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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.8 no.1 Botucatu  2002 




1 Laboratorio de Biología Molecular, Instituto de Ciencias Biológicas “Antonio Raimondi” (ICBAR), Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Av. Venezuela cuadra 34, Lima, Perú.



ABSTRACT: Micrurus spixii venom was studied after fractionation by Sephadex G-100 SF gel filtration chromatography. Several enzymatic activities and biological effects were investigated in whole venom and fractions. The venom was resolved in four peaks in a range of about 73.2-10.7 kDa molecular weight.

Alkaline phosphatase and acetylcholinesterase activities were found in peak I, and procoagulant activity was seen in peak II. Phospholipase A2, hemorrhagic, and proteolytic activities were detected in peak III. A second procoagulant factor and proteinase were present in peak IV. Thrombin-like enzyme and direct hemolytic activities were not found in any assayed samples.
KEY WORDS: Phospholipase A2, acetylcholinesterase, alkaline phosphatase, venom, Micrurus spixii.




In Peru, Micrurus spixii (Elapidae Family) is called “Peruvian coral snake”, “Amazonian coral snake”, or "Naca Naca". This snake inhabits a large area of the Peruvian jungle, and many native communities consider M. spixii the most dangerous snake due to the potent and rapid biological action of its venom on humans, which could be related to apparent neurotoxicity. Unfortunately, there have been no reliable statistical studies or biochemical research on its venom up until now. Meneses (12), Carrillo, and Icochea (2) described some details referring to its morphological characteristics and geographical distribution. We have also observed interesting behavioral aspects in our serpentarium; for instance, this snake is an ophiophage that eats venomous and non-venomous snakes. Despite its low level of aggression, only a small amount of venom can be milked.

We have initiated a research program on the biochemical and biological characterization of the venom. To ensure venom quality, appropriate milking procedures are performed under captivity conditions.

In this study, four adult Micrurus spixii (70–80 cm) from Pucallpa, Ucayali were kept for several months in the “Oswaldo Meneses” serpentarium of San Marcos University, Lima. The venom was milked, lyophilized immediately, and kept at -10°C until used. Sephadex G-100 SF, all other reagents, and substrates for enzyme assays were purchased from Sigma Chemical Company (St. Louis, USA).

Chromatographical fractionation was carried out using 28.5 mg crude venom dissolved in 0.05M Tris HCl buffer, 0.01M MgCl2, pH 7.5, and applied to a Sephadex G-100 SF gel filtration column (38 x 1.1 cm) equilibrated with the same buffer. The flow rate was adjusted to 6 ml/h and 1-ml fractions were collected. Fractions were analyzed for protein by absorbance measurement at 280 nm.

Alkaline phosphatase activity was measured using p-nitrophenylphosphate as per Sulkowski et al. (13). Phospholipase A2 activity was determined using a spectrophotometrical assay with egg yolk L-a-phosphatidylcholine as substrate, as described by De Oliveira and Palma (5). Acetylcholinesterase activity was examined according to Ellman et al. (6), using acetylthiocholine iodide as substrate. Proteolytic activity was tested on casein as substrate, as described by Takahashi and Ohsaka (14). Thrombin-like activity was determined using bovine serum fibrinogen according to Copley et al. (4). Procoagulant activity was measured using critated human plasma as per Williams et al. (16). Hemorrhagic activity was determined according to Gutiérrez et al. (9) using mice; and direct hemolytic activity was tested on human erythrocyte (3). Molecular weight range was calculated using bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12,4 kDa) on a Sephadex G-100 SF column (1).

In this study, we found some enzymatic and biological activities when venom was fractionated on a Sephadex G-100 SF column (Table 1). Alkaline phosphatase and acetylcholinesterase are present in peak I, corresponding to approximately 73.2 kDa molecular weight. In peak II (42.2 kDa), a procoagulant factor was found, which produced blood coagulation on citrated human plasma but did not affect bovine fibrinogen and BApNA chromogenic substrate. A strong phospholipase A2 activity was observed in peak III (22.4 kDa), as well as a hemorrhagic principle and proteolytic activity on casein. Finally, peak IV (10.7 kDa) corresponded to a second procoagulant fraction and proteolytic activity (Figure 1).


Table 1. Enzymatic activities of Micrurus spixii crude venom and fractions obtained by Sephadex G-100 SF gel filtration chromatography.

a One unit of activity was defined as mmoles of acetylthiocholine hydrolyzed per minute.
b One unit of activity was defined as mmoles of p-nitrophenol released per minute.
c One unit of activity was defined as mmoles of fatty acid released from L-a-phosphatidylcholine per minute.
d One unit of activity was defined as mg of L-tyrosine released per minute.
e One unit of activity is defined as an arbitrary clotting unit where 1 unit/ml was defined as causing clotting of pooled normal citrated plasma in 30 sec.
* The MHD was defined as the smallest quantity of fraction (mg) that produced a hemorrhagic lesion (10 mm diameter) after 24 hours.



Figure 1. Gel filtration chromatography of Micrurus spixii venom on Sephadex G-100 SF column (38 x 1.1 cm) 0.05 M Tris HCL buffer, 0.01 M MgCl2, pH 7.5, as eluent.


According to several authors, most elapid venoms contain neurotoxic peptides while their enzymatic composition is very poor (10). However, our study of M. spixii venom showed a high phospholipase A2 activity of 338.85 Units/mg crude venom and 900.6 Units/mg in peak III. Specific activity of crude venom was 16.4 times higher than Lachesis muta and 20.2 times higher than Bothrops atrox (11). Hemorrhagic and proteolytic activities are also present in this venom.

 It is interesting to note that alkaline phosphatase is a monophosphate esterase found in several elapid venoms, such as Micrurus dumerilii carinicauda and M. frontalis frontalis (15). In this study, 10mM Mg2+ was necessary to stabilize alkaline phosphatase during fractionation, and this did not modify the chromatographic pattern. Similarly, a hemorrhagic effect detected in this venom (peak III) was only found in Ophiophagus hannah, Notechis scutatus scutatus, M. fulvius, and M. frontalis frontalis (7,15). It has also been reported that M. fulvius and M. frontalis venoms show high phospholipase A2 associated with hemorrhagic effect, which are very similar to M. spixii venom. No coagulant thrombin-like action is found in elapid venoms, but procoagulant factors were detected in some Australian elapid venoms. For instance, a procoagulant factor was isolated from Notechis ater niger and this type of protein was isolated from four Pseudonaja Australian species (16). This is in agreement with a general differentiation between two typical elapid groups in that continent; one of them showing procoagulant venom that could have a lethal effect on mammals (8). We assume that M. spixii venom is related to elapid Australian venoms because both show phospholipase A2 and hemorrhagic activities, as well as procoagulant factor. Our future studies will focus on the two different procoagulant factors (peaks II and IV), as they produce blood coagulation without addition of Ca2+ ions and phospholipids.

Since the procoagulant factors, and hemorrhagic, phospholipase A2, and alkaline phosphatase activities are the main components of this venom, they could be related to damage and deadly effects, as neurotoxic action was not detected by IV injection into caudal veins of mice. There is a strong need to complete the characterization of this venom, as other Peruvian elapid snakes, such as M. surinamensis show a potent neurotoxic action in our initial assays. These results show the general composition of this venom, however, further research is required to locate the lethal fraction.



This research was supported by LANBIO and San Marcos University through Consejo Superior de Investigaciones (CSI).



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Received December 12, 2000
Accepted April 20, 2001

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