Thioredoxn Redox State Determination硫氧还蛋白的氧化还原状态的确定.pptVIP

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Thioredoxn Redox State Determination硫氧还蛋白的氧化还原状态的确定.ppt

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Thioredoxn Redox State Determination硫氧还蛋白的氧化还原状态的确定

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 restores oxidatively inactivated enzymes to their reduced active state Thioredoxin exists in either the reduced or oxidized form Trx H S H S Reduced Thioredoxin Oxidized Thioredoxin LEGEND SH = thiol S-S = disulfide Trx S S Thioredoxin participates in dithiol-disulfide exchange reactions H S S S S S HS HS RNR oxidized RNR reduced reduced Trx oxidized Trx H S Trx thioredoxin S H S H Trx S S ribonucleotide reductase S H S H RNR S S thioredoxin reductase RNR NADPH NADP+ Thioredoxin reductase thioredoxin 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 SH IAA DTT IAM Protein electrophoretic mobility shift assay (PEMSA) Iodoacetic acid Dithiothreitol Iodoacetamide LEGEND SH = thiol = Thioredoxin A- = negative charge M = neutral state = Disulfide S S acetone wash SH SH SH S S SH SH SH SH SH SH SA- SA- SA- SA- S S SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SA- SH SH SA- SA- SA- SA- SM SM SA- TRX ox TRX ox TRX ox TRX ox TRX red TRX red TRX red TRX red Trx red SH SH SH SH SH SH Trx ox SH SH SH SH S S SA- SA- SA- SA- SA- SA- SH SH SH SH SH SH SM SM SM SM SM SM SM or SA- SM or SA- SM or SA- SM or SA- SM or SA- SM or SA- DTT IAA IAM IAA/IAM Protein electrophoretic mobility shift assay 0 -1 -2 -3 -4 -5 -6 1 2 3 4 TrxSS TrxSH urea + Representative results IAA only 1 2 3 4 5 37 200 201 229 230 Ladder Wild Type Mutant 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 Mut 26% 27% 47% mixed disulfide Trxred Trxox Trxox mixed disulfide Trxred IA