Fig. 3.
(A) Time course studies of native chemical ligation of H4 thioester 3 (2 mM) with H4 thiol 4 (2 mM) in phosphate buffer solution (10 mM, pH 7.5) containing6MGnd·HCl and 2% thiophenol (vol/vol) at room temperature (left trace). Under these HPLC conditions, thiol 4 and ligation product 6 coeluted at 57 min. (B) Data after 16 h (right trace) indicated near complete consumption of the starting thioester 3 as well as partial hydrolysis of the unreacted thiophenol adduct (peaks at 18 and 19.5 min). The reaction proceeded to 60% completion in 16 h as judged by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis. Thiol 4 and the ligation product 6 were subsequently separated by an additional preparative HPLC step under different gradient conditions. (C) Mass analysis of the reactants and products of the native chemical ligation synthesis and desulfurization of acetylated X. laevis histone H4. Experimentally determined and actual average masses are indicated on each figure panel. The italicized number after a Mr value indicates the mass difference (in units of 1.00235 Da) between that peak and the monoisotopic peak. This MALDI-TOF MS spectrum depicts the average mass of the purified thioester 3. (D) FT-ESI MS spectrum of H4 C-terminal recombinant protein thiol 4.(E) FT-ESI MS spectrum of the native chemical ligation product 6.(F) FT-ESI MS spectrum of the desulfurization reaction product 7 containing a native histone H4 primary sequence.