Frog skins keep redox homeostasis by antioxidant peptides

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Background of the Study:

Background of the Study Rana Odorrana livida

Radicals:

Radicals These active radicals may cause damage to cells or tissues easily by lipid peroxidation, denaturation of proteins, or nucleic acid damage with severe consequences to overall metabolism.

Antioxidant Systems:

Antioxidant Systems Enzymes: Superoxide dismutase, catalase, peroxidase , and glutathione reductase Many low molecular weight antioxidant (LMWA) such as GSH, NADPH, carnosine , uric acid, carotene, polyphenols , and lipoic acid

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1. Collection of frog skin secretions 2. Peptide fractionation Sephadex G-50 (Superfine, 2.6 x 100 cm; Amersham biosciences) 3. Structural analysis Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, Model 491

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4. cDNA Synthesis - Trizol -SMART PCR

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5. cDNA Cloning -Applied Biosystems DNA sequencer, Model ABI PRISM 377. Materials and Methods

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6. Free Radical Scavenging Test ABTS radical (ABTS + ) Antioxidin -RL Butylated Hydroxytoluene (BHT) Ultrospec 2100 Pro UV/ Visible Spectrometry Materials and Methods

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Determination of Reducing Power BHT a- tocopherol Materials and Methods

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pH Studies Materials and Methods

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Nuclear Magnetic Resonance (NMR) Materials and Methods

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Alkylation of Cysteine Materials and Methods

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Synthetic peptides Materials and Methods

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Purification of Antioxidant Peptide Sephadex G-50 column RP-HPLC column (15 peaks)

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Purification of Antioxidant Peptide Sephadex G-50 column RP-HPLC column (15 peaks)

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Structural Characterization antioxidin -RL amino acid sequence is AMRLTYNRPCIYAT MALDI-TOF-MS analysis Result

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cDNA Cloning Result

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Free Radical Scavenging Activity Result

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Free Radical Scavenging Activity Result

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Free Radical Scavenging Activity Result

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NMR titration indicated that tyr6, cys10, and the tyr12 are crucial residues for radical elimination Result

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Cys10 is responsible for rapid radical scavenging Result

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Cys10 is responsible for rapid radical scavenging Result

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Cys10 is responsible for rapid radical scavenging Result

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Cys10 is responsible for rapid radical scavenging Result

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Cys10 is responsible for rapid radical scavenging Result

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Cys10 is responsible for rapid radical scavenging Result

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Discussion

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Discussion

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