SPECTROSCOPIC PROPERTIES OF THE STATES OF PIG PANCREATIC PHOSPHOLIPASE-A(2) AT INTERFACES AND THEIR POSSIBLE MOLECULAR-ORIGIN

The near-UV absorption and fluorescence spectroscopic properties of Trp-3 of pig pancreatic phospholipase A2 (PLA2) in aqueous solution (E form) or at the interface without (E* form) or with a ligand at the active site (E*L form) are characterized. In the E form, the single tryptophan residue is exposed on the protein surface to the aqueous environment, as it is freely accessible to aqueous quenchers such as succinimide and acrylamide. The fluorescence quantum yield of E is about one-third that of N-acetyl-tryptophanamide, indicating significant intramolecular quenching processes including charge-transfer reactions, as seen by the D2O effect. Upon binding of PLA2 to micelles of 1-hexadecylpropanediol-3-phosphocholine (E*), a positive difference spectrum with a shoulder at 284 nm (DELTAepsilon = 370 M-1 cm-1) is observed. Similar difference spectra are also observed upon binding of sulfate ion to the E form. The fluorescence emission of E* is blue-shifted by about 10 nm to 336 nm, with a 2-fold higher quantum yield. Trp-3 in E* is significantly shielded from aqueous quenchers, and the D2O effect on the quantum yield is still present. The UV difference spectrum for the E*-to-E*L transition is of large amplitude, with peaks at 292 (DELTAepsilon = 2540 M-1 cm-1) and 284 nm (DELTAepsilon = 2100 M-1 cm-1), which suggests transfer of tryptophan from an aqueous to a less polar environment. Upon conversion to the E*L form, there is a further blue shift to 333 nm, with about a 20% increase in the fluorescence quantum yield. The frequency domain fluorescence intensity decays of Trp-3 in all three forms of the enzyme are complex and require up to four fluorescence lifetime components of about 0.1-0.3, 0.6-1.5, 2.3-3.2, and 6-7.5 ns. A significant shift in the population from the two short-lived components to the 3.2-ns component seems to account for the higher quantum yield on the E-to-E* change. The frequency domain anisotropy decays indicate a highly hindered Trp-3 in the E form whose limited motional freedom is lost upon the transition to E* and E*L forms. Compared to that in E*, Trp-3 in E*L is only marginally more shielded from the solvent. Most of the decrease in the accessibility of Trp-3 to the bulk aqueous phase occurs during the change from E to E*, while the dehydration of the enzyme-lipid microinterface occurs primarily during the E*-to-E*L transition. In conclusion, the spectral changes in the E-to-E* step are due to changes in the ionic environment of Trp-3 resulting from dampened segmental motions of the interfacial binding region, while the changes in the E*-to-E*L step are primarily due to the dehydration of the enzyme-lipid microinterface.