Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
J Proteomics. 2013 Mar 27;80:250-67. doi: 10.1016/j.jprot.2013.01.021. Epub 2013 Feb 4.
Bothrops pirajai snake venom was analyzed by a proteomic strategy. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI-TOF/TOF mass spectrometry, and assignment to known protein families by similarity. Proteins belonging to six families were found in B. pirajai venom, including abundant PLA2s and metalloproteinases, with the remaining proteins distributed among l-amino acid oxidase, serine proteinase, disintegrin and lectin-like families. A P-I class metalloproteinase, named BpirMP, was isolated from this venom by three chromatographic steps. The enzyme has a molecular mass of 23.1kDa, as determined by mass spectrometry. Its proteolytic activity on azocasein was inhibited by chelating and reducing agents, with optimum activity at higher pH values and 37°C. BpirMP presented weak hemorrhagic activity, with an MHD of 50μg, and was able to hydrolyze basement membrane components in vivo and in vitro. The toxin cleaved both Aα and Bβ chains of fibrinogen and was also able to degrade fibrin and blood clots in vitro. The primary sequence analysis indicates that BpirMP contains a zinc ligand motif and a CVM motif that is associated with a Met-turn structure. These results demonstrate that BpirMP is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activities.
This manuscript describes the diversity of protein components present in the venom of Bothops pirajai, a threatened snake species from northeastern Brazil, as well as the isolation and biochemical properties of a PI-SVMP. The results showed distinct mechanisms of action that should contribute in the elucidation of the differences in the hemorrhagic potential of SVMPs, allowing a better understanding of this class of enzymes and of the biology of Bothrops pirajai species.
使用蛋白质组学策略分析了矛头蝮蛇（Bothrops pirajai）蛇毒。使用反相高效液相色谱法（RP-HPLC）分离蛋白质，然后进行 SDS-PAGE、胶内胰蛋白酶消化、基质辅助激光解吸/电离飞行时间/飞行时间质谱（MALDI-TOF/TOF MS）鉴定，并根据相似性将其分配到已知蛋白家族。在 B. pirajai 毒液中发现了属于六个家族的蛋白质，包括丰富的 PLA2 和金属蛋白酶，其余蛋白质分布在 L-氨基酸氧化酶、丝氨酸蛋白酶、解整合素和凝集素样家族中。通过三步色谱法从这种毒液中分离出一种 P-I 类金属蛋白酶，命名为 BpirMP。该酶的分子量通过质谱法测定为 23.1kDa。其对偶氮酪蛋白的蛋白水解活性被螯合剂和还原剂抑制，在较高 pH 值和 37°C 时具有最佳活性。BpirMP 表现出较弱的出血活性，MHD 为 50μg，能够在体内和体外水解基底膜成分。该毒素裂解纤维蛋白原的 Aα 和 Bβ 链，并且还能够在体外降解纤维蛋白和血栓。初级序列分析表明，BpirMP 含有一个锌配体基序和一个与 Met-转弯结构相关的 CVM 基序。这些结果表明，BpirMP 是一种锌依赖性出血性金属蛋白酶，具有纤维蛋白（原）水解和溶栓活性。
本文描述了来自巴西东北部受威胁蛇种矛头蝮（Bothrops pirajai）毒液中存在的蛋白质成分的多样性，以及 PI-SVMP 的分离和生化特性。结果表明存在不同的作用机制，这应该有助于阐明 SVMP 出血潜力的差异，从而更好地理解这一类酶和 Bothrops pirajai 物种的生物学。
