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298 Lee and Aguilar
2.2. Equipment and Supplies
1. HPLC solvent delivery system equipped with quaternary gradient capability and
a variable wavelength UV detector. Typically, the detector is set to a range of a
0.08 bandwidth and a response time of 1.0 s.
2. IAM.PC.DD2 guard column 12-μm particle size, 300Å pore size, 3.0 mm i.d. × 1
cm length (Regis Technologies Inc., Morton Grove, IL, USA).
3. IAM.PC.DD2 12-μm particle size, 300Å pore size, 4.6 mm i.d. × 15 cm length
(Regis Technologies Inc.).
4. Solvent filtration apparatus equipped with a 0.22-μm Durapore filter (Millipore,
Billerica, MA, USA).
5. Sample filter, 0.22 μm cellulose acetate membrane.
6. Buffer A: 0.1 M Tris–HCl (pH 7.2), 0.2 M KCl, 20% ethylene glycol, 0.05%
NaN 3 (see Note 1).
7. Buffer B: 1% sodium octanesulfonate in Buffer A.
8. Buffer C: 4% acetonitrile in Buffer B.
9. A programmable fraction collector.
3. Methods
3.1. Sample Preparation
1. Solubilize 100 mg lyophilized Crotalus artox venom powder from Sigma (St.
Louis, MO, USA) containing phospholipase A 2 in 20 mL sample buffer (25
mM CaCl 2 , 50 mM Tris–HCl, pH 7.6) and filtered through a 0.22-μm cellulose
acetate membrane syringe filter. The protein concentration of this solution is
approximately 4 mg/mL.
2. For the preparation of phospholipase A 2 directly from pancreatic tissue, homogenize
300 g tissue in 300 mL of tissue homogenizing solution 0.1 M NaCl using
a blender for 30 s at 4ºC. After homogenization, adjust the tissue homogenate
solution to pH 4.0 with concentrated HCl and heat the solution at 70ºC for 2–3 min.
Cool the homogenate solution in an ice water bath for 30 min and readjust the pH
to 7 with concentrated NH 4 OH. Centrifuge the sample at 3500 × g for 5 min at 4ºC
and then gradually add solid (NH4) 2 SO 4 to the supernatant with constant stirring
until the concentration of (NH4) 2 SO 4 reaches 60% saturation at room temperature.
Precipitate the proteins in an ice bath for 1 h and collect the precipitate by
centrifugation at 5000 × g for 10 min at 4ºC. Dissolve the pellet in 2.5 mL Milli-Q
water followed by 50 μL of a 0.1 M solution of PMSF in isopropanol. Incubate
the sample further on ice for 1 h and lyophilize. To lyophilize the proteins, the
solution is kept in a –75ºC deep-freezer or placed in a dried ice/acetone bath till
the solution completely frozen. The sample is then lyophilized overnight at –75ºC
in a vacuum lyophilizer. Dissolve the lyophilized proteins in sample buffer with
volume which gives the protein concentration approximately 4 mg/ml (see Note
2). Before use, activate the lyophilized sample by adding Trypsin relative to the
total protein. Trypsin converts the inactive phospholipase A 2 to its active form by
selectively cleaving an N-terminal octapeptide.