Development of a Quantitative Assay
for Glutathione Reductase
Paul Hagey,
Eamonn F. Healy,
Chemistry
Department,
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St. Edward’s
University,
Austin TX 78704
Abstract
Glutathione Reductase has been implicated as the critical enzyme
for maintenance of GSH during oxidative stress. We report here
steps towards development of quantitative assay for Glutathione
Reductase using biotin. Biotinylation has achieved widespread
and general utility in a variety of bioanalytical applications due to
the ready formation of biotin-avidin complexes (1015 M-1).
We have succeeded in coupling a biotin to GSSG (reduced
glutathione), with detection by HPLC at 260nm. The GSSGbiotin is then complexed with Glutathione Reductase, and
subsequently complexed in a Glutathione Reductase :GSSGbiotin : avidin triplex. By purifying the resulting complex and
spectroscopically determining the concentration, Glutathione
Reductase can be accurately assayed.
Glutathione Reductase
Glutathione reductase (GR, EC 1.6.4.2)
is an ubiquitous enzyme which catalyzes
the reduction of oxidized glutathione
(GSSG) to glutathione (GSH).
Glutathione reductase is essential for the
glutathione redox cycle that maintains
adequate levels of reduced cellular GSH.
GSH serves as an antioxidant, reacting
with free radicals and organic peroxides,
in amino acid transport, and as a
substrate for the glutathione peroxidases
and glutathione S-transferases in the
detoxification of organic peroxides and
metabolism of xenobiotics, respectively
Role of Glutathione
NH2
O
O
O
NH
NH
OH
NH2
OH
O
Glutathione
Reductase
S
O
NH
NH2
O
NH
Oxidized Glutathi one : GSSG
OH
O
Reduced Glutathione : GSH
O
O
OH
SH
OH
NH
O
O
NH
S
OH
O
Biotinylated Probes
O




Biotin (B) is a small molecule that
covalently attaches to selected residues of
bioactive peptides and proteins , termed
probes (P), without causing loss of
function
O
Biotin is useful because its high affinity for
avidin (Av) and streptavidin allows the HN NH
biotin-peptide complex to form a triplex
(Av:B-P) with the protein, thus isolating
S
the probe
By incubating this triplex with the probe’s
natural target molecule (T) a complex of
composition Av:B-P:T is now isolated
Biotinylated GSSG
An assay of the avidin present is now also
a quantitative assay the target protein
HN
NH
O
S
OH
Biotin
O
O
NH
O
O
NH
NH
OH
OH
O
S
S
OH
O
OH
NH
O
NH
NH2
O
O
Project




Our first requirement was to
identify the particular form of
biotin (B) suitable for attachment
to our GSSG peptide. We have
achieved success with a NHSester derivative with a 6-C spacer
arm, shown on the right.
After developing a suitable
HPLC separation method we are
currently focusing on using
preparative HPLC to isolate the
purified B-GSSG probe.
After suitable characterization we
will proceed to form a triplex
(Av:B-GSSG) of a out
biotinylated probe with Avidin
Finally we hope to form our
GRase:GSSG-B:Av complex
O
HN
NH
O
Biotin
S
OH
O
HN
O
NH
O
NHS- bioti n
N
S
O
O
HN
O
O
NH
O
N
(CH2)n
S
O
O
O
NHS- bioti n with
spacer arm
HPLC
HPLC instrumentation includes a pump,
injector, column, detector and recorder or data
system, connected as shownon the right. The
heart of the system is the column where
separation occurs. The chromatographic
process begins by injecting the solute onto the
top of the column.Separation of components
occurs as the analytes and mobile phase are
pumped through the column.
Eventually, each component elutes from the
column as a narrow band (or peak) on the
recorder. Analyte molecules, while moving through the porous packing bead,
tend to interact with the surface adsorption sites. All these interactions are
competitive. Analyte molecules are competing with the eluent molecules for
the adsorption sites. So, the stronger analyte molecules interact with the
surface, and the weaker the eluent
interaction, the longer analyte will be retained on the surface.
UV-Vis Absorbance of GSSG
Biotinylation Methodology





The probe to be biotinylated is dissolved in a phosphate,
pH=7.6 buffer at a concentration of 10mg/mL
The biotinylation reagent is dissolved in
dimethylformamide at a concentration of 25mg/mL, and
stored as a stock solution at 2-8 oC (stable for approx. 24
hrs)
The biotinylation reagent is added slowly to the probe
solution in a 10-30 molar excess and gently mixed for 1-4
hours
The reaction is terminated by adding 1mL of 20% TFA
The products are analyzed using reverse phase HPLC and
a C-18 column
Results
Biotin
GSSG
Time : 1hour
Time :1 hour
Phosphate pH=7.6
Phosphate pH=7.6
Solvent A: ACN
Solvent A: ACN
Solvent B: ACN/1%TFA
Solvent B: ACN/1%TFA
Detector : 260nm
Detector : 260nm
Flow Rate : 1ml / min
Flow Rate : 1ml / min
Results
GSSG / Biotin
GSSG / Biotin
Time : 1 day
Time : 4 days
Phosphate pH=7.6
Phosphate pH=7.6
Solvent A: ACN
Solvent A: ACN
Solvent B: ACN/1%TFA
Solvent B: ACN/1%TFA
Detector : 260nm
Detector : 260nm
Flow Rate : 1ml / min
Flow Rate : 1ml / min
Results
Lys-Tyr-Lys / Biotin
Lys-Tyr-Lys / Biotin
Time :initial
Time : 1 day
Phosphate pH=7.6
Phosphate pH=7.6
Solvent A: ACN
Solvent A: ACN
Solvent B: ACN/1%TFA
Solvent B: ACN/1%TFA
Detector : 260nm
Detector : 260nm
Flow Rate : 1ml / min
Flow Rate : 1ml / min
Discussion




The peak at 7.6 min on the chromatogram of the GSSG / NHS-spacer-biotin
indicates to use the successful biotinylation of our peptide probe. However
definitive characterization will require purification using a semi-preparative C18 column and analysis by mass spectroscopy.
As a preliminary check we biotinylated a Lys-Tyr-Lys tripeptide, similar in
size to GSH, and found a peak at a retention time of 8.1 mins. Since biotin is
known to attach easily to the e-amino group of a lysine residue this result does
seem to support our hopes of a successful biotinylation of GSSG. It remains to
bee seen whether this 7.6 min peak represents a mono-or di-biotinylated
adduct.
Upon purification of the B-GSSG complex we intend to incubate it with avidin
horseradish peroxidase(AvHpr), an avidin derivative capable of simple and
quantitative assay
Final formation of our Grase:GSSG-B:AvHpr complex will then allow a quick,
colorimetric, quantitive assay of Glutathione Reductase
References

Brian T. Miller, et al.;Peptide Biotinylation with AmineReactive Esters;Peptides. 1997, 18, 1586
Acknowledgements

We gratefully acknowledge the support of the Welch
Foundation in the form of a Departmental Research Grant