Most are characterized by having a sharp canthus rostralis and an unelevated snout. Usually there are supralabials and sublabials. There are rows of dorsal scales at midbody, ventral scales , and subcaudals , which are generally divided. Thus, older writings, as well as popular and sometimes scientific writings including the American Heritage, Merriam-Webster, and New Shorter Oxford dictionaries , still often call them fer-de-lance French , "iron of the lance". However, many scientists and hobbyists now restrict this name to the Martinican species, B.
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Published23 Jun Abstract The venom of snakes is composed of a heterogeneous mixture of simple and complex substances, with inflammation and hyperalgesia being the first symptom caused by the action of Bothrops venom, generating processes such as leukocyte infiltration, hemorrhage, and the intravascular formation of thrombi. Within the simple substances, we have free amino acids, peptides, nucleotides, carbohydrates, lipids, and biogenic amines organic molecules as well as cations and anions inorganic constituents.
Of the ions, we can highlight calcium, which is an important cofactor of some proteolytic enzymes as well as phospholipases A2. And magnesium and zinc are important cofactors of venom metalloproteases. Complex substances are related to proteins and enzymes. In the present study, a fraction with serinoprotease and coagulant activity has been purified on fibrinogen, called TLBpic, using a cationic ion exchange chromatographic system coupled to an HPLC system.
The main characteristic of our protocol is the speed, and the high recovery of the fraction with optimal terms gave result of evidence in the SDS-PAGE gel. Introduction Fifty years ago, when the first serpent venom protease was purified, several researchers have been reporting the purification of this kind of enzyme, finding in the biochemistry the relevant information in the understanding of certain metabolic regulations and the specific inhibition of fibrinogen coagulation and the ability to degrade to TLBm [ 1 ].
The venom of snakes causes processes such as hemorrhages that can lead to severe tissue destruction [ 2 ].
The hypotension, the incoagulability of the blood, with the consumption of the fibrinogen, and the signal of increase in breathing are simultaneous systemic symptoms. Depending on the amount of poison injected, the kidney may be compromised with irreversible results [ 3 ].
The proteases of snake venom act in the conversion of fibrinogen [ 4 ], in factors I and X of the blood coagulation cascade, as well as in enzymes that activate the coagulation protein factors, being probably the most important inducers of damage and symptoms caused by Bothrops poison such as thrombin that induces the direct coagulation of fibrinogen and prothrombin activators and factor X of metalloproteinases [ 5 ]. Snake venom possesses several enzymes [ 7 ], and at least 26 types of enzymes have already been described, many of which are hydrolytic in nature.
Of this total, 12 are commonly found in varying proportions in the different groups of snakes, and the rest is distributed in a timely manner. There are complex substances referred to as proteins and enzymes similar to the thrombin of other snakes that show coagulant activities [ 5 ].
And simple compounds are referred to as organic molecules and inorganic constituents are referred to as cations and anions, highlighting calcium within the ions, being an important cofactor of some proteolytic enzymes as well as phospholipases A2 and magnesium and zinc that are also important cofactors of metalloproteases of poison [ 8 ].
Citrate is found in high concentrations in poisons of certain snakes, and experimentally, it was shown that citrate acts as an inhibitor of several enzymes, such as nucleotidases, esterases, proteases, and phospholipases A2 [ 9 ] noting that poisons are not individual compounds but a complex mixture of proteins and their activity varies between each species of snake [ 10 ]; in this case, it would act as a cofactor of endogenous neutralization of the snake against the present enzymes of the venom.
In the present investigation, a fraction with serinoprotease-coagulant activity has been purified on fibrinogen, a fraction denominated TLBpic, using a cationic ion exchange chromatographic step coupled to an HPLC system.
This methodology guarantees that the purified material has a high degree of purity. The main feature of our protocol is the speed and high recovery of the fraction, which can be translated into optimal cost-benefit terms.
The column and chromatographic system were previously equilibrated with initial buffer 0. The elution of the samples was performed using a linear gradient of ammonium bicarbonate concentration 1. The chromatographic run was monitored at nm, and the fractions were collected in a Foxy automatic collector. Polyacrylamide gel electrophoresis was performed according to the methodology described by Laemmli [ 11 ].
The concentration gel was made using a 0. Samples and molecular mass markers were dissolved in the sample buffer 0. The electrophoretic run was performed at 30 mA. The gels were colored with 0. The synthetic substrate BApNA was used to measure the amidase activity of proteolytic enzymes such as trypsin, chymotrypsin, factor Xa, human plasma kallikrein, thrombin, and human plasmin.
