Mast cell-lysosomal cationic protein interaction: Use of 1-anilinonaphthalene-8-sulfonate as a fluorescent probe

Abstract

Band 2 protein (B2), a lysosomal cationic protein from neutrophils, interacts with mast cells to stimulate non-cytotoxic histamine release. This interaction appears to initiate a biochemical sequence of events similar to that initiated by the antigen-antibody reaction. 1-Anilinonaphthalene-8-sulfonate (ANS) is almost non-fluorescent in aqueous solutions, but becomes highly fluorescent when bound to B2. The number of binding sites per mole of B2 is approx 4, and the statistical binding constant, K ̄ app , is 2.3 × 10 −5 M. It should be noted that when ANS was mixed with band 3 protein (B3), a lysosomal cationic protein with molecular weight and basicity similar to that of B2, but lacking the ability to stimulate histamine release, there was no increase in fluorescence. When ANS was interacted with band 2 protein in the presence of 5 × 10 5 mast cells, the fluorescence was enhanced and the number of binding sites per mole of B2 was increased to 5. The fluorescence was not enhanced when an equal number of lymphocytes or total rat peritoneal cells (6–7% mast cells) were reacted with B2. There was no increase in fluorescence when B3 plus ANS was mixed with mast cells. The data presented here suggest that the interaction of B2 with mast cells exposes an additional binding site for ANS which may be on the cell surface, or associated with both the stimulus and the cell. These investigations demonstrate that ANS can be used as a fluorescent probe in the study of the interaction of band 2 protein and mast cells during activation of mast cells for histamine release.