The Purification, Crystallization and Preliminary Structure Analysis by X-ray Diffraction of Phospholipase A2
Davis, Alita A.
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Phospholipases A2 catalyze the calcium-requiring hydrolysis of the C2 ester bond of the l,2-diacyl-3-sn-phosphoglycerides which are intrinsic components of cell membranes and serum lipoproteins. Although much information exists about phospholipases A2, their mechanism of action is still unknown. The most promising method of determining this mechanism is by building structural models of the enzyme based on its crystallographic analyses by X-ray diffraction that, coupled with information obtained from chemical and kinetic modification studies, allow functional conclusions to be made. Any diffraction analysis requires that the protein first be isolated from its source, purified to a high level of homogeneity and crystallized in a form suitable for data collection. This project therefore consisted of three sections: purification of a phospholipase A2 from its source (copperhead venom), crystallization (via vapor diffusion) of phospholipases A2from three different sources (honeybee, copperhead and water moccasin venoms) and preliminary structure analysis of the phospholipase A2 that produced the best crystal. Of the three, the water moccasin crystals (grown in 73 to 75 % ethanol and 2-methyl-2,4-pentandiol [MPD] mixtures [85 parts ethanol to 15 parts MPD]) were the most suitable for analysis. The preliminary structure analysis (using precession photography) performed on these crystals revealed amonoclinic space group (P2j) with an estimated two molecules per asymmetric unit (four molecules per unit cell) and unit cell dimensions of: a=42.58(1) A, b=57.60(3) A, c=45.00(2) A, and 6=96.36(3) °. This corresponds to a cell volume of 109,700 A3.