Characterisation of the dominant oxidative folding intermediate of hen lysozyme

Reduced denatured lysozyme has been oxidised and refolded at pH values clos e to neutral in an efficient way by dilution from buffers containing 8.0 M urea, and refolding intermediates were separated by reverse-phase HPLC at p H 2. By using peptic digestion in combination with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tande m MS/MS the dominant intermediate was identified to be des-[76-94]. This sp ecies has three of the four native disulphide bonds, but lacks the Cys76-Cy s94 disulphide bond which connects the two folding domains in the native pr otein. Characterisation of des-[76-94] by 2D H-1 NMR Shows that it has a hi ghly native-like structure. This provides an explanation for the accumulati on of this species during refolding as direct oxidation to the fully native protein will be restricted by the burial of Cys94 in the protein interior. (C) 1999 Academic Press.