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THIOREDOXIN REDOX STATE DETERMINATION

THIOREDOXIN REDOX STATE DETERMINATION. Hong Nguyen Mentor: Dr. Gary Merrill. Thioredoxin. protein essential for life in mammals acts as an antioxidant to prevent oxidative stress acts as an electron donor in a variety of cellular reactions

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THIOREDOXIN REDOX STATE DETERMINATION

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  1. THIOREDOXIN REDOX STATE DETERMINATION Hong Nguyen Mentor: Dr. Gary Merrill

  2. Thioredoxin • protein essential for life in mammals • acts as an antioxidant to prevent oxidative stress • acts as an electron donor in a variety of cellular reactions • restores oxidatively inactivated enzymes to their reduced & active state

  3. S S H S H S Trx Trx Thioredoxin exists in either the reduced or oxidized form Oxidized Thioredoxin Reduced Thioredoxin LEGEND SH = thiol S-S = disulfide

  4. Thioredoxin participates in dithiol-disulfide exchange reactions HS HS S S RNR oxidized RNR reduced H S H S S S reduced Trx oxidized Trx

  5. S S H H H H S S S S Thioredoxin reductase & thioredoxin thioredoxin ribonucleotide reductase Trx RNR NADP+ thioredoxin reductase S S Trx RNR NADPH

  6. Hypothesis • Thioredoxin reductase is a major determinant of thioredoxin redox state • If true, then deletion of thioredoxin reductase should shift the redox state of thioredoxin from primarily reduced to primarily oxidized

  7. Protein electrophoretic mobility shift assay (PEMSA) SA- SA- SA- SH TRX red TRX red TRX red TRX red SA- SA- SH SA- SA- SA- SH SA- SA- SA- SH SA- SA- SA- SH SA- SA- SA- acetone wash SH SA- IAM DTT IAA Iodoacetamide Iodoacetic acid Dithiothreitol SH SA- SA- SA- TRX ox TRX ox TRX ox TRX ox SH SA- SA- SA- SH SA- SA- SA- SH SA- SA- SA- S S SH SM S S SH SM LEGEND A- = negative charge = Thioredoxin M = neutral state S = Disulfide SH = thiol S

  8. Protein electrophoretic mobility shift assay SH SH Trx red Trx ox SH SH SH SH SH SH S SH 0 SH S - -1 DTT -2 urea PAGE SH -3 SH SH -4 TrxSS SH SH SH -5 IAA/IAM + -6 TrxSH IAM IAA 1 2 3 4 SM or SA- SM SA- SM or SA- SM SA- SM or SA- SM SA- SM or SA- SM SA- SM or SA- SM SA- SM or SA- SM SA-

  9. Representative results Wild Type Mutant 1 3 4 5 200 201 2 37 229 230 Ladder IAA only IAM only Trxox mixed disulfide Trxred Trxox mixed disulfide Trxred

  10. Conclusion • Thioredoxin reductase is not the major determinant of thioredoxin redox state • Another pathway must be able to reduce thioredoxin

  11. Acknowledgement • Dr. Gary Merrill • Biochemistry & Biophysics Lab • Dr. Kevin Ahern • Howard Hughes Medical Institute • Undergraduate, Research, Innovation, Scholarship & Creativity

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