A single-step purification of bothropstoxin-1

Abstract

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A 2 -like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Purification and Preliminary Crystallographic Analysis of a New Lys49-PLA2 from B. Jararacussu

“…Similarly to other Lys49-PLA2s, such as PrTx-I, PrTx-II [25] and BthTx-I [21], BjVIII does not show significant enzymatic activity and induces similar myonecrosis as BthTx-I, when assayed at the same conditions as Barbosa et al [26]. However, an atypical effect presented by BjVIII, and not by other Lys49-PLA2s, is a strong human platelet aggregation activity.…”

“…Bothrops toxin I (BthTx-I) was the only Lys49-PLA2 purified from Bothrops jaracussu venom by one chromatographic step [21]. We modified the original protocol, using a two-step chromatographic procedure (section Materials and methods ) and observed that BthTx-I fraction from the one-step protocol is composed of two closely related isoforms.…”

Purification of Phospholipases A2 from American Snake Venoms

“…Spencer et al [31] described the purification of BthTX-I with the use of Resourse S® (methyl-sulphonate Ion-exchange chromatography on CM-Sephadex® column.…”

Study of irradiated bothropstoxin-1 with60Co gamma rays: immune system behavior

“…The column was subsequently washed with 10 mL of B buffer, followed by 10 mL of A buffer to wash NaCl out of the column (13). …”