Novel in vitro assays for assessing the haemorrhagic activity of snake venoms and for demonstration of venom metalloproteinase inhibitors

A rapid and sensitive fluorometric method for the quantitative analysis of snake venom metalloproteases and their inhibitors

“…Overall, activity of Viperinae and Crotalinae venoms are consistent with the known metalloprotease content, physiological effects, and biochemical properties of the venoms tested (Soto et al, 1988; Bjarnson and Fox, 1994), and correlate with the gelatin degradation activity reported by Bee et al (2001) (Figure 5). The eight elapid venoms tested here showed low levels of metalloprotease activity, as expected (Table 1).…”

“…The sensitivity of the assay is comparable to the ELISA method of Bee et al (2001). We were able to successfully detect SVMPs in two of the most active venoms when using less than 2 ng of whole venom protein (Table 2).…”

“…In addition to their role in producing hemorrhage (Baramova et al, 1989), SVMP also contribute to local myonecrosis, edema, ischemia, and skin lesioning (Gutiérrez and Rucavado, 2000; Matsui et al, 2000; see Gutiérrez et al, 2009 for a recent review). Therefore, researchers have used a variety of in vitro assays of metalloprotease activity as proxy measures of venom hemorrhagic activity, ranging from classic measures such as spot production on the gelatin emulsion on X-ray film to the gelatin-degradation ELISA method (Bee et al, 2001). These assays have advantages over in vivo methods because they don't require the use of large numbers of laboratory animals and yield measures with lower variability.…”

Purification and characterization of five snake venom metalloproteinases from Egyptian <i>Echis pyramidum </i><i>pyramidum</i> venom

“…All enzymes with hemorrhagic activity (EpyB1, B2, B4 and B5) recognized clear zones on a blue background indicate the gelatinase activity, whereas EpyB3 and EpyB6 had no effect on gelatin, which confirms the results obtained as described in section (Substrates specifi city). Previously Bee et al (2001), demonstrated that gelatin zymogram was an efficient means of identifying proteolytic enzymes in venoms and appeared to be a reasonable of venom hemorrhagic activity. In addition, potent gelatinase activity in venoms from snakes of the family Viperidae was detected and little or no activity was evident in the venoms of snakes that induce neurotoxic pathology.…”

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

“…Gelatin zymography (SDS-PAGE incorporating gelatin, that is denatured collagen) was demonstrated as an efficient and reasonable method for identifying proteolytic enzymes and measuring gelatinolytic activity as infallible measure of venom haemorrhagic activity (Bee et al, 2001;Hasson et al, 2004). The uncontrolled bleeding is the result of gelatinolytic (some of them 2-mercaptoethanol sensitive!)…”