Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-nitro-1-naphthyl-β-D-galactopyranoside as an example.

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-β-D-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli β-D-galactosidase is 3-fold of that on para-nitrophenyl-β-D-galactopyranoside and about 40% of that on ortho-nitrophenyl-β-D-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.