Deconvoluted Average Masses (Da) of various BPTI species.

Masses were determined by electrospray ionization mass spectrometry. Data marked * are means of 3 independent determinations all falling within +/−1 Da; the remaining data are single determinations. Example deconvoluted spectra for the refolded species are shown in Figure S1.
The observed mass of the refolded wild-type corresponds to the theoretical mass of fully-oxidised wild-type BPTI containing 3 disulfide bonds (6643 Da), while that of the refolded double mutant corresponds to the mass expected for the species containing 2 disulfide bonds (6613 Da). The mass differences between the refolded and reduced species confirm the formation of 3 and 2 disulfide bonds respectively. The mass of a carboxamidomethyl group (-CH2-CO-NH2) is 58 Da, hence the masses observed for reduced/alkylated wild-type and mutant are as expected for alkylation of 6 and 4 Cys residues respectively.
Alkylations of the refolded species were carried out in presence of 8M urea. No alkylated species were detected and hence any protein molecules containing free –SH groups must represent <1–2% of the total refolded material.