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
