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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
This work was funded by an ARC Discovery Project Grant to P. W. K. and Assoc. Prof. J. I.
Vandenberg. Dr. A. Torres, and Dr. P. Bansal are thanked for vital contributions to the work on platypus
venom peptide isomerase. Mr. A. Hallen is thanked for valuable discussions on enzyme purification.
J. M. S. K. was supported by an Australian Postgraduate Award.
Experimental Part
Synthetic Hexapeptides. Customer-specified synthetic hexapeptides were purchased from EZBiolab
(Indiana, USA). To make up the peptide soln., 1 mg of peptides was dissolved in 1 ml of 50 mm
phosphate buffered saline (PBS; 100 mm NaCl, 50 mm Na2H2PO4, pH adjusted with NaOH to 7.4).
Mouse Tissue Extracts. Eight tissues were dissected from wild type C57/BL6 mouse; these tissues
were brain, heart, kidney, liver, lung, small intestine, spleen, and stomach. Each tissue was finely sliced
and then homogenised in 5 ml of ice cold 1% (w/v) trehalose soln. with a Bamix cutter using short bursts
to prevent enzyme denaturation by heating. The homogenate was then centrifuged at 3000g at 48 for
30 min. After centrifugation, the solid material was separated and discarded while the supernatant was
kept and filtered through a 0.2 mm Minisart filter (Sartorius, D-Gçttingen). The filtrate was concentrated
by centrifugal ultrafiltration using a Macrosep 30-kDa molecular weight cut-off membrane (Pall, New
York, USA) and then frozen for later use.
Isomerase Assays. Frozen tissue extract was thawed and further concentrated by centrifugal
ultrafiltration using a Nanosep 30 kDa molecular-weight cut-off membrane (Pall). It was spun at ca.
5000g at 48 for 10 min and then washed with 50 mm PBS (pH 7.4) and 100 mm EDTA (pH 7.0). The
retentate (MW >30 k) was resuspended in 50 mm PBS, pH 7.4. For each assay, 40 ml of the concentrated
tissue extract was mixed with 40 ml of peptide soln. and 40 ml of Complete protease inhibitor cocktail, 5ꢀ
concentration (Roche, D-Mannheim). The mixture was incubated at 378; aliquots of mixture were
withdrawn for RP-HPLC assays at 0, 2, 4, and 24 h.
RP-HPLC. Isomerase assays mixtures were analysed using RP-HPLC with an Agilent Eclipse XDB-
C18 column (4.6 mmꢀ250 mm) on an Agilent Technologies 1200 series system (California, USA). The
solvent system was made up of H2O containing 0.1% (v/v) F3CCOOH (TFA; solvent A) and 90% MeCN
containing 0.1% (v/v) TFA (solvent B). The solvent gradient for the assay was 5–45% B for 15 min, 45–
60% B for 1 min, 60–5% B for 1 min, 5–0% B for 2 min and 0% B for 1 min, at the rate of 1 ml minꢁ1
.
)
Mass Spectrometry. MALDI-TOF-MS was used to determine the mass-to-charge ratio ([MþH]þ
of peptides on a QSTAR XL mass spectrometer equipped with a MALDI source (Applied Biosystems,
California, USA).
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