Description
2-Nitrophenyl-β-D-galactopyranoside (ONPG) is a colorimetric and spectrophotometric substrate for detecting β-galactosidase activity. This compound is usually colorless. However, if β-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and ortho-nitrophenol. The latter compound has a yellow color that can be used to check for enzyme activity utilizing a colorimetric assay (at 420 nm wavelength). β-Galactosidase is required for lactose utilization, so the intensity of the color produced can be used to measure the enzymatic rate. Though ONPG mimics lactose and is hydrolyzed by β-galactosidase, it cannot act as an inducer for the lac operon. Without another lactose analog that can act as an inducer, such as isopropyl β-D-1-thiogalactopyranoside (IPTG), β-galactosidase will not be transcribed and ONPG will not be hydrolyzed.
Chemical Properties
White Crystalline Solid
Uses
2-Nitrophenyl-beta-D-galactopyranoside is a β-Galactosidase substrate for colorimetric and EIA applications; counterpart of widely employed pNPP/alkaline phosphatase substrate.
Definition
ChEBI: A beta-D-galactoside having a 2-nitrophenyl substituent at the anomeric position.
General Description
ONPG (2-Nitrophenyl β-D-galactopyranoside) is a colorimetric substrate for β-galactosidase.
Biochem/physiol Actions
β-galactosidase, breaks down lactose into galactose and glucose. β-Galactosidase is not lactose specific and can act on simple galactosides. 2-Nitrophenyl β-D-galactopyranoside hydrolysis results in the release of galactose and a yellow chromogenic compound. The test substrate does not depend on an induced or constitutive permease enzyme to enter the cell, therefore reactions are rapid and occur within a 24-hour period.
Purification Methods
Purify 2-nitrophenyl--D-galactopyranoside by recrystallisation from EtOH. [Seidman & Link J Am Chem Soc 72 4324 1950, Snyder & Link J Am Chem Soc 75 1758 1953]. It is a
chromogenic substrate for -galactosidases [Jagota et al. J Food Sci 46 161 1981]. [Beilstein 17/7 V 52.]
