A, α4V8-16, produced by in-gel digestion of [125I]TID-labeled α4 subunit with V8 protease (Figure 3A), was eluted and purified by reversed-phase HPLC. B, 125I (●) and PTH-amino acids released (□) when pooled fractions 37–40 were sequenced with sequencing interrupted for OPA treatment before cycle 15. The primary amino acid sequence detected began at α4-Ile201 (Io, 6 pmol; R, 94%; 45,560 cpm loaded in the filter and 26,850 cpm left after 40 cycles). The peak of 125I release in cycle 26 (148 cpm) and 31 (74 cpm) correspond to labeling of α4-Cys226 and α4-Cys231, respectively. The amino acid sequence of the detected fragment is shown, with a line indicating the M1 region.