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

H
S

H
S

Trx

Trx

Reduced Thioredoxin

Oxidized Thioredoxin

LEGEND
SH = thiol
S-S = disulfide

Thioredoxin participates in dithioldisulfide exchange reactions

S

RNR
oxidized

H
S

H
S

reduced Trx

H
S

H
S

RNR
reduced

oxidized Trx

Thioredoxin reductase & thioredoxin

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thioredoxin
H
S
NADP+
thioredoxin
reductase
NADPH

H
S

Trx

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
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Protein electrophoretic mobility shift assay (PEMSA)

SASASASH
SASASH
SASASASH
SASASASH
SASASA- acetone
SH
SASAIAM
DTT
SASHIodoacetic
SAIAA
wash
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

SH
SH
SH
SH
SH
SH

IAA/IAM

SASASASASASA-

IAM

-1
-2

urea PAGE

DTT

IAA

-3
-4

TrxSS

-5
+

-6

TrxSH
1

SM
SM
SM
SM
SM
SM

SM or SA
SM or SASM or SASM or SASM or SASM or SA-

Representative results
La
dd
er

23
0

22
9

20
1

37

20
0

Mutant

on
ly
IA
M

IA
A

on
ly

Wild Type

Trxox

mixed disulfide

Trxred
Trxoxidized

12 16

21 23 21

Mixed
disulfide
Trxreduced

12 10 14 13 15 17 17

20 21 18

27 22 27 23 29 29 30

34 36 35

WT

Mut

Trxox

16%

26%

mixed
disulfide

28%

27%

Trxred

56%

47%

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
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