Enzymatic and toxinological activities of Hypnale hypnale (hump-nosed pit viper) venom and its fractionation by ion exchange high performance liquid chromatography

Abstract

Abstract:Hypnale hypnale (hump-nosed pit viper) has been recently identified as one of the medically important venomous snakes in Sri Lanka and on the southwestern coast of India. The characterization of its venom is essential for understanding the pathophysiology of envenomation and for optimizing its management. In the present study, the biological properties of Hypnale hypnale venom and venom fractions obtained using Resource Q ion exchange chromatography were determined. The venom exhibited toxic activities typical of pit viper venom, comparable to that of its sister taxon, the Malayan pit viper (Calloselasma rhodostoma). Particularly noteworthy were its high activities of thrombin-like enzyme, proteases, phospholipase A 2 , L-amino acid oxidase and hyaluronidase. The thrombin-like enzyme was mainly acidic and distributed over several chromatography fractions, indicating its existence in multiple isoforms. The hemorrhagic and necrotic activities of the venom were likely associated with the proteolytic enzyme found mainly in the basic fraction. Phospholipase A 2 and phosphomonoesterase exist in both acidic and basic isoforms, while L-amino acid oxidase and hyaluronidase are highly acidic. The venom clotting activity on fibrinogens showed distinct species specificity in the following increasing order for clotting time: bovine < rabbit < goat < human < horse < < dog, and was comparable to that of C. rhodostoma venom. Its clot formation on human fibrinogen is gradual and prolonged, a phenomenon suggestive of consumptive coagulopathy as a complication observed clinically. At an intramuscular sublethal dose, the venom did not cause acute kidney injury in a rodent model, contrary to the positive control group treated with Daboia russelii venom. Nephrotoxicity may result from higher venom doses in the context of coagulopathy, as a complication provoked by venom hematoxicity.

Proteomic investigation of Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom

“…The study also revealed that L-amino acid oxidases (LAAO) exist in high abundance (11.9%) in H. hypnale venom, consistent with the venom's yellow coloration (due to high content of flavins) and potent enzymatic activity (Tan et al, 2011b). L-amino acid oxidases usually constitute 1e4% in most snake venoms (Tan, 1998), but venoms from mambas and sea snakes contain either no or only trace amount of LAAO, while the enzyme is typically absent in colubrid venoms (Mackessy, 2002).…”

“…The SVSP shares homologous sequences with ancrod, the major thrombinlike enzyme in Calloselasma rhodostoma venom, however their abundance in H. hypnale venom is relatively lower (3.3%) than that of ancrod (7%) (Esnouf and Tunnah, 1967). This is congruent to the fact that C. rhodostoma venom exhibits a much stronger procoagulant effect by 2 folds compared to that of H. hypnale venom (Tan et al, 2011b). SVSP are important coagulant enzymes that can induce defibrinogenation leading to consumptive coagulopathy that potentially leads to renal hypoperfusion and ischemia (Sitprija, 2008).…”

“…The content and enzymatic activity of venom PLA 2 are however generally higher for elapids especially the Asiatic cobras, for instance, Naja sumatrana, with activity exceeding 300 mmol/min/ mg (Yap et al, 2011) and abundance of up to 30% venom mass (Yap et al, 2014). In contrast, the venom of H. hypnale exhibits a relatively lower PLA 2 enzyme activity much like the Malayan pit viper venom (Tan et al, 2011b); nonetheless, the current study clearly showed that PLA 2 constitutes an exceptionally large proportion of the venom content (40.1%), suggesting the presence of nonenzymatic PLA 2 in a substantial amount. The sequential separation method by reverse-phase HPLC and SDS-PAGE revealed four PLA 2 isoforms from the pooled venom, each with N-terminal sequence consistent with one of two PLA 2 subtypes reported by Wang et al (1999b) based on their 6th amino acid residue: Glu 6 (E6-PLA 2 ), or Trp 6 (W6-PLA 2 ).…”

“…Unfortunately, specific antivenom against this species is not available, while the heterologous Indian polyvalent antivenoms (raised against the venoms of Indian Big Four: Indian cobra, Indian krait, saw-scaled viper, Russell's viper) were proven ineffective but risking hypersensitive reactions in the patients (Sellahewa and Kumararatne, 1994;Premawardena et al, 1996Premawardena et al, , 1998Joseph et al, 2007;Ariaratnam et al, 2008). Tan et al (2011aTan et al ( , 2012a demonstrated that Thai Hemato polyvalent antivenom (raised against Malaysian pit viper, white-lipped green pit viper and Siamese Russell's viper) conferred paraspecific protection against H. hypnale venom's various toxicities in mice; the findings of efficacy however still await clinical validation. Laboratory studies thus far confirmed the activities of enzymatic toxins like phospholipases A 2 (PLA 2 ), thrombin-like enzymes (TLE), proteolytic enzymes (snake venom metalloproteinases, SVMP) and Lamino acid oxidases (LAAO) in the venom (Wang et al, 1999a;Tan et al, 2011b).…”

“…Tan et al (2011aTan et al ( , 2012a demonstrated that Thai Hemato polyvalent antivenom (raised against Malaysian pit viper, white-lipped green pit viper and Siamese Russell's viper) conferred paraspecific protection against H. hypnale venom's various toxicities in mice; the findings of efficacy however still await clinical validation. Laboratory studies thus far confirmed the activities of enzymatic toxins like phospholipases A 2 (PLA 2 ), thrombin-like enzymes (TLE), proteolytic enzymes (snake venom metalloproteinases, SVMP) and Lamino acid oxidases (LAAO) in the venom (Wang et al, 1999a;Tan et al, 2011b). Recently, Ali et al (2013) reported the identities of major proteins from the venom of a single H. hypnale specimen using 1-dimensional (1D) and a 2-dimensional (2D) gel electrophoresis.…”