A comparison of native and covalently crosslinked creatine kinases: denaturation and reassembly

Creatine kinase from rabbit muscle, a dimeric protein of molecular weight 81,000, was crosslinked with glutaraldehyde. The time course of the reaction was monitored by inhibition of activity, fluorescence polarization, and electrophoretic mobility in polyacrylamide slab gels containing sodium dodecyl sulfate. Inactivation is complete in 40 min. ADP (1.2 mM) decreases the rate but does not prevent complete inactivation. Fluorescence polarization analysis of N-dansylaziridyl creatine kinase suggests the formation of high molecular weight oligomers. This is confirmed through electrophoretic molecular weight analysis, which demonstrates the formation of products with molecular weight greater than 150,000 throughout the course of reaction with glutaraldehyde. During crosslinking at least three other products appear with apparent molecular weights of 150,000, 95,000, and 85,000. The 150,000 and 95,000 molecular weight products are transient, disappearing after 40 min of exposure to glutaraldehyde. Monomers of 40,000 molecular weight have also been eliminated by 40 min. The 85,000 molecular weight product, which is not present until 10 min after crosslinking, is the only product other than oligomers present at 60 min of crosslinking. These two products are separated by gel filtration. The isolated 85,000 molecular weight protein exhibits absorption and fluorescence spectra identical to those of noncrosslinked creatine kinase. Upon denaturation in 8 M urea, the noncrosslinked and crosslinked preparations undergo a 2.8-fold and 2.0-fold increase, respectively, in intrinsic protein fluorescence. Since crosslinked enzyme is incapable of dissociation upon denaturation, the difference in fluorescence (28%) between crosslinked and noncrosslinked protein in denaturant may be attributed to the contribution of dissociation to the total fluorescence change resulting from denaturation. The kinetics of reassembly of both preparations exhibit a rapid, initial decrease in fluorescence, followed by a slower, more protracted decline until the level of fluorescence of the undenatured proteins is achieved. The decrease in fluorescence during the first phase of reassembly is 93 and 72% of the total fluorescence change for crosslinked and noncrosslinked enzyme, respectively. Since, the magnitude of these changes is considerably greater than the fluorescence change attributed to association of subunits, the first kinetic phase cannot reflect dimerization, and most probably constitutes the refolding of subunits. The second, slower phase of reassembly, which is approximately 28% of the fluorescence change observed in the reassembly of noncrosslinked enzyme, but only 7% for crosslinked species, may be assigned to reassociation.