The absorbance changes were read at nm. Automatic Amino Acid Analysis The amino acid analysis was performed in an automatic amino acid analyzer Pico-Tag Waters System , and the phenylthiocarbamyl-amino acid product was identified through the HPLC equipment, product derived from the derivatization with phenylisothiocyanate of the amino acids obtained by acid hydrolysis with reference to the activity of substrates hydrolyzed by poisons to proteinase activity the influence of metal ions [ 13 ].
This form of chromophores can be detected in concentrations of 1 pmol according to the methodology described [ 14 ]. A vacuum of approximately torr was then made until the start of HCl bubbling.
Created the vacuum was allowed to enter nitrogen SS ultra pure for 5 seconds. This stage was repeated three times. After this period, the reaction tube was placed in vacuum up to 65 millitorr to dry the hydrolyzed sample. Each tube was agitated and centrifuged at for 15 minutes and placed in vacuum up to 65 millitorr for its evaporation.
This procedure removed the salts and solvents adsorbed by the amino acids. After derivatization, the sample was dried under vacuum until 50 millitorr to complete the removal of the entire PITC. The identification of each amino acid was made in relation to a run pattern of amino acids-PITC [ 15 ]. For the quantification of cysteine and methionine, the samples were previously oxidized with performic acid.
The hydrolysis and derivatization of the oxidized samples of these amino acids followed the described methodology. The elution of the samples was carried out using a linear gradient of concentration of ammonium bicarbonate 1. Determination of Proteolytic Activity The synthetic substrate BApNA was used to measure the amidase activity of proteolytic enzymes such as trypsin, chymotrypsin, factor Xa, human plasma kallikrein, thrombin, and human plasmin.
Determination of Fibrinogenolitic Activity It was determined by the method described in [ 18 ] and the genetic regulation of fibrinogen synthesis and its assembly of clot properties and its variability [ 19 ], adapted to our experimental condition. The purified TLBpic fibrinogenolytic fraction was diluted in 0.
According to the results, it was shown that it is an alpha-type thrombin-like. The time of the linear relationship between the intervals of the coagulation times and the respective protein concentrations in the fibrin network formation reaction was after 2 minutes. The number of coagulation units is defined as the amount of enzyme that coagulates 1.
Determination of the Molecular Mass of the Thrombin-Like by Mass Spectrometry Electrospray ESI Referring to ionization sources by mass spectrometry, identification by relevant electron ionization was established [ 19 ]. An aliquot of the sample 4. The parameters of processing were a strip of mass of exit — Da with a resolution of 0. The automatic sequencer uses the degradation technique [ 21 ], to remove and identify amino acids from the N-terminal portion of a polypeptide.
After the activation of a filter composed of paper and fiberglass, the protein was covalently bound to this support and then placed in the reaction chamber. At the end of each degradative cycle, the N-terminal amino acid is removed from the side chain in the form of a more stable derivative, such as phenylthiohydantoin of the corresponding amino acid PTH. The PTH-amino acid is transferred to a high efficiency liquid chromatography system, where the identification is made by comparison with the chromatography of a PTH-amino acid standard.
Reagents and buffer were transferred to the reaction chamber and converted by automatic control through a microprocessor, allowing automatic sequences with a high sensitivity between 10 and picomoles of protein or peptides. Results and Discussion 3. Chromatographic Fractionation of Bothrops pictus Total Venom by Column Ion Exchange As a first chromatographic step for the purification of thrombin-like fraction from the total venom of Bothrops pictus coastal pallet.
From total snake venom, mg was dissolved in 25 ml of a buffer solution of 0. Figure 1 Ion exchange cationic chromatography. The run was performed with bicarbonate ammonium buffer 0. Electrophoresis in SDS-Page To determine the molecular mass of the enzyme, polyacrylamide gel electrophoresis was performed at a concentration of Figure 2 Electrophoresis analysis.
Electrophoresis is observed in Fibrinogenolitic Activity of the TLBpic Fraction The TLBpic fraction exhibited a fibrinogenolytic activity against bovine fibrinogen, revealing that it is an alpha-type thrombin-like, being able to degrade the fibrinopeptide alpha of the fibrinogen molecule, evidenced in Figure 3.
Figure 3 Fibrinogenolytic activity. Measurement of Proteolytic Activity The proteolytic activity was determined according to the methodology described in the previous chapter. It was observed that the TLBpic fraction from ion exchange cationic chromatography was the one that exhibited a significant proteolytic activity, as shown in Figure 4. Figure 4 Determination of proteolytic activity. The overall composition analysis showed a high concentration of amino acids such as Asp and Glu, a high concentration of hydrophobic amino acids such as Pro, Val, and Tyr, and a low content of basic amino acids like His and Arg.
The residue that corresponds to Trp due to the method Pico-Tag was not determined.
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