Snake Venom Hemotoxic Enzymes: Biochemical Comparison between Crotalus Species from Central Mexico

Abstract

Snakebite envenoming is a serious medical problem in different areas of the world. In Latin America, the major prevalence is due to snakes of the family Viperidae, where rattlesnakes (Crotalus) are included. They produce hemotoxic venom which causes bleeding, tissue degradation and necrosis. Each venom has several enzymatic activities, producing different effects in the envenoming, doing its clinical effects difficult to study. Comparison between venom molecules is also difficult when different techniques are used, and therefore, their identification/characterization using the same methodology is necessary. In this work, a general biochemical characterization in snake venom of serine proteases (SVSP), phospholipases A₂ (PLA₂), metalloproteases (SVMP) and hyaluronidases (SVH) of Crotalus aquilus (Ca), Crotalus polystictus (Cp) and Crotalus molossus nigrescens (Cmn) was done. Differences in protein pattern, enzyme content and enzymatic activities were observed. All the venoms showed high PLA₂ activity, high molecular weight SVSP, and a wide variety of SVMP and SVH forms. Ca and Cp showed the highest enzymatic activities of SVMP and SVSP trypsin-like and chymotrypsin-like, whereas Cmn showed the highest SVH and similar PLA₂ activity with Ca. All the venoms showed peptides with similar molecular weight to crotamine-like myotoxins. No previous biochemical characterization of C. aquilus has been reported and there are no previous analyses that include these four protein families in these Crotalus venoms.

Keywords: Crotalus; hyaluronidases; metalloproteases; phospholipases A2; serine proteases; snake venom.

Figure 1

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein banding pattern for C. aquilus (Ca), C. polystictus (Cp) and C. molossus nigrescens (Cmn) venoms. Twenty micrograms of lyophilized venoms were analyzed by non-reducing (NR) and reducing conditions (R) in 10% polyacrylamide gels. MWM: molecular weight markers (kDa). Variation in the bands with molecular weight ~15 kDa and ~50 kDa are marked with red boxes.

Figure 2

Protein pattern by SDS-PAGE, and Serine proteases zymography for C. aquilus (Ca), C. polystictus (Cp) and C. molossus nigrescens (Cmn) venoms. (A) 10% SDS-PAGE and (B) zymography. Trypsin-like zymography was performed using 50 μg of venom samples protein with BApNA substrate. Chymotrypsin-like zymography was determined using 100 μg of venom protein with SAAFpNA substrate. After electrophoresis, a cellulose membrane with each serine protease was placed on top of the gel and incubated for 5 h and 2.5 h at 37 °C, respectively. MWM: molecular weight markers (kDa). Variability in proteases bands is marked with red boxes.

Figure 3

Snake venom serine proteases activities for C. aquilus (Ca), C. polystictus (Cp) and C. molossus nigrescens (Cmn) venoms. (A) Trypsin-like proteases, using bovine trypsin as positive control; (B) chymotrypsin-like proteases, using bovine chymotrypsin as positive control; (C) elastase-like proteases, using porcine elastase as a positive control. Enzymatic activity expressed in activity units (AU/µg). Small letters show significant differences (Tukey p < 0.05) between samples for each enzyme, positive controls (Pc) were not compared.

Figure 4

Gelatin zymography in 10% polyacrylamide gels and proteolytic activity using casein substrate. (A) Ten micrograms of venom protein from C. aquilus (Ca), Crotalus polystictus (Cp) and C. molossus nigrescens (Cmn) were incubated after electrophoresis for 2.5 h at 37 °C. MWM—molecular weight markers (kDa). (B) Proteolytic activity of 100 μg of venom samples in the presence/absence of 50 mM EDTA incubated with casein as a substrate for 2.5 h at 37 °C. Negative control (Nc). Small letters (a,b,c,d,e) show statistical difference (Tukey p < 0.05). Variability in proteases bands is marked with red boxes. Small letters show significant differences (Tukey p < 0.05) between samples, either with or without ethylendiaminetetraacetic acid (EDTA).

Figure 5

Zymography in 10% polyacrylamide gels co-polymerized with gelatin. Ten microgram of venom protein from (A) C. aquilus (Ca), (B) C. polystictus (Cp), and (C) C. molossus nigrescens (Cmn). In addition, 10 μg of the same samples were tested with 10, 50 and 100 mM EDTA in gels that were incubated after electrophoresis for 2.5 h at 37 °C. Red arrows indicate inhibition of snake venom metalloproteases (SVMP) by EDTA, red asterisk (*) show bands not shared between the venoms, red boxes show proteases bands with gelatinolytic activity unaffected by EDTA. Molecular weight markers (MWM) in kDa.

Figure 6

Zymography and enzymatic activity of PLA2. (A) 12% SDS-PAGE, (B) 4% agarose-egg yolk co-polymerized gel zymography using 20 μg of protein from C. aquilus (Ca), C. polystictus (Cp), and C. molossus nigrescens (Cmn) venoms incubated after electrophoresis, for 2 h at 37 °C. MWM—molecular weight markers (MW) in kDa. (C) Enzymatic activity of 10 μg of bee venom phospholipase (BPLA2) as a positive control and 100 ng from Ca, Cp and Cmn venoms. Small letters (a,b) show statistical differences (Tukey p < 0.05) between samples, positive control was not compared. Red arrows show bands with PLA₂ activity, and red asterisk show bands not shared between the venoms.

Figure 7

Hyaluronidase activity. (A) Zymography on SDS-PAGE co-polymerized with hyaluronic acid using 100 μg venom protein, incubated after electrophoresis for 2.5 h at 37 °C. Molecular weight markers (MWM) in kDa. (B) Enzymatic activity using 5 to 50 μg of C. aquilus (Ca), C. polystictus (Cp), and C. molossus nigrescens (Cmn) venoms as well as hyaluronidase from bovine testes used as positive control (BHA). Red boxes show variability between the venoms. Small letters show statistical differences (Tukey p < 0.05) between samples, positive control was not compared.

Figure 8

Venoms fractionation by RP–HPLC. Pooled crotalus venoms from (A) C. aquilus (Ca), (B) C. polystictus (Cp) and (C) C. molossus nigrescens (Cmn). Red asterisk show peaks not shared between the venoms. Snake venom serine proteases (SVSP), snake venom metalloproteases, (SVMP) and snake venom hyaluronidases (SVH). Absorbance units (AU) at 280 nm. Enzymatic activities were not determined, the family proposed for the enzymes are based on similarity of the elution profiles with reported data.