Proteomic and Functional Comparison of Active-Season and Overwintering-Stage Venoms from Naja atra

Seasonal changes in physiological state and metabolism may influence snake venom composition and biological activity, yet these relationships remain poorly understood. This study investigated seasonal variation in venom composition and functional properties in Naja atra. Venom samples were collected from the same ten adult snakes during the active season and after approximately two months of overwintering. Venom yield, protein composition, and multiple biological activities were compared using quantitative proteomic profiling and functional assays. Proteomic quantification was based on scaled protein abundance values exported from Proteome Discoverer (version 3.3), with peptide and protein identification performed using the Sequest HT search engine. Overwintering was accompanied by body-weight loss and reductions in venom yield, with wet and dry venom yields decreasing by 37.75% and 30.06%, respectively. Proteomic analysis identified 178 protein entries in active-season venom (AV) and 163 in overwintering venom (HV), with 142 shared entries, indicating substantial conservation of the detectable venom protein repertoire alongside compositional changes. Three-finger toxins showed lower relative abundance during overwintering, whereas snake venom metalloproteinases and L-amino acid oxidase exhibited relatively higher representation. Functional assays revealed increased phospholipase A₂ (PLA₂) activity and decreased proteolytic activity in overwintering venom, whereas L-amino acid oxidase activity, nicotinic acetylcholine receptor (nAChR)-binding activity, cytotoxicity, and hemolytic activity remained largely comparable between groups. These findings indicate that overwintering is associated with reduced venom yield and selective remodeling of venom composition and function rather than uniform suppression of venom activity. Although the underlying mechanisms remain unresolved, this study highlights physiological state as an important consideration in understanding snake venom plasticity.