**5. Phospholipasic activity**

## **5.1. 4N3OBA Substrate enzymatic hydrolysis**

Phospholipase A2 activity can be measured according to the technique described by Holzer and Mackessy [65], modified for 96 wells plate [74].

Prepare aliquots of 100 µL of 4N3OBA 0.1% solution in acetonitrile and lyophilize. Keep the aliquots at -20° C until ready to use. The color reagent is prepared solubilizing the contents of one aliquot of 4N3OBA in 1ml of reagent containing Tris 10 mM, CaCl2 10 mM, NaCl 100 mM, and pH 8.0. For the test in micro plates, add 180 µL of color reagent and 20 µL of sample or water (blank), incubate the mixture at 37 °C for 5 minutes, measuring the optical density at 425 nm and 600 nm (to correct sample turbidity) at 30 second intervals. The activity will be expressed according to the equation (1) where 1 unit of phospholipase activity corresponds to the production of 1 µmol of 4-nitro-3-hydroxy-benzoic acid per minute.

( ) <sup>2</sup> PLA activity mol • min 1• g 1 OD425nm OD 600nm / min x 0.07862 umol / OD425nm / 1 / protein 1 / g *µ µ µ* − −= − (1)

## **5.2. Enzymatic hydrolysis of fluorescent substrates (NBD)**

The phospholipase activity can also be assayed with the used of chromogenic substrates, using acyl-NBD reagents: NBD-PC (Phosphatidylcholine), NBDPG (phosphatidylglycerol), NBD-PE (phosphatidylethanolamine) or NBD-PA (phosphatidic acid). A solution of fluorescent lipids should be previously prepared in a 1 mg/ml concentration in chloroform. 100 µL aliquots are distributed and then dried under nitrogen flow. The dried lipid will be solubilized in 1 ml of NaCl 0.15 M and sonicated until the obtention of a limpid solution. For

the test, the lipids should be diluted in a solution containing Tris-HCl 50 mM, CaCl2 1 mM pH 7.5. Initially, incubate the solution at 37 °C and, after 2 minutes, make an initial reading, configuring the equipment for excitation at 460 nm and emission at 534 nm. Following, apply the sample and make a second reading after 12 minutes. The change in fluorescence intensity is converted to nanomoles of product per minute (nmoles/min) using a calibration curve, prepared by hydrolyzing completely a substrate solution through sodium hydroxide treatment. The fluorescence intensity unit was converted to nmoles/min [33].

Purification of Phospholipases A2 from American Snake Venoms 23

minutes. Then, centrifuge, collect the supernatant and determine the optical density at 405 nm, using PBS as blank. The results will be expressed in % of hemolysis compared with the

Hemoglobin dosage present in solution with the use of the Drabkin reagent (potassium ferrocyanide in buffered environment) [77] can be done by comparing the optical density of the samples with the standard curve made with the hemoglobin cyanide solution, according

To assay the hemolytic activity in agarose gel, carefully heat the suspension containing agarose 2% in PBS until complete fusion. After partial cooling (45 °C), add an equal volume of PBS containing CaCl2 0.02 M; egg yolk suspension (1:30 m/v), erythrocytes washed in PBS (1:30 m/v), pouring over Petri plate until the formation of a layer approximately 2 mm thick. After solidification of the gel, orifices of uniform diameter (0.2 cm diameter) to apply the sample are made. The gel is incubated for 12 hours, at 37 °C and humid environment. The formation of a translucid halo around the gel application point is indicative of phospholipase activity, contrasting with the rest of the gel which remains with a reddish

tone due to the presence of integral red blood cells retained in the gel net.

PAF-AH: acetyl-hydrolases from platelet activating factors and liposomal

SDS-PAGE: Sodium dodecyl sulfate – PolyAcrylamide Gel Electrophoresis

positive control.

