Figure 3.

Total synthesis of sPLA₂ by native chemical ligation. (A) Reaction at t = 0. HPLC chromatogram of the starting synthetic peptide segments (Cys⁵⁹-124) and (1-Gly⁵⁸)-^αCOSR, dissolved at 9 mg/ml in phosphate buffer (pH 7.5) containing 6 M GuHCl. ESMS spectra show the m/z ratios of the (1–58)-^αCOSR peptide (3rd–10th ionized states) observed mass 6,996.5 ± 0.7 Da (calculated 6,997.7 Da, average isotope composition of the reduced peptide), and the (59–124)-peptide (4th–11th ionized states) observed mass of 7,510.1 ± 2.6 Da (calculated 7,510.6 Da, average isotope composition of the reduced peptide. (B) HPLC chromatograms of the ligation reaction, started by the addition of 1% thiophenol and 1% benzylmercaptan. At t = 5 hr (Upper), ligated product (1–124) is shown, as well as the C-terminal segment starting material. The unreacted N-terminal material can be accounted for as multiple intermediates eluting between 15 and 20 min (see text). Peak 1 represents a N-terminal peptide derivative; peaks 2 and 3 represent the ligated material (1–124) with, respectively, one and two DNP groups still attached to His side chains (see text). At t = 24 h (Lower), an almost quantitative ligation has occurred. The m/z pattern of the ligated material is shown (6th–21th ionized states) observed mass of 13,919.5 ± 1.6 Da (calculated 13,918.0 Da, average isotopic mass of the reduced peptide). (C) Folding and disulfide formation of the sPLA₂ purified 124 residue polypeptide. HPLC chromatograms are shown of the purified reduced sPLA₂ polypeptide (t = 0), the crude folded material (t = 48 hr), and the purified final product (sPLA₂). The m/z pattern from the material in peak 4 is shown (6th–12th ionized state), observed molecular mass of 13,904.7 ± 0.8 Da (calculated 13,904.0 Da, average isotopic mass of the sPLA₂ containing seven disulfides). Note the shift in m/z intensities between the reduced and folded sPLA₂ (see text).