Use of Thiazole in the Synthesis of Hydroxy-aldehydes Leading to Peptidomimetic Inhibitors

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Development of a Quantitative Assay for
Glutathione Reductase
Paul Hagey, Eamonn F. Healy, Chemistry
Department, St. Edwards University, Austin TX
78704
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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 GSSG-biotin is then complexed
with Glutathione Reductase, and subsequently
complexed in a Glutathione Reductase GSSG-biotin
avidin triplex. By purifying the resulting
complex and spectroscopically determining the
concentration, Glutathione Reductase can be
accurately assayed.
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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
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Role of Glutathione
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Biotinylated Probes

• 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
• Biotin is useful because its high affinity for
  avidin (Av) and streptavidin allows the
  biotin-peptide complex to form a triplex (AvB-P)
  with the protein, thus isolating the probe
• By incubating this triplex with the probes
  natural target molecule (T) a complex of
  composition AvB-PT is now isolated
• An assay of the avidin present is now also a
  quantitative assay the target protein

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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 NHS-ester 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 (AvB-GSSG) of a out
  biotinylated probe with Avidin
• Finally we hope to form our GRaseGSSG-BAv
  complex

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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.
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UV-Vis Absorbance of GSSG
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Biotinylation Methodology

• The probe to be biotinylated is dissolved in a
  phosphate, pH7.6 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

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Results
GSSG Time 1 hour Phosphate pH7.6 Solvent A
ACN Solvent B ACN/1TFA Detector 260nm Flow
Rate 1ml / min
Biotin Time 1hour Phosphate pH7.6 Solvent A
ACN Solvent B ACN/1TFA Detector 260nm Flow
Rate 1ml / min
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Results
GSSG / Biotin Time 1 day Phosphate
pH7.6 Solvent A ACN Solvent B
ACN/1TFA Detector 260nm Flow Rate 1ml / min
GSSG / Biotin Time 4 days Phosphate
pH7.6 Solvent A ACN Solvent B
ACN/1TFA Detector 260nm Flow Rate 1ml / min
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Results
Lys-Tyr-Lys / Biotin Time initial Phosphate
pH7.6 Solvent A ACN Solvent B
ACN/1TFA Detector 260nm Flow Rate 1ml / min
Lys-Tyr-Lys / Biotin Time 1 day Phosphate
pH7.6 Solvent A ACN Solvent B
ACN/1TFA Detector 260nm Flow Rate 1ml / min
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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 C-18 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 GraseGSSG-BAvHpr complex
  will then allow a quick, colorimetric, quantitive
  assay of Glutathione Reductase

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References

• Brian T. Miller, et al.Peptide Biotinylation
  with Amine-Reactive EstersPeptides. 1997, 18,
  1586

Acknowledgements

• We gratefully acknowledge the support of the
  Welch Foundation in the form of a Departmental
  Research Grant