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 Tr p-3 of pig pancreatic phospholipase A2 (PLA2) in aqueous solution (E f orm) or at the interface without (E form) or with a ligand at the act ive site (EL form) are characterized. In the E form, the single trypt ophan residue is exposed on the protein surface to the aqueous environ ment, as it is freely accessible to aqueous quenchers such as succinim ide and acrylamide. The fluorescence quantum yield of E is about one-t hird that of N-acetyl-tryptophanamide, indicating significant intramol ecular quenching processes including charge-transfer reactions, as see n by the D2O effect. Upon binding of PLA2 to micelles of 1-hexadecylpr opanediol-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 t he 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 sign ificantly shielded from aqueous quenchers, and the D2O effect on the q uantum yield is still present. The UV difference spectrum for the E-t o-EL transition is of large amplitude, with peaks at 292 (DELTAepsilo n = 2540 M-1 cm-1) and 284 nm (DELTAepsilon = 2100 M-1 cm-1), which su ggests transfer of tryptophan from an aqueous to a less polar environm ent. Upon conversion to the EL 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 thr ee 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 compon ents to the 3.2-ns component seems to account for the higher quantum y ield on the E-to-E change. The frequency domain anisotropy decays ind icate a highly hindered Trp-3 in the E form whose limited motional fre edom 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 aqueo us 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 d ampened 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.