.5μM (low concentration). In this study, we compared the antioxidant capacity ofthree kinds of cytochrome c from yeast, pigeon chest and bovine heart respectively.The redox state of cytochrome c was also discussed.The results are as follows:1) In low concentration, and in the natural state, these three kinds of cytochromec showed different efficiency on the elimination of O2.-and H2O2. Yeast cytochrome c,the worst performance, only can suppress43%of H2O2and63%of O2.-, but pigeon chest cytochrome c, the best one, can remove80%~90%ROS (both H2O2and O2.-).The sequence of the antioxidant ability is: pigeon breast cytochrome c> bovine heartcytochrome c> yeast cytochrome c. If high concentration of cytochrome c wasreconstituted, no significant difference was shown because of the strong ability.2) In the presence of antimycin A (AA), the electron transfer was blocked, andROS generation increase. Due to the increase of baseline, high concentration ofreconstituted natural cytochrome c shows the species difference. The antioxidantcapacity sorting is: pigeon breast cytochrome c> bovine heart cytochrome c> yeastcytochrome. Under the condition of low reconstituting concentration, there is nosignificant difference on the antioxidant ability between three kinds of cytochrome c.All of them only can suppress less than40%ROS.3) If only reduced cytochrome c was added to the system, besides no significantdifference was shown when high concentration of cytochrome c was reconstituted, theresults were different with that of natural cytochrome c. When low concentration ofcytochrome c was used, yeast reduced cytochrome c get high antioxidant ability, assame as pigeon chest reduced cytochrome c. Bovine reduced cytochrome c is the lastone in sorting.4) In the presence of AA, yeast reduced cytochrome c and pigeon chest reducedcytochrome c show powerful antioxidant capacity, and there is no significantdifference between them whether high concentration or low concentration. In anycondition, the antioxidant capacity of bovine heart cytochrome c is the weakest.Based on above results, we can get some conclutions: A: The antioxidant abilityof cytochrome c obtained from different species show significant difference, whetherthe suppression of O2.-or the elimination of H2O2. The difference of antioxidantability is based on the changes of cytochrome c’s microstructure. B: From this study,we can not say that the antioxidant capacity of cytochrome c is related to the lifespanof species even some relationship was suggested. In further studies, we need choosemore species and make them in same classis, for example, Mammalia. C: Yeast reduced cytochrome c still play antioxidant role in c-dHMP system even though thereis no cytochrome c peroxidase in the reactive system. The specific mechanismremains to be further discussed.