Among the serpents that inhabit Peru, TLE has been identified in the poisons of Lachesis muta ⁽⁵⁾i>Bothrops bilineatus ⁽⁶⁾, B. barnetti ⁽⁷⁾, B. atrox ⁽⁸⁾, and B. andianus ⁽⁹⁾. As for B. pictus, there are reports about a thrombinlike activity ^{(10, 11)} suggesting that it is one of the weakest in the genus Bothrops ^(12-14).

Bothrops pictus, the “desert lancehead,” causes ophidism in the central coast of Peru. Its poisoning pattern is characterized by phlogosis, erythema, ecchymosis, and loss of function. Among laboratory findings, hypoprothrombinemia and prolongation of partial thromboplastin time occur in most cases ⁽¹⁵⁾. Recently, our group reported partial characterization of the clotting protein of B. pictus; this protein has unusual features in the TLE group, such as a higher pH range for its activity and enhancement of the enzymatic activity by the Mn²⁺ion ⁽¹⁶⁾.

The interest in the studies on TLEs lies in their potential application to the fields of clinical diagnosis and treatment of cardiovascular diseases such as deep vein thrombosis, acute myocardial infarction, peripheral arterial thrombosis, and sudden sensorineural hearing loss as well as the use for prevention of thrombus formation after a surgical procedure ⁽¹⁷⁾. Some of these enzymes are widely used in diagnostic laboratories to detect fibrinogen in heparinized blood samples ⁽¹⁷⁾. A vivid example is the drugs Ancrod® and Reptilasa® TLE produced from the poisons of Agkistrodon rhodostoma and Bothrops atrox, respectively ⁽¹⁸⁾

In this study, we assessed biochemical features of the clotting enzyme of B. pictus poison and characterized it at the molecular level to determine its functions.

MATERIALS AND METHODS

POISON

The poison was obtained from Bothrops pictus specimens from the city of Pachacamac (Lima region) that are kept in the Oswaldo Meneses Serpentarium (MHN-UNMSM). A part of the extracted venom was lyophilized for purification purposes, while a smaller amount was immediately processed to obtain RNA.

CLONING AND SEQUENCING OF THE GENE

The cDNA was synthesized, and the gene was later amplified in accordance with the method of Vivas-Ruiz et al.⁽⁷⁾, using the F primer: 5′-ATGGTGCTGATCAGAGTG-3′ and R primer: 5′-CTGCAATAATGCTCTGGA-3′ designed manually based on the multiple alignment of the batroxobin (J02684.1), bothrombin (AB178321.1), BjussuSP-I (AY251282.1), BITS01A (AF490536.1), Bothrops asper-TLE (DQ2447724.1), and barnettobin (JX499027) sequences. The amplified product was examined on a 1% agarose gel after electrophoresis. The gene sequencing was conducted on an ABI 3730 XL automated sequencer (Macrogen, Inc., South Korea). The multiple alignment was performed by means of the Clustal W algorithm of the BioEdit software, version 7.2.5. The protein sequence was deduced in the Translate Tool software, and the biochemical properties were predicted in the Protparam software. Both programs were obtained from http://web.expasy.org/. Both the cDNA sequence and the deduced protein sequence were deposited in the databases GenBank and UniProt.

ENZYME PURIFICATION

We resuspended 100 mg of lyophilized venom in 0.05 M ammonium acetate buffer pH 5.0 and centrifuged the mixture at 2000 rpm. The supernatant was loaded onto a CM-Sephadex-C50 (1.2 × 47.5 cm) ion exchange column equilibrated with the aforementioned buffer, and the enzyme was eluted with a 0.1 to 1 M NaCl linear gradient in the same buffer at a flow rate of 14 mL/h.

The enzymatic activity was monitored by means of fibrinogen and the BApNA chromogenic substrate. The active fractions were concentrated using Microcom tubes. The resulting concentrate (33.9 mg) was applied to a Sephadex G-100 (1.4 × 64 cm) filtration column, equilibrated with the buffer from the first step at a flow rate of 14 mL/h (elution involved the same buffer). The active fractions from the previous step were concentrated again in a volume of 0.6 mL (6.4 mg). This sample was applied to a Sephadex G-75 column (1 × 30 cm) equilibrated with the same buffer at a flow rate of 15 mL/h (elution involved the same buffer).

