The protein was dissolved in 6 M guanidine–Tris (pH 8.5) and
then reduced using 0.1 M dithiothreitol for 3 h at 50C in the dark
under argon. Iodoacetamide was added to a final concentration of
0.15 M and the alkylation was allowed to proceed for 3 h at room
temperature. The method for HPLC purification of the reduction–
alkylation reaction mixture was the same as described above but
included 4 min at the initial conditions to remove salts.