Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.