Figure 2.

Separation by size-exclusion chromatography of tryptic peptides derived from HEWL labeled with methylene carbene. After the clean-up step described above (see Fig. 1), HEWL samples were reduced, carbamidomethylated, and digested with TPCK-trypsin (see Materials and Methods). The resulting soluble peptide mixtures corresponding to HEWL labeled in its free (a) or complexed form (b) were subjected to size-exclusion chromatography on a Superdex Peptide HR 10/30 column. Elution was monitored by the UV absorption at 280 (thin line) and 215 nm (thick line), and by measurement of the radioactivity (gray bars). In c, the horizontal bars represent the relative differential labeling of each peak (named A–D). This parameter is defined as the numerical difference between the label incorporation for free and complexed HEWL expressed relative to the value for the complexed form. The elution profile (absorbance at 215 nm) of peptides is plotted alongside.