PRELIMINARY INVESTIGATION ABOUT THE ANTI-COMPLEMENT PROPERTIES OF PARTIALLY SEPARATED Vipera ammodytes VENOM

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PRELIMINARY INVESTIGATION ABOUT THE ANTI-COMPLEMENT PROPERTIES OF PARTIALLY SEPARATED Vipera ammodytes VENOM

S. STANILOVA , Z. ZHELEV , I. MATEV

1 Department of Molecular Biology and Immunology, Medical Faculty, Armeiska 11 Street, 6000 Stara Zagora, Bulgaria, 2 Department of Biology, Zootechnic Faculty, Thracian University of Stara Zagora, Bulgaria.

ABSTRACT. The present study demonstrates that Vipera ammodytes venom is a potent inhibitor of human complement activation. Crude venom was fractionated by ion-exchange chromatography and each fraction was then tested for proteolytic, hemolytic and anti-complement activities. Three peaks decreased the hemolytic activity of human serum when measured using assays for the classical and alternative pathways of activation. One of the anti-complement peaks contains a protease capable of cleaving the C3 component of complement.

KEY WORDS: Anti-complement, venom, Vipera ammodytes.

INTRODUCTION

The anti-complement properties of cobra venom have been known since the first decade of this century. Using highly purified preparation of the anti-complement cobra venom factor (CVF), it has been shown that in the presence of Mg this protein binds to factor B of the alternative complement pathway. Once bound to CVF, factor B can continuously and uncontrollably hydrolyze C3 and C5, resulting in a depletion of serum complement activity(8). In contrast to cobra venom, little is known about the anti-complement properties of Vipera venoms (4). In the present study, we report a preliminary investigation about the anti-complement activity of partially fractionated Vipera ammodytes venom and provide some evidence as to the mechanism of action involved.

Lyophilized Vipera ammodytes venom was fractionated by ion-exchange chromatography on a column of QAE-Sephadex A-50 using a discontinuous gradient of NaCl (0.1, 0.2, and 0.3 M). Phospholipase A2 (PLA2) activity was estimated by an indirect hemolytic test(1). Assays for anti-complement activity via the alternative and classical pathways were carried out as described by Ballow and Cochrane(2). Proteolytic activity was measured using casein as a substrate(6). The hemolytic assay for CVF was performed according to Vogel and Muller-Eberhard(8).

Figure 1 shows the elution profile of the venom as well as the distribution of PLA2 and proteolytic activities. Seven peaks were obtained under these experimental conditions. Four of these peaks 1, 3, 5 and 7 contained protease activity while three contained PLA2 (peaks 2, 3 and 5). These results are comparable to those reported by Gubensek et al.(5) for this same species. Inhibition of the complement activity of human serum via both pathways was observed with peaks 5, 6 and 7 (Figure 2). The effects were dose-dependent and were most pronounced in

FIGURE 1. QAE-Sephadex A50 ion-exchange chromatography of Vipera ammodytes venom. Crude venom (250 mg) dissolved in 10 ml of 0.01M phosphate buffer, pH 7.4, was dialyzed (72h against above mentioned buffer) and applied to a column (2.6 x 10 cm) of resin equilibrated with the same buffer. Fractionation was carried out at a flow rate of 36 ml/h using a discontinuous NaCl gradient as shown on the elution profile. Fractions of 6 ml were collected.

FIGURE 2. Changes in the hemolytic complement activity of human serum (0.5 ml) following a 30-min incubation at 37ºC with venom fractions (0.5 ml of each fraction). Data represent the means ± SD of triplicate assays.

the alternative complement pathway. These results suggest that the loss of the complement activity is due to a common initial pathway of complement (C3 and/or C5) consumption. Peak 7 contained the most pronounced complement inhibiting activity and also possessed marked protease activity. This observation suggests that there exists a correlation between the two activities of V. ammodytes venom as reported for V. lebetina venom by Gasmi et al. (4). Peak 6 had no detectable protease activity, thus suggesting a different mechanism from that of peak 7. One possible explanation is that a protein in fraction 6 may form an inactive complex with C3 as has already been reported for another anti-complement factor(9). A second explanation could be that this peak contains an inhibitor of C3 convertase, as reported by Lasure et al. (7) and Blondin et al. (3).

To investigate the nature of the anti-complement action of the various peaks obtained, we studied their ability to lyse human red blood cells in the presence of fresh homologous serum (hemolytic assay for CVF). No hemolysis was detected in crude V. ammodytes venom or in any of the peaks. Thus, the anti-complement activity of peaks 5, 6 and 7 is unlikely to result from the formation of a convertase-like complex similar to that shown for CVF.

These preliminary results therefore provide evidence for at least two different mechanisms of anti-complement action of V. ammodytes venom.

09 ZHELEV Z., STANILOVA S., CARPENTER B. Isolation, partial characterization and complement inhibiting activity of a new glycoprotein from Cuscuta europea. Biochem. Biophys. Res. Commun., 1994, 202, 186-94.

Received 14 March 1996.

Accepted 30 May 1996.

CORRESPONDENCE TO:

ZHIVKO ZHELEV - Department of Molecular Biology and Immunology, Medical Faculty, Thracian University, Armeiska 11 Street, 6000 Stara Zagora, Bulgaria.

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• 09 ZHELEV Z., STANILOVA S., CARPENTER B. Isolation, partial characterization and complement inhibiting activity of a new glycoprotein from Cuscuta europea Biochem. Biophys. Res. Commun., 1994, 202, 186-94.  

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997

History

• Accepted
  30 May 1996
• Received
  14 Mar 1996