A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification, proteomic, functional and structural characterizations

A phospholipase A2 was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA2. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA2 was a new snake venom acidic PLA2. Seven peptides were sequenced from Akbu-PLA2 by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA2 shared homolog peptides of phospholipases A2 from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA2 has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA2 showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA2 was shown to contain one Ca2+ per monomer by ICP-AES measurement. The Ca2+ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA2. Ca2+ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA2. The hydrolytic activity of Akbu-PLA2 was accelerated due to the addition of Ca2+ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA2 was found to be difficult to eliminate for the purification of Akbu-PLA2. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA2, which indicated that it should be a homodimer of Akbu-PLA2. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA2 monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA2 monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca2+ ion was found to be able to promote the dimer formation of Akbu-PLA2. We conclude that a novel PLA2 was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA2 reveal new threads and provide valuable inputs for the study of snake venom phospholipases A2.