Fluorometric determination of heparin based on self-quenching of fluorescein-labeled protamine

A new signaling technique for a fluorometric heparin assay has been developed using the self-quenching of fluoresceinisothiocyanate-labeled protamine (F-protamine). The binding between F-protamine and heparin led to a significant fluorescence quenching due to enhancing the proximity of the F-protamine molecules. The fluorescence of F-protamine (5.9 μg/mL) decreased to 13% in the presence of 2.0 μg/mL heparin. An advantage of this self-quenching system is that the detection can be carried out without using other indicators. With the present system, we could monitor the therapeutic level of heparin in bovine plasma, and the response was quick. These properties of the self-quenching system are suitable for practical use.