Rapid and highly sensitive fluorescent detection of protamine and trypsin activity with Eosin B

A highly sensitive detection system for protamine and trypsin activity was developed using a commercially available anionic fluorescent dye, Eosin B (EB), as the probe. EB exhibited a highly sensitive colorimetric and fluorescent response for visual detection of protamine with a good selectivity over other interfering substances in the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (10 mM, pH 7.4). A linear relationship between the fluorescence intensity and protamine concentration was obtained ranging from 0 to10 mu g/mL with a detection limit of 29.25 ng/mL. The presence of trypsin hydrolyzed protamine, leading to the release of EB from EB-protamine complex. The fluorescence recovery of EB can monitor the trypsin activity with satisfactory selectivity and sensitivity. The introduction of a trypsin inhibitor affected the recovery of the fluorescence. Thus, this detection system supplied a method for trypsin inhibitor screening.