Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotrans ferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroe thanol (TFE) have been studied. Significant aggregation was observed when C K was denatured at TFE concentrations between 10% and 40% (v/v). 50%;, TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational chang es during denaturation followed by fluorescence emission spectra and far-ul traviolet CD spectra. DTNB modification and size exclusion chromatography w ere used to find that CK dissociated and was in its monomer state after den aturation with 50% TFE. Reactivation and refolding were observed after 80-f old dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The d enatured CK recovered about 38%; activity following a two phase course (k(1 )=4.82+/-0.41X10(-3) s(-1), k(2) =0.60+/-0.01 x 10(-3) s(-1)). Intrinsic fl uorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1) = 4.34+/-0.27X10(-3) s(-1), k(2) = 0.76 /- 0.02 X 10(-3) s(-1)) after dilution into the proper solutions. The far-u ltraviolet CD spectra ellipticity changes at 222 nm during the refolding pr ocess also showed a two phase course (k(1) = 4.50 + 0.07 x 10(-3) s(-1), k( 2) = 1.13 +/- 0.05 X 10(-3) s(-1)). Our results suggest that TFE can be use d as a reversible denaturant like urea and GuHCl, The 50% TFE induced CK de naturation state, which was referred to as the 'TFE state', and the partial ly refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper. (C) 2001 Elsevier Science B.V. All rights reserved.