Relation between ONP absorption at 420nm and concentration (used for calculation of β-Galactosidase activity)

Value 4.5E-09 M^-1 cm^-1
Organism Generic
Reference J. H. Miller, Experiments in Molecular Genetics. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972). Unit VII Assays of the lac operon enzymes, Experiment 48 Assay of ß-Galactosidase, pp. 352-355
Method (from ref, pp. 352) ß-Galactosidase is an enzyme which hydrolyzes ß-D-Galactosides. It can easily be measured with chromogenic substrates, colorless substrates which are hydrolyzed to yield colored products. An example is o-nitrophenyl-ß-D-Galactoside. This compound is colorless, but in the presence of ß-Galactosidase it is converted to galactose and o-nitrophenol. The o-nitrophenol is yellow and can be measured by its absorption at 420 nm. If the o-nitrophenyl-ß-D-Galactoside (ONPG) concentration is high enough, the amount of o-nitrophenol produced is proportional to the amount of enzyme present and to the time the enzyme reacts with the ONPG. In order for the assay to be linear, the ONPG must be in excess. For best results, the amount of enzyme should be such that it takes between 15 min and 6 hours for a faint yellow color to develop. The reaction is stopped by adding a concentrated Na2CO3 solution, which shifts the pH to 11. At this pH ß-Galactosidase is inactive.
Comments The specific activity of ß-Galactosidase is defined in terms of units/mg protein. Many investigators define a unit of ß-Galactosidase as the amount of enzyme which produces 1 nmole o-nitrophenol/min at 28°, pH 7.0. Under these conditions, 1µM o-nitrophenol has an optical density (420nm) of 0.0045 using a 10mm light path. From this value the Units of ß-Galactosidase may be measured according to Units=1000×(OD(420)-1.75OD(550))/(t×v×OD(600)) where OD(420) and OD(550) are read from the reaction mixture, OD(600) reflects the cell density just before assay, t=time of the reaction in minutes, v=volume of culture used in the assay, in ml. Wallenfels and Weil report a ß-Galactosidase turnover rate of 1.38×10^9[ONP]M/([LacZ]M×[ONPG]M×min) The reference is Kurt Wallenfels and Rudolf Weil, The Enzymes 7, 617 (1972). These sources courtesy of by H. Garcia
Entered by Uri M
ID 105140