TRX Force Tactical Kit

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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

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Thioredoxin exists in either the reduced or oxidized form

Trx

HS

HS

Reduced Thioredoxin Oxidized Thioredoxin

LEGEND

SH = thiolS-S = disulfide

Trx

S S

Thioredoxin participates in dithiol-disulfide exchange reactions

HS S S

S SHS

HS

RNRoxidized

RNRreduced

reduced Trx

oxidized Trx

HS

Trx

thioredoxin

SH

SH

Trx

S S

ribonucleotidereductase

SH

SH

RNR

S S

thioredoxinreductase

RNRNADPH

NADP+

Thioredoxin reductase & thioredoxinwww.prosuspensiontrainer.com

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

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SH

IAA DTT IAM

Protein electrophoretic mobility shift assay (PEMSA)

Iodoacetic acid Dithiothreitol Iodoacetamide

LEGEND

SH = thiol

= Thioredoxin

A- = negative charge

M = neutral state

= DisulfideS

S

acetone wash

SHSHSHSS

SHSHSHSHSHSH

SA-SA-SA-SA-SS

SA-SA-SA-SA-SA-SA-

SA-SA-SA-SA-SA-

SA-SA-SA-SA-SA-

SA- SA-

SA-SA-SA-SHSH

SA-SA-SA-SA-SMSM

SA-

TRXox

TRXox

TRXox

TRXox

TRXred

TRXred

TRXred

TRXred

Trxred

SHSHSHSHSHSH

Trxox

SHSHSHSHSS

SA-

SA-

SA-

SA-

SA-

SA-

SHSHSHSHSHSH

SMSMSMSMSMSM

SM or SA-

SM or SA-

SM or SA-

SM or SA-

SM or SA-

SM or SA-

DTT

IAA IAMIAA/IAM

Protein electrophoretic mobility shift assay

0

-1

-2

-3

-4

-5

-6

1 2 3 4

TrxSS

TrxSH

urea

PA

GE

-

+

Representative resultsIA

A onl

y

1 2 3 4 5 37 200

201

229

230

Ladd

er

Wild Type Mutant

Trxoxidized6 7 8 7 9 1

216 2

123

21

Mixed disulfide

12

10

14

13

15

17

17 20

21

18

Trxreduced27

22

27

23

29

29

30 34

36

35

Mut

26%

27%

47%

mixed disulfide

Trxred

Trxox

Trxox

mixed disulfide

Trxred

IAM

onl

y

WT

16%

28%

56%

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 www.prosuspensiontrainer.com