Fluorescence properties of Savinase®: the X-ray structure in the region of the tryptophyl residues

Fluorescence properties of the alkaline proteinase Savinase® are described and related to the enzyme X-ray crystal structure. The intrinsic protein emission is dominated by the ‘exposed’ Trp 241. The fluorescence decay of the phenylmethanesulfonyl (PMS)-proteinase excited at 297 nm was well fitted by three exponentials with lifetimes of 0.34±0.06 ns (39.4%), 2.80±0.17 ns (42.3%) and 6.80±1.15 ns (18.3%). Savinase® and the closely related proteolytic enzyme Esperase® are an attractive couple of proteins for fluorescence studies. Trp 6 and Trp 113, common to both proteinases, are located in identical microenvironments in the two globular proteins. Iodide ions are efficient quenchers of the PMS-Savinase® fluorescence. Caesium had almost no effect on the indole emission. An electrostatic parameter E=12.4 was obtained as the ratio K SV −/ K SV +. This means a positive microenvironment of the emitting tryptophans. Acrylamide quenching proceeds via both dynamic and static processes. The activation energy for the thermal deactivation of the excited indole groups is 59.2 kJ mol −1 in the absence of extraneous calcium and 76.4 kJ mol −1 in the presence of 100 mM Ca 2+. The T m values in the absence and presence of added Ca 2+ are 61 and 78°C, respectively; saturation of the ‘weak’ calcium-binding site, Ca2, dramatically increased the PMS-proteinase thermostability. A good correlation between the spectroscopic properties and the crystallographic structure of Savinase® was observed.