Unraveling the Proteome Composition and Immuno-profiling of Western India Russell’s Viper Venom for In-Depth Understanding of Its Pharmacological Properties, Clinical Manifestations, and Effective Antivenom Treatment Bhargab Kalita, Aparup Patra, and Ashis K. Mukherjee* Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur 784028, Assam, India * S Supporting Information ABSTRACT: The proteome composition of western India (WI) Russell’s viper venom (RVV) was correlated with pharmacological properties and pathological manifestations of RV envenomation. Proteins in the 5-19 and 100-110 kDa mass ranges were the most predominate (∼35.1%) and least abundant (∼3.4%) components, respectively, of WI RVV. Non-reduced SDS-PAGE indicated the occurrence of multiple subunits, non-covalent oligomers, self-aggregation, and/or interactions among the RVV proteins. A total of 55 proteins belonging to 13 distinct snake venom families were unambiguously identified by ESI-LC-MS/MS analysis. Phospholipase A 2 (32.5%) and Kunitz-type serine protease inhibitors (12.5%) represented the most abundant enzymatic and non-enzymatic proteins, respectively. However, ATPase, ADPase, and hyaluronidase, detected by enzyme assays, were not identified by proteomic analysis owing to limitations in protein database deposition. Several biochemical and pharmacological properties of WI RVV were also investigated. Neurological symptoms exhibited by some RV-bite patients in WI may be correlated to the presence of neurotoxic phospholipase A 2 enzymes and Kunitz-type serine protease inhibitor complex in this venom. Monovalent antivenom was found to be better than polyvalent antivenom in immuno- recognition and neutralization of the tested pharmacological properties and enzyme activities of WI RVV; nevertheless, both antivenoms demonstrated poor cross-reactivity and neutralization of pharmacological activities shown by low-molecular-mass proteins (<18 kDa) of this venom. KEYWORDS: venom proteome, ESI-LC-MS/MS, pro-coagulant, anti-coagulant, venom-antivenom cross-reactivity, neurotoxicity ■ INTRODUCTION Russell’s viper (Daboia russelii) is a medically important snake in many South Asian countries, including India (Figure 1). RV bites account for a large amount of snakebite morbidity and mortality and show complex clinico-pathological manifestations in human victims. 1-4 Russell’s viper venom (RVV) primarily targets blood coagulation in victims and promotes tissue damage, edema, necrosis, hypotension, hemostatic imbalance, hemorrhage, and acute renal failure. 2,4,5 In addition, a few RV bite patients in western India (WI) showed neurological symptoms like flaccid paralysis, 3,4 although such clinical symptoms are uncommon in eastern India. 2 The RVV toxin or toxins accountable for neurotoxic symptoms post RV bite in WI remain to be identified. Further, marked differences in clinical manifestations of RV bites in different parts of the Indian subcontinent 2,4,6-8 may not be well explained without analyzing in detail the proteome composition of venom of RVs from particular geographical locations. About 35 000-50 000 snakebite deaths have been reported in India, of which the “Big Four” species (Naja naja, D. russelii, Echis carinatus, and Bungarus caeruleus) account for the maximum share. 9 Therefore, Indian polyvalent antivenom is raised in equines against venoms from the “Big Four”. Nonetheless, efficacy, potency, and safety of antivenoms are of immense concern 10-12 because geographical, seasonal, and ontogenic variations in snake venom composition may affect the antivenom treatment. 12-15 Consequently, knowledge on the composition of the venom of a snake from a particular geographical location, as well as assessment of efficacy of commercial antivenom to neutralize the enzymatic activities and pharmacological properties of RVV, is necessary for efficient hospital management of snakebites and appraisal of antivenom efficacy. Recent proteomics studies have documented significant qualitative as well as quantitative variations in venom composition of RV from southern India, Sri Lanka, and Pakistan. 16-18 This suggests further characterization of RVV from different regions of the Indian peninsula would be valuable for a better understanding of the pharmacological Received: July 27, 2016 Article pubs.acs.org/jpr © XXXX American Chemical Society A DOI: 10.1021/acs.jproteome.6b00693 J. Proteome Res. XXXX, XXX, XXX-XXX