Photoaffinity labeling was used to evaluate optimal conditions for purification of I-Ak histocompatibility molecules in functionally active form. We assessed the biological activity of I-Ak primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [125I]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-Ak on B cell hybridoma membranes and their detergent solubilisates, at the α chain. Following extensive detergent dialysis, the intensity of this labelling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-Ak from various monoclonal antibodies and by determining the denaturation of I-Ak under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-Ak purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus, I-Ak was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-Ak on cell membranes, purified I-Ak in detergent showed extensive labelling of the β chain. The overall labelling intensity and the extent of β chain labelling substantially changed upon addition of certain lysophosphatides.
- Histocompatibility molecule
- I-A molecule
- Major histocompatibility complex