FLUORESCENCE OF EOSINOPHIL LEUKOCYTE GRANULES INDUCED BY 1-HYDROXY-3,6,8-PYPENETRISULFONATE - VISUALIZATION OF DIFFERENCES IN PROTEIN ISOELECTRIC POINTS

After treatment of horse, rat and human blood smears with alkaline sol utions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucoc yte granules were the unique cell components which showed a bright gre en fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished afte r washing or staining in salt solutions or by using blocking methods f or amino groups. Using HPTS at pH 12, the fluorescence reaction of hor se granules was specifically located in the peripheral region, appeari ng as fluorescent rings. These microscopic observations, which indicat e differences in the isoelectric point of proteins within the eosinoph il granule, were also confirmed by HPTS staining of protein blots as m odel substrates. Spectral analysis of HPTS at pH 10 and 12 showed prac tically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic bind ing occurs between cationic proteins and HPTS in alkaline solution, an d that the most cationic proteins (with isoelectric points at pH highe r than 12) are located in the peripheral annular region of horse eosin ophil granules.