N-TERMINAL SEQUENCING

The N-terminal sequencing of the enzyme was conducted on a Shimadzu PPSQ-21A automated sequencer based on Edman degradation, using a ~1 mg/mL solution of the purified enzyme. The procedure was performed in accordance with the report of Magalhaes et al. ⁽¹⁹⁾.

DETERMINATION OF THE LINKED CARBOHYDRATES

The presence of carbohydrates linked to the protein was detected by means of hexoses and hexosamines according to the method of Winzler R. ⁽²⁰⁾, and the presence of sialic acid was identified by the method of Warren L. ⁽²¹⁾ by means of an initial protein solution at the concentration of 0.5 mg/mL.

ENZYMATIC ACTIVITY

The clotting activity was measured by quantifying human fibrinogen (5 mg/mL) in a Tris-HCl buffer (0.05 M, pH 7.4) or citrated human plasma (0.2 mL) with appropriate amounts of the enzyme (0.5 to 12 µg). One unit of clotting activity was considered equivalent to one NHI unit of thrombin. The specific activity was defined as the amount of NHI units of thrombin per mg of protein. Likewise, the minimum clotting dose (MCD) was determined ⁽²²⁾. The amidolytic activity was determined by means of BApNA at 37ºC in 50 mM Tris-HCl pH 8.1 after an increase in absorbance at 405 nm and 37ºC.

FIBRINOGENOLYTIC ACTIVITY

This activity was determined by incubating the purified enzyme with 0.1 mL of 0.2% fibrinogen in 0.05 M Tris-HCl pH 7.5 at 37°C for 10, 20, 30, 60, or 120 minutes. The incubation was stopped by addition of electrophoresis sample buffer and subsequent heating at 100ºC for 3 minutes. The results were examined by means of SDS-PAGE ⁽²³⁾.

DEFIBRINOGENATING ACTIVITY

This activity was tested in albino mice (BALB/c strain, weight 18–22 g) grouped randomly into six groups (four mice per group), which were injected via the tail vein with decreasing doses of the purified enzyme starting with 50 µg diluted in 0.1 mL of saline. The minimum defibrinogenating dose (MDD) was defined as the minimum dose of the enzyme that does not cause total blood clotting within 60 minutes of intravenous injection ⁽²²⁾.

EFFECTS OF pH AND TEMPERATURE

Both parameters were evaluated by means of BApNAusing 20 µL of the enzyme. The effect of pH was determined by means of the following buffers: 50 mM ammonium acetate (pH: 4.0–6.0), 50 mM sodium phosphate (pH: 6.0–8.0), and 50 mM Tris-HCl (pH: 8.0–10.0). The effect of the temperature was evaluated by preincubating the enzyme for 15 minutes in the range 4°C to 95ºC.

EFFECTS OF IONS AND INHIBITORS

The effects of various ions in the form of chlorides (cf.: 25 mM) on the amidolytic activity were tested by preincubating a solution of the relevant salt with 20 µL of the enzyme at 37ºC for 20 minutes. The inhibitors that were tested were soybean trypsin inhibitor (STI) 1 mg/mL, ethylenediamine tetraacetic acid (EDTA), tosyl-lysyl chloromethyl ketone (TLCK), phenylmethyl sulfonylfluoride (PMSF), and dithiothreitol (DTT) at the final concentration of 10 mM. In the case of heparin, three final concentrations were tested: 250, 175, and 87.5 IU (referential values in the literature). These results were compared to those obtained with bovine thrombin (100 IU) as the reference protein.

STATISTICAL ANALYSIS

The data on the enzymatic activity were expressed as mean ± standard deviation (SD). Evaluation of the differences was done by analysis of variance (ANOVA) and Student’s t test. Differences with p values below 0.05 (p < 0.05) were considered significant.

RESULTS

We obtained a nucleotide sequence of 754 base pairs (GenBank: KF410948) encoding a protein with 250 amino acids (Figure 1). The mature protein, named TLE-Bp (GenBank: AGZ87932), corresponds to 233 aa with the N-terminal sequence V-I-G-G-D-E typical of serine proteases. The molecular weight and isoelectric point were found to be 25.9 kDa and 8.2, respectively. In addition, five sites for N-glycosylation were identified.