**4. Functional characterization**

18 Chromatography – The Most Versatile Method of Chemical Analysis

The proteomic analysis of snake components has made use of the 2D electrophoresis as a tool, due to its high-resolution capability that allows, in a single process, the determination of apparent molecular mass and isoelectric point of the venom constituents. Fernandez et al. [22] described the determination of the isoelectric point and apparent molecular mass of Basp-PLA2-II using this technique. In order to do it, the protein was focused in IPG Immobiline® Dry Strip of 7 cm and pH 3-10, under a 200 V tension for 1 min, followed by a second stage of 3500 V for 120 minutes. The second dimension was done in SDS-PAGE 12% and then subsequently dyed with Coomassie blue. It was demonstrated that Basp-PLA2 –II had a pI of 4.9, which is close to the theoretical isoelectric point value (pI 5.05) defined by the primary sequence, evaluated using the Compute pI/MW tool (www.expasy.ch/tools) software and apparent molecular weight between 15 and 16 kDa, consistent with the molecular weight (MW

The advantage of this technique is the high resolution. Alape-Giron [63] working with *B. asper* venom, performed an ontogenic analysis and an analysis based on the snake's capture location in different regions of Costa Rica. Using tryptic digestion, MALDI-TOF mass fingerprinting analysis and aminoacid sequencing by MALDI-TOF submitted to similarity search by BLAST, the author showed the intra-specific variability in venom composition. It was hence evidenced that among the venoms obtained from adult species collected in the Caribe area and the Pacific area, there are around 30 proteins that are found in a snake

In our lab, this technique has been used as follows: The proteins are separated by the isoelectric point in 13 cm strips with pH values varying between 3 and 10 in a nonlinear form. These strips contain polyacrylamide gel, where the gradient pH is formed by the presence of ampholytes. To re-hydrate the strips, 250 µL of sample [400 µg of proteins plus re-hydration solution (7 M of urea, 2 M of Thiourea, 2% of Triton X-100 (v/v/), 1% of IPG *Buffer*® (v/v) and DTT)] is applied in a channel of the apparatus over which the strips are set. The strip's gel is re-hydrated at room temperature for about 12 hours. After this period, the strips are taken to the focusing system in the following conditions: (1) 500 V step until accumulates 500 Vh; (2) 500 to 1000 V gradient until it accumulates 800 Vh; (3) 1000 to 8000 V gradient until it accumulates 11300 Vh and (4) 8000 V step until it accumulates 3000 Vh. In average, the program run during 5.5 hours, but the time of the final step can be lengthened, if the sample does not reach to the end of the strip during the running according the initial program, it could be confirmed by a bromophenol blue line. At the end of focusing, the strips are equilibrated in two steps. On the first, 10 mL of the solution containing 6 M of urea, 2% of SDS (m/v), 30% of glycerol (v/v), 75 mM of Tris-HCl (pH 8,8), 0,002% of bromophenol blue and 1% DTT (m/v) for each strip is used. In the second, the same solution is used, but DTT is replaced by 2.5% of iodoacetamide (m/v). Each strip equilibrium step run during 15 minutes, under light stirring. Following that, the strips are applied on 10 % polyacrylamide gels previously prepared on 180 X 160 X 1.0 mm plates. After each strip and the standard stay appropriately accommodated in the polyacrylamide gel, a 0,5% agarose (m/v) heated (40 °C) solution is added. The agarose polymerization, provides an effective contact between the strip and the gel, thus avoiding the appearance of air bubbles. Protein

14,212±6 Da) obtained by ESI/MS (Electrospray Ionization/Mass Spectrometry).

group from a place which find no correspondents in the other.

Many biological activities are related to myotoxins with PLA2 structure obtained from snake venoms. In bothropic snake bite accidents and in experimental models with the use of these venoms, the noxious activity induced by these toxins on the striated muscles is striking [64]. The detection of the myotoxic activity associated to the phospholipase activity detection (in the case of Snake venom PLA2 Asp49) is used as an important auxiliary biological marker in the purification procedures, monitoring its presence.

The myotoxic activity assay can be done in two ways: *in vivo* and *in vitro*. The analysis can be done through the quantification of the released intracellular enzymes activity to the

periphery blood or to the supernatant of the culture medium of cellular lineages. There are two main enzymes used to this end:

Purification of Phospholipases A2 from American Snake Venoms 21

( )

*µ*

(1)

concentration is determined according to manufacturer's instructions and expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute [26,70-72].

