A novel phospholipase A 2 from Agkistrodon blomhoffii ussurensis venom: Purification, proteomic, functional and structural characterizations

Abstract

A phospholipase A 2 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-PLA 2. 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-PLA 2 was a new snake venom acidic PLA 2. Seven peptides were sequenced from Akbu-PLA 2 by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA 2 shared homolog peptides of phospholipases A 2 from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA 2 has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA 2 showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA 2 was shown to contain one Ca 2+ per monomer by ICP-AES measurement. The Ca 2+ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA 2. Ca 2+ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA 2. The hydrolytic activity of Akbu-PLA 2 was accelerated due to the addition of Ca 2+ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA 2 was found to be difficult to eliminate for the purification of Akbu-PLA 2. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA 2, which indicated that it should be a homodimer of Akbu-PLA 2. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA 2 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-PLA 2 monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca 2+ ion was found to be able to promote the dimer formation of Akbu-PLA 2. We conclude that a novel PLA 2 was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA 2 reveal new threads and provide valuable inputs for the study of snake venom phospholipases A 2.