Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and I-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (1 5 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.Keywords: creatine kinase; dimerization of subunits; folding intermediate; kinetics; molten globule; protein foldingIn spite of the accumulation of a large number of experimental studies, protein folding remains one of the most challenging subjects in structural biology. Characterization of folding intermediates is considered an important strategy for the elucidation of the mechanism of protein folding. A common equilibrium intermediate, the "molten globule" (MG) state, has been detected between the native (N) and the fully unfolded (U) states for many proteins (Fink, 1995; Ptitsyn, 1995). The MG state is characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface, and the absence of rigid side-chain packing

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R g value of about 17.3 A î in 8 M urea. The reduced ribonuclease A is more expanded, its R g value is about 20 A î in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996)

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH 4 ) 2 SO 4 precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single-stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgCl 2 above 150 mM, MnCl 2 above 200 mM, ZnCl 2 above 150 mM, CaCl 2 above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other wellcharacterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.