A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A 2 and their interaction with substrate analogues and a transition-state inhibitor

Abstract

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A 2 (PLA 2) and the zymogen of porcine PLA 2, prophospholipase A 2 (proPLA 2), in both H 2O and D 2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% α-helix, 15% /gb-sheet and 23% β-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A 2 in the presence or absence of Ca 2+ ions, or in the temperature range 10–45°C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no signifficant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of β-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA 2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the α-helical structure and a decrease in the β-sheet, and/or possibly β-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA 2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy- sn-glycero-3-phosphoglycol in micellar form.