**Abbreviations**

22 Chromatography – The Most Versatile Method of Chemical Analysis

**5.3. Potentiometric titration of fatty acids** 

acid per minute, in the reaction conditions.

**5.4. Phenol red** 

described by Radvanyi [67].

**5.5. Indirect hemolysis** 

convert 0.001 UA 558 nm per minute.

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

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

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

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

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

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

a hemolysis halo present in agarose gel containing intact red blood cells [76].

treatment. The fluorescence intensity unit was converted to nmoles/min [33].

PLA2s: Phospholipases A2 sPLA2: secreted PLA2 cPLA2 : cytosolic PLA2 iPLA2: Ca2+ independent PLA2 PAF-AH: acetyl-hydrolases from platelet activating factors and liposomal Mr: relative mass PAF: platelet activating factors svPLA2s: phospholipase A2 found in snake venoms SDS-PAGE: Sodium dodecyl sulfate – PolyAcrylamide Gel Electrophoresis MW: Molecular weight RP-HPLC: reverse-phase chromatography DEAE-Sepharose: Dietylaminoetyl Sepharose tEnd cells: endothelial cells CNBr: cyanogen bromide PBS: phosphate buffer saline IEF: isoelectric focusing 2DE: bi-dimensional electrophoresis IPG: immobilized pH gel pIs: isoelectric points DTT: Dithiothreitol TBP: Tributyl phosphine ESI: Electrospray Ionization

MS: Mass Spectrometry CK-MM: Creatine Kinase - skeletal muscle tissue CK-MB: Creatine Kinase – cardiac CK-BB: Creatine Kinase - brain CK: Creatine Kinase LDH: lactate dehydrogenase NBD: N-4-Nitrobenzo-2-Oxa-1,3-Diazole NBD-PC: N-4-Nitrobenzo-2-Oxa-1,3-Diazole Phosphatidylcholine NBD-PG: N-4-Nitrobenzo-2-Oxa-1,3-Diazole NBD-PE: N-4-Nitrobenzo-2-Oxa-1,3-Diazole phosphatidylglycerol NBD-PA: N-4-Nitrobenzo-2-Oxa-1,3-Diazole Phosphatidic acid
