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

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 approximately 45 degrees 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).