Characterization, crystallization and preliminary X-ray diffraction analysis of acutohaemolysin, a haemolytic toxin fromAgkistrodon acutusvenom

Abstract

Acutohaemolysin, a phospholipase A(2) (PLA(2)) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA(2) proteins from other snake venoms. Although its PLA(2) enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 A were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 A, beta = 117.69 degrees. The asymmetric unit contains one molecule.

The Crystal Structure of a Novel, Inactive, Lysine 49 PLA2 from Agkistrodon acutus Venom

“…Overall Structure-The amino acid residues of acutohaemolysin were sequenced from the crystallographic electron density, and the identities of the 27 N-terminal residues were determined by Edman degradation as well (25). These results were found to be in agreement with the sequence of a cDNA clone of unknown functions from the same species (31).…”

“…Crystallization and Diffraction Data Collection-Acutohaemolysin was purified and crystallized using previously reported procedures (25). Crystals with a size of ϳ0.8 ϫ 0.8 ϫ 1.0 mm were soaked in a 0.05 M sodium citrate solution (pH 5.6) containing 18%(v/v) isopropanol, 18% (w/v) polyethylene glycol 4000, and 5% (v/v) glycerol and then subjected to cryogenic flash freezing.…”

“…Although all of the required catalytic apparatus (including His 48 , Tyr 52 , Tyr 73 , and Asp 99 , and the catalytic water molecule WAT2010) is conserved in the acutohaemolysin structure, no measurable activity is exhibited in standard assays (25). When the catalytic triad of acutohaemolysin is superimposed with the corresponding residues of four structurally homologous Lys 49 PLA2s, at least one of which possesses catalytic activity (9), it is apparent that all the triads have nearly identical conformations and are anchored tightly to the scaffold composed of two ␣ helices (Fig.…”

Study on the molecular mechanism of Chinese herbal medicine <italic>Cynanchum paniculatum</italic> for treating snakebites

“…1MG6为碱性肌肉毒素, 属于Lys49-PLA 2 , 具有较 强的间接出血活性和抗聚集活性, 能够引起骨骼肌纤 维不可逆性损伤 [17,42] . 对接结果显示(图9D), 丹皮酚原 [43,44] , 丹皮酚原苷可能通过O12与His48残基 相互作用而起到抑制1MG6活性的作用.…”