In order to assay myotoxic *in vitro* activity, myoblast lineage cells are used, such as murine skeletal muscle C2C12 myoblasts (ATCC CRL-1772) as described by Lomonte et al. [73], cultivated in modified Dubelco Eagle medium, supplemented with 1% bovine fetal serum. PBS solubilized sample, negative control (PBS) and positive control (Triton X-100) should be filtered through 22 µm pore filters immediately prior to use. Reagents for LDH activity

In 96 well plate, 2X105 cells/150 µL are set, sample and/or control (50 µL) are incubated in humid atmosphere at 37 °C and 5% CO2 for a 3 hour period. Afterwards, collect supernatant aliquot and quantify LDH activity released by cells with cytoplasmic membrane integrity compromised, according to manufacturer's instructions and expressed in U/I, where one

Phospholipase A2 activity can be measured according to the technique described by Holzer

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

OD425nm OD 600nm / min x 0.07862 umol / OD425nm / 1 / protein 1 / g

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

dosage are prepared and used according to manufacturer's instructions.

unit corresponds to the production of 1 mmol of lactate per minute.

the production of 1 µmol of 4-nitro-3-hydroxy-benzoic acid per minute.

 − −= −

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

**5.1. 4N3OBA Substrate enzymatic hydrolysis** 

and Mackessy [65], modified for 96 wells plate [74].

<sup>2</sup> PLA activity mol • min 1• g 1

*µ µ*

**4.2.** *In vitro* **myotoxic activity assay** 

**5. Phospholipasic activity** 

Creatine Kinase (EC 2.7.3.2): is a dimeric protein formed by the combination of subunits (B and M) and in its cytosolic form is found in many tissues, especially in skeletal muscle tissue (CK-MM), cardiac (CK-MB) and in the brain (CK-BB).

Lactate dehydrogenase (EC 1.1.1.27): is an enzyme widely distributed in many tissues and organisms. It is presented in the form of homo or hetero tetramers of subunits M and H, being present in muscular tissue in the homotetrameric form of subunit M.

*In vivo*, the CK activity quantification in murine models has been the most used to assay the presence of myotoxic PLA2, especially due to their low cost, ease of performance and high specificity as skeletal muscular tissue lesion markers when exposed to myotoxins.

As for the *In vitro* assays, myoblast lineages C2C12 (ATCC CRL-1772), differentiated until the formation of myotubules, have been used as models to assay the cellular toxicity, through the quantification of LDH levels in the supernatant of cell cultures exposed to toxins.

Regarding the phospholipase activity detections, it can be done by direct and indirect methods. Directly, it is possible to detect the presence of PLA2s with the use of chromogenic substrates, such as 4N3OBA(4-nitro-3-octanoyloxybenzoic acid) that induce the formation of detectable product at 425 nm [65] and fluorescent substrates (NBD) coupled to phospholipids that are used to quantitively and qualitatively survey the PLA2s activity isolated from snake venom [23].

Indirectly, the approach used consists in the potentiometric assay of the fatty acids released after the enzymatic hydrolysis of the phospholipids, through the quantification with standard alkaline solution [66]. Moreover, fatty acids released by the enzymatic degradation can be quantified through the alteration of the optical density of the pH indicator solution, such as phenol red [67], brilliant yellow [68] and bromothymol blue [69]. Another indirect method to assay PLA2 activity present in samples consists in the detection of hemolysis induced by lysophospholipids derived from phospholipids submitted to enzymatic digestion. This can be done through the quantification of hemoglobin present in solution or through the visualization of hemolytic halo in agarose matrix with immobilized red blood cells.

## **4.1.** *In vivo* **assay of the myotoxic activity**

Mice is used for the *in vivo* assay of the myotoxic activity. Swiss males weighing between 18 g and 22 g, kept in controlled environment (12 h in the light and 12 h in the dark), with food and water *ad libitum* up to the moment of use. PBS solubilized sample and control (PBS) are filtered through 44 µm pores immediately prior to use. Reagents for CK activity dosage are prepared and used according to manufacturer's instructions.

A Sample (50 µL) or control (50 µL) will be injected in mice gastrocnemic muscle using adequate device in order to guarantee a precise volume control. After a time lap (3 and/or 6 h), blood sample is collected in heparinized tubes and centrifuged to separate plasma. CK concentration is determined according to manufacturer's instructions and expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute [26,70-72].
