Epidemiology: Open Access
Open Access

Poisonous; Miniaturization; Venom

Introduction

Modern analytical techniques have developed strongly in recent decades with respect to the growth of sensor miniaturization, data digitization and computing power to enable the use of high performance mathematical tools with equipment less and less bulky [5,6].

Biochemists currently have a wide variety of techniques for analyzing biomolecules such as snake venom proteins. The frenetic research of scientists for the high precision that must characterize the results of their work always pushes them to perfect their analysis techniques on the one hand and on the other hand, to combine them for a better performance [7]. Mass spectrometry has become a powerful analytical tool over the last 20 years. The discovery of new ionization modes (Electrospray ionization (ESI), matrix-assisted desorptionionization or MALDI), which earned their authors part of the Nobel Prize in Chemistry in 2002, made it possible to implement this family, and in almost all types of samples, and in particular to biological macromolecules [8]. In this study, we are interested in the development of an approach based on mass spectrometry to allow characterizing progressively the venoms of Congolese snakes. The results of this study will make it possible, on the one hand, to build a database of Congolese snake venom proteins and, on the other hand, to understand the differences between these two serpents in terms of the biochemical constitution of their respective venoms.

Biological material

The venoms of Puff Adder and African Bush Viper are taken manually from live specimens of the Lwiro serpentarium (South Kivu Province) in DR Congo. The venoms are then frozen and freeze-dried and stored at 4ºC.

Method

Our samples were analyzed using the Waters process with the assistance of MassLynx software (c: \ masslynx \ snake venom \ snake venom.pro \ acqudb \ 20151209_mse_60min) for data processing. The details of the different steps are as follows:

Run method parameters

Pump

Waters Acquity SDS

Run Time: 60.00 min

Comment:

Solvent Selection A: A1

Solvent Name B: 0.1% FA Acetonitrile

Switch 1: No Change

Switch 2: No Change

Switch 3: No Change

Seal Wash: 20.0 min

Chart Out 1: System Pressure

Chart Out 2: %B

System Pressure Data Channel: Yes

Flow Rate Data Channel: No

%A Data Channel: No

%B Data Channel: No

Primary A Pressure Data Channel: No

Accumulator A Pressure Data Channel: No Primary B Pressure Data Channel: No

Accumulator B Pressure Data Channel: No

Degasser Pressure Data Channel: No

Gradient Table

Time (min) Flow Rate %A %B Curve

1. Initial 0.300 98.0 2.0 Initial

2. 2.00 0.300 98.0 2.0 6

3. 40.00 0.300 50.0 50.0 6

4. 40.10 0.300 20.0 80.0 6

5. 50.00 0.300 20.0 80.0 6

6. 50.10 0.300 98.0 2.0 6

7. 60.00 0.300 98.0 2.0 6

Run Events: Yes

Gradient Start (Relative to Injection): 0 uL

Participate in pre-analysis: No

2D Repeat: No

Detector

Waters Acquity TUV

Run Time: 30.00 min

Wavelength Mode: Single Wavelength

Lamp On: On

Channel A..

Comment:

Wavelength: 205 nm

Sampling Rate: 20 points/sec

Data Mode: Absorbance

Time Constant: 0.1 sec

Auto Zero on Wavelength Change: Maintain Baseline

Auto Zero On Inject Start: Yes

Analog 1...

Sensitivity: 2.000 AUFS

Chart Polarity: Positive (+)

Voltage Offset: 0 mV

Enable Chart Mark: Yes

Run Events: Yes

Pulse Width: 1.0 sec

Rect Wave Period: 0.2 sec

Auto sampler

Waters ACQUITY FTN Auto Sampler

Run Time: 60.00 min

Comment:

Load Ahead: Disabled

Loop Offline: Automatic min

Wash Solvent Name: Water

Pre-Inject Wash Time: 0.0 sec

Post-Inject Wash Time: 6.0 sec

Purge Solvent Name: Water

Dilution: Disabled

Dilution Volume: 0 uL

Delay Time: 0 min

Dilution Needle Placement: 4 mm

Target Column Temperature: Off C

Target Sample Temperature: 8.0 C

Sample Temperature Alarm Band: Disabled

Syringe Draw Rate: Automatic

Needle Placement: Automatic

Pre-Aspirate Air Gap: Automatic

Post-Aspirate Air Gap: Automatic

Column Temperature Data Channel: No

Room Temperature Data Channel: No

Sample Temperature Data Channel: No

Sample Organizer Temperature Data Channel: No

Sample Pressure Data Channel: No

Preheater Temperature Data Channel: No

Seal Force Data Channel: No

End detector

Run Events: No

Sample Run Injection Parameter

Injection Volume (uL) - 4.00

End autosampler

End of experimental record..