**Abbreviations** 

Mr: relative mass

MW: Molecular weight

tEnd cells: endothelial cells CNBr: cyanogen bromide PBS: phosphate buffer saline IEF: isoelectric focusing

IPG: immobilized pH gel pIs: isoelectric points DTT: Dithiothreitol TBP: Tributyl phosphine ESI: Electrospray Ionization

PLA2s: Phospholipases A2 sPLA2: secreted PLA2 cPLA2 : cytosolic PLA2

iPLA2: Ca2+ independent PLA2

PAF: platelet activating factors

svPLA2s: phospholipase A2 found in snake venoms

RP-HPLC: reverse-phase chromatography DEAE-Sepharose: Dietylaminoetyl Sepharose

2DE: bi-dimensional electrophoresis

to manufacturer instructions.

## **5.3. Potentiometric titration of fatty acids**

The phospholipase activity can be assayed by potentiometric titration as described by de Haas [75], using as substrate an egg yolk emulsion in the presence of sodium deoxycholate 0.03 M and CaCl2 0.6 M. Fatty acids released enzymatically are titrated with a standard solution of NaOH 0.1 N at pH 8.0 at room temperature. The phospholipase activity is generally done with different concentrations of toxin, and calculated per amount of microequivalents of alkali consumed per minute, by mg of protein. One unit of phospholipase activity can be defined as the quantity of enzyme that releases 1 µmol of fatty acid per minute, in the reaction conditions.

## **5.4. Phenol red**

The spectrophotometric detections of phenol red solution, induced by the increase of free fatty acids concentration can also be used to assay the phospholipase activity in samples, as described by Radvanyi [67].

In order to use this technique, prepare the reagent solution containing Phosphatidylcholine 0.25% (w/v) TritonX-100 0.4% (v/v), phenol chloride 32 mM. In a thermostatic cuvette at 37 °C, add 1mL of reagent solution and 10 µL of sample. After stabilization for 20 seconds, determine the optical density measuring at 558 nm for 3 minutes, in kinetic intervals of 15 seconds. One unit of phospholipase can be defined as the quantity of enzyme necessary to convert 0.001 UA 558 nm per minute.

## **5.5. Indirect hemolysis**

In this test, phospholipids (from egg yolk, soy lecithin or other sources) are used as substrates, with the production of fatty acid and corresponding lysophospholipids. These lysophospholipids have membrane activity over red blood cells, producing hemolysis that can be detected through the quantification of hemoglobin present in solution or through a hemolysis halo present in agarose gel containing intact red blood cells [76].

For the test, collect blood in a heparinized tube, wash the red blood cells with PBS, centrifuging at 800 xg for 5 minutes and prepare the suspension at 3%. Prepare solution containing phosphate buffer 20 mM, sodium chloride 100 mM and CaCl2 10 mM, erythrocyte suspension 3% (1:30 v/v) and egg yolk solution 0.1% (1:30 v/v). Add 10 µL of sample or PBS (control 0%) or Triton X-100 0.1% (control 100%) and incubate at 37 °C for 30 minutes. Then, centrifuge, collect the supernatant and determine the optical density at 405 nm, using PBS as blank. The results will be expressed in % of hemolysis compared with the positive control.

Hemoglobin dosage present in solution with the use of the Drabkin reagent (potassium ferrocyanide in buffered environment) [77] can be done by comparing the optical density of the samples with the standard curve made with the hemoglobin cyanide solution, according to manufacturer instructions.

To assay the hemolytic activity in agarose gel, carefully heat the suspension containing agarose 2% in PBS until complete fusion. After partial cooling (45 °C), add an equal volume of PBS containing CaCl2 0.02 M; egg yolk suspension (1:30 m/v), erythrocytes washed in PBS (1:30 m/v), pouring over Petri plate until the formation of a layer approximately 2 mm thick. After solidification of the gel, orifices of uniform diameter (0.2 cm diameter) to apply the sample are made. The gel is incubated for 12 hours, at 37 °C and humid environment. The formation of a translucid halo around the gel application point is indicative of phospholipase activity, contrasting with the rest of the gel which remains with a reddish tone due to the presence of integral red blood cells retained in the gel net.
