Fig. 8.

[³H]dFBr photolabeling within α subunit. ³H (○, ⬤) and phenylthiohydantoin amino acids (⬜, ▪) released during sequence analyses of rpHPLC fractions 30–32 (A) and 27–29 (B) from the fractionation of Endolys-C digests of αV8-20 (Fig. 7B) and fractions 17–19 (C) from the purification of the Endolys-C digest of αV8-18 (Fig. 7C). (A) The primary sequence began at the N-terminus of αM2 (αMet²⁴³; 4 pmol each condition) with a contaminating peptide beginning at βM²⁴⁹ (∼0.5 pmol each condition). The peaks of Carb-enhanced ³H release in cycles 2, 5, and 9 indicate [³H]dFBr photolabeling −Carb/+Carb, in cpm/pmol) at αThr²⁴⁴ (αM2-2, 4/15), αIle²⁴⁷ (αM2-5, 11/36), αSer²⁴⁸ (αM2-6, 7/31), and αIle²⁵¹ (αM2-9, 15/101). (B) The only sequence began at αHis¹⁸⁶ (⬜−Carb/▪+Carb, 8/10 pmol), with Carb-inhibitable peaks of ³H release at cycles 5 and 13 indicating [³H]dFBr photolabeling at αTyr¹⁹⁰ and αTyr¹⁹⁸ (−Carb/+Carb, 12/1 and 19/2 cpm/pmol, respectively). (C) The pool of material in rpHPLC fractions 17–19 from Fig. 7C was sequenced with the sequencing filter treated before cycle 12 with o-phthalaldehyde (OPA), which prevents sequencing of peptides not containing a proline at that cycle (Middleton and Cohen, 1991). After OPA treatment, sequencing continued only for the fragment beginning at αLys⁷⁷ (⬜-Carb/▪+Carb, 22/31 pmol) that contains αPro⁸⁸ in cycle 12. The peak of ³H release in cycle 17 indicated [³H]dFBr photolabeling of αTyr⁹³ (−Carb/+Carb, 3/0.1 cpm/pmol).