THIOREDOXIN REDOX STATE DETERMINATI ON Hong Nguyen Mentor: Dr. Gary Merrill Thioredoxin protein essential for life in mammals acts as an antioxidant to prevent oxidative stress acts.
Download ReportTranscript THIOREDOXIN REDOX STATE DETERMINATI ON Hong Nguyen Mentor: Dr. Gary Merrill Thioredoxin protein essential for life in mammals acts as an antioxidant to prevent oxidative stress acts.
THIOREDOXIN REDOX STATE DETERMINATI ON 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 restores oxidatively inactivated enzymes to their reduced & active state Thioredoxin exists in either the reduced or oxidized form H S H S S S Trx Trx Reduced Thioredoxin Oxidized Thioredoxin LEGEND SH = thiol S-S = disulfide Thioredoxin participates in dithioldisulfide exchange reactions S S RNR oxidized H S H S reduced Trx H S H S RNR reduced S S oxidized Trx Thioredoxin reductase & thioredoxin thioredoxin H S NADP+ thioredoxin reductase NADPH H S Trx S S Trx ribonucleotide reductase S S RNR H S H S RNR 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 Protein electrophoretic mobility shift assay (PEMSA) SASASASH SASASH SASASASH SASASASH SASASA- acetone SH SASAIAM DTT SA- wash SHIodoacetic SAIAA Iodoacetamide acid Dithiothreitol TRX ox TRX red TRX red TRX red SH SH SH SH S S TRX ox SASASASAS S TRX ox TRX red SASASASASH SH LEGEND A- = negative charge = Thioredoxin SH = thiol M = neutral state S = Disulfide S TRX ox SASASASASM SM Protein electrophoretic mobility shift assay SH SH SH SH SH SH Trx red SH SH SH SH S S Trx ox urea PAGE DTT SH SH SH SH SH SH IAA/IAM IAA IAM 0 -1 -2 -3 TrxSS -4 -5 + -6 TrxSH 1 SASASASASASA- SM SM SM SM SM SM SM or SM or SM or SM or SM or SM or SASASASASASA- 2 3 4 Representative results Wild Type Mutant Trxox mixed disulfide Trxred Trxoxidized 6 7 8 7 9 12 16 21 23 21 Mixed disulfide 12 10 14 13 15 17 17 20 21 18 Trxreduced 27 22 27 23 29 29 30 34 36 35 WT Mut Trxox 16% 26% mixed disulfide 28% 27% 56% 47% Trxred Conclusion Thioredoxin reductase is not the major determinant of thioredoxin redox state Another pathway must be able to reduce thioredoxin Acknowledgement Dr. Gary Merrill Biochemistry & Biophysics Lab Dr. Kevin Ahern Howard Hughes Medical Institute Undergraduate, Research, Innovation, Scholarship & Creativity