TIME-RESOLVED FLUORESCENCE STUDY OF A CALCIUM-INDUCED CONFORMATIONAL CHANGE IN PROTHROMBIN FRAGMENT-1

The wavelength dependent fluorescence decay properties of bovine proth rombin fragment 1 have been investigated employing a picosecond time-c orrelated single photon counting technique, All observations are discu ssed with using the crystal structure (Soriano-Garcia ct al,, Biochemi stry 31:2554-2566, 1992). Fluorescence lifetimes distribution and conv entional multiexponential analysis, as well as acrylamide quenching st udies lead to the identification of six distinguishable tryptophan exc ited-states, Accessibility to the quencher and the known structure are used to associate a fluorescence decay of the tryptophan present in t he Gla domain (Trp42) with two red shifted components (2.3 and 4.9 ns) . The two kringle domain tryptophans (Trp90 and Trp126) exhibit four d ecay times (0.06, 0.24, 0.68, and 2.3 us), which are blue shifted. The calcium induced fluorescence quenching is a result of static quenchin g: the five decay times remain unchanged, whereas the fluorescence int ensity of Trp42 is decreased, The static quenching process is a conseq uence of a ground state interaction between the Cys18-Cys23 disulfide bridge and Trp42, The monomolecular equilibrium constant for this disu lfide-pi-electron interaction is found as 4.8. (C) 1996 Wiley-Liss, In c.