The structure of fibrinogen in solution has been investigated by the technique of steady state fluorescence polarization. Factor XIII a has been employed to catalyze the incorporation of approximately 3 moles dansylcadaverine per mole fibrinogen without disruption of its structure or function. Two γ-chain and one α-chain crosslink acceptor sites have been labelled, according to SDS-polyacrylamide gel electrophoresis. Light scattering and electron microscopy verify that this derivative forms normal fibrin upon thrombin activation; the modified protein is 93% clottable. The fluorescence emission maxiumum is shifted to 500 nm (vs. 540 nm for dansylcadaverine) and a substantial increase in fluorescence intensity is noted. Measurements of the fluorescent lifetime, by a phase shift technique, show an average lifetime of 14.5 ±2.5 nanoseconds (vs. 3 ns for dansylcadaverine). The rotational relaxation time, determined from a Perrin plot of polarization vs. temperature, is 165 ±13 ns. No change in rotational relaxation time was found in 0.005 M CaCl2 or at high ionic strength. The limiting polarization (extrapolated to infinite viscosity) agrees with that determined with dansylcadaverine fibrin (coarse gel conditions) as well as with the vlaue measured in 75% glycerol at 0°C. These controls confirm that overall macromolecular motion, not probe rotation,is being measured.The measured rotational relaxation time for dansylcadaverine fibrinogen is substantially shorter than values calculated for an anhydrous rigid sphere (300 ns) or a prolate ellipsoid (1000 ns), as well as other models based on hydrodynamic data. These results indicate that the rotating unit is smaller than the entire fibrinogen molecule, i.e. that the structure of fibrinogen in solution is flexible. This observation is consistent with earlier polarization data reported for dansylchloride labelled fibrinogen, as well as structural data indicating fibrinogen to be a multidomain protein, whose globular portions are connected by helical chains.
Lifetime and rotational relaxation time of dansylgalactoside bound to the lac carrier protein
