Interaction of procion red HE-3B and other reactive dyes with alkaline phosphatase: a study by means of kinetic, difference spectroscopic and chromatographic methods.

The interaction of alkaline phosphatase (EC 3.1.3.1) from calf intestine with different dyes, especially with Procion Red HE-3B was studied by several methods. From the kinetic analysis a nonlinear noncompetitive type of inhibition with an inhibition constant Ki = 0.03 mM for Procion Red HE-3B and Cibacron Blue F3G-A was estimated. The extent of inhibition of the two dyes at constant substrate and inhibitor concentration is 10 to 20 times higher than that of natural inhibitors like L-phenylalanine and NADH. Difference spectroscopic measurements with Procion Red HE-3B showed that the enzyme dimer possesses two binding sites for the dye. The dissociation constant of the dye-enzyme complex was estimated to be Kd = 0.01 mM. The binding of Procion Red HE-3B to the enzyme is mainly stabilized by electrostatic interactions. Large aromatic parts of a dye molecule like a combination of two naphthol ring systems or an anthraquinone ring flanked by spatially arranged charged substituents are important for the extent of specificity. The elution of the enzyme from the immobilized dye and the quenching of the dye-protein difference spectral signal by the competitive inhibitor phosphate and by substrates suggest the involvement of the active center of the enzyme in the dye binding region.