Mechanism of Interference and Azur A Response in the Heparin Assay

Abstract

The heparin determination method of dye-heparin assay is popular. Assay interference by base, SDS, and other compounds was explained in terms of the effect upon the equilibria between dye and heparin. To gain insight into the mechanism of activation of the interference, we used cationic dye-azur A. It was found that increased salt concentration caused a change in (n_max - n_min) and ▵ε values, thus decreasing the sensitivity of the azur A-heparin assay. Azur A dye binding heparin was not affected by pH between 2 and 10. Dye binding required a macromolecular form with sulfate-rich groups. The carboxyl groups without sulfate groups gave no responses. The binding behaviour was attributed to electrostatic attractions and Van der waals forces.

Key Words:

• Heparin
• Azur A
• Heparin-dye assay
• Interference
• Spectrophotometry