Generic Instrument Postrun Report

Software Version: 1.60.2782

Firmware Version: 1.60.2274 (Aug 10 2013)

Checksum: 0xd71e0eda

Serial Number: E10TUV015A

Lamp On/Off Event: No

Lamp Life: 1744.00

Lamp Serial Number: 1F8ABD2

Flow Cell Type: Analytical LG

Flow Cell Path Length: 10.000 mm

Flow Cell Volume: 0.50 micro liters

Flow Cell Serial Number: 10346

Flow Cell Part Number: 205015016

Optics Temperature Stabilization Setting: Normal Temperature

Waters Acquity SDS Postrun Report

IcsVersion: 1.60.1872

Firmware Version: 1.60.267 (Aug 19 2013)

Checksum: 0x36f220a9

Serial Number: C14BUR100G

Minimum System Pressure: 2890.5

Maximum System Pressure: 4731.5

Average System Pressure: 4248.0

Minimum Degasser Pressure: 0.5

Maximum Degasser Pressure: 0.5

Average Degasser Pressure: 0.5

Software Version: 1.60.1774

Firmware Version: 1.60.364 (Sep 20 2013)

Checksum: 0x35d5392b

Serial Number: B14USM471G

Needle Size: 15.0

Minimum Sample Temperature: 8.0

Maximum Sample Temperature: 8.3

Average Sample Temperature: 8.2

Minimum Column Temperature: -0.2

Maximum Column Temperature: 0.0

Average Column Temperature: -0.2

Results

HRMS Analysis of samples from snake venom

This data contains an overlay of total ion chromatogram of all two samples [Figures 1 and 2].

Figure 1: Mass Chromatogram - TIC of Puff Adder.

Figure 2: Mass Chromatogram - TIC of African Bush Viper.

• Peptides have eluted from ~ 6 min to ~ 35 min.

• Due to presence of high number of peptides, the TIC gives a bellshaped curve.

•Every scan of the TIC gives mass spectral information of eluted/ co-eluted peptides.

• This data contains an overlay of mass spectra of combined scan [Figures 3 and 4]

Figure 3: Mass Spectra of Puff Adder.

Figure 4: Mass Spectra African Bush Viper.

At ~24 min of the mass chromatogram, of all two samples.

• Mass spectral information is obtained for peptide present

Discussion

In the present study, snake venom toxin proteins of Puff Adder and African Bush Viper were analyzed with the help of Mass Spectrometric. The venom of Puff Adder contains 44 % of proteins against 36 % for African Bush Viper. Generally, snake venoms are considered to contain mainly proteins, ranging from 70 to 90 % (10). In their study by using the two-dimensional gel electrophoresis, Vijayan J. et al were reported the protein content of eight Malaysian snake venom was between 30 and 80% (11). According the molecular weight (Mw) of proteins, the Puff Adder venom contains 15, 9% of Mw (1000-9000 Da), 68, 2% of Mw (10000-49000 Da), 15, 9% of Mw (50000-99000 Da) and 0, 0% of Mw (100000-190000 Da). However, African Bush Viper venom comprise 2,8% of Mw (1000-9000Da), 44,4% of Mw (10000-49000 Da), 27,8% of Mw (50000-99000 Da) and 25,0% of Mw (100000-190000 Da). Both venoms under study belong to the Viperidae family. Several studies show that enzymes are high molecular weight proteins [9] this is what we find in this work. It should be noted that the African Bush Viper venom contains many enzymes of very high molecular weight compared to that of Puff Adder. The hydropathy index is a measure that allows knowing the hydrophilic or hydrophobic character of a region of a protein through the amino acid sequence. A positive value of the index corresponds to a hydrophobic behavior and a negative value corresponds to a hydrophilic behavior.

The GRAVY (medium hydropathy) of these two venoms shows that 18.2% of the Puff Adder venom proteins are hydrophobic and 81.8% are hydrophobic. However, in the African Bush Viper venom, 5.6% is hydrophobic and 94.4% hydrophilic. The amino acid profiles of the venom proteins examined show that Adder Puff possesses a venom rich in negatively charged amino acids (Asp + Glu) while that of African Viper has many positively charged amino acids (Arg + Lys). Lysine and arginine and their adequate presence help to become effective in the bio-molecule [10]. The most striking element in this study is that both venoms are made up of proteins of different natures. This leads some authors to talk about the variability of venoms that can be observed even within the same family. This variability of the venoms would be observed on the symptomatology related to the local necrosis for example, presented during an envenomation by these two different vipers. We also find that the African Bush Viper venom is free of phospholipase A2. However one of the most important protein superfamilies present in snake venoms are the phospholipasesA2 (PLA2, E.C. 3.1.1.4), a class of heat-stable and highly homologous enzymes, which catalyze the hydrolysis of the 2-acyl bond of cell membrane phospholipids releasing arachidonic acid and lysophospholipids.

Table 1: Physicochemical characterization of venom toxin proteins of Puff Adder.

Table 2: Physicochemical characterization of venom toxin proteins of African Bush Viper.

Table 3: Amino acid composition profile (in %) of Puff Adder venom toxin proteins.

Table 4: Amino acid composition profile (in%) of African Bush Viper venom toxin proteins.

Conclusion

At the end of our analysis, we realize that although both snakes belong to the same family of vipers, they have fundamentally different venoms from the point of view of their biochemical compositions. This leads specialists to talk about the variability of venom. This variability is genetic, that is, specific to each individual. The variations concern both the concentration of the different fractions and their biochemical structures [11]. Mass spectrometry has been an undeniable aid to us. This allowed us to scrutinize the secrets of the venoms of these two snakes. As our samples came from the eastern part of the country, it perhaps would be useful to extend this study to all geozoological areas of the Democratic Republic of Congo but also to other venomous species that this vast territory has.

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