Synthesis, Characterization, Kinetic Parameters, and Diagnostic Application of a Sensitive Colorimetric Substrate for β-Galactosidase (2-Chloro-4-Nitrophenyl-β-D-Galactopyranoside)

The synthesis and characterization of 2-chloro-4-nitrophenyl β-D-galactopyranoside, an improved chromogenic substrate for β-galactosidase, is described. The important kinetic parameters (Km, Vmax and Kp) for this substrate were compared with those of other substrates. The diagnostic utility of this substrate in a digoxin liposome immunoassay is discussed. The new substrate offers at least four times the sensitivity enhancement as that with ortho-nitrophenyl β-D-galactopyranoside in the assays for β-galactosidase. This substrate should find use in enzyme immunoassays where β-galactosidase is used as a label.