Chapter 3:

Procedure 3.2
The ß-Galactosidase Assay

Background

The basis of this assay is the biological activity of the enzyme: in this case, cleavage of the bond between the two sugars found in a family of disaccharides known as ß-galactosides. Because it has ß-galactosidase, E. coli is able to utilize lactose, the sugar found in milk, as an energy source. As a result of cleaving lactose, two monosaccharides are produced that can then be metabolized to produce energy through glycolysis and the citric acid cycle. When ß-galactosidase cleaves lactose, its natural substrate in E. coli, the reaction looks like this:

ß-galactosidase
lactose => glucose + galactose

The natural products of this reaction are colorless and not readily detectable. Therefore, an assay system has been developed in which a synthetic sugar is cleaved, producing a colored product that absorbs light in the visible range. The artificial sugar is o-nitrophenyl-ß-galactoside (ONPG). The product, o-nitrophenol (ONP), is yellow and can be quantitated spectrophotometrically at 420 nm.

ß-galactosidase
o-nitrophenyl-ß-galactoside => o-nitrophenol (ONP) + galactose

When more enzyme is present, more product will be formed and more yellow color will be seen. Enzyme assays must be performed at temperatures at which the enzyme is active and, for the sake of comparison, must always be performed at the same temperature; in this case, 37°C. Since measuring the activity of the enzyme is dependent on having an ample supply of substrate, you want the substrate to always be present in excess so that it is not limiting your ability to measure the enzyme. Therefore, if the assay tubes turns bright yellow immediately, the substrate is being used too quickly and the assay will not be accurate. Because the reaction continues as long there is substrate available, you will need to stop the reaction after a designated time in order to measure the absorbance consistently. This is done by adding NaCO₃, a strong base (pH 10-11) that denatures the enzyme and renders it inactive.

Figure 3.2: Reactions catalyzed by ß-galactosidase.

Overview of assay

Using disposable glass tubes for these assays, you will add 10-50 µl of sample to 1 ml of Z buffer, a buffer in which ß-galactosidase has full activity. The Z buffer containing the sample and the ONPG substrate will then be separately pre-incubated to 37°C. The enzymatic reaction will begin as soon as you add the substrate to the samples. After you incubate the tubes for a sufficient length of time, you will terminate the reaction in all the tubes, using the STOP solution (NaCO₃ ). Read the sample absorbance on the spectrophotometer after transferring the contents of the tube to a plastic cuvette.

1. Make a Flow Chart. Begin your preparations by making a flow chart or outline that details exactly what you are going to do in this assay. Save this flow chart as you did for the Bradford Assay and modify it as you refine and streamline your technique.
2. Turn On The Spec. Turn on the spectrophotometer and set the wavelength to 420 nm.
3. Dilute The Sample(s). In your first assay you will be using a sample of ß-galactosidase provided by your instructor. It will be your job to determine the activity of this sample. (After starting the purification, you will be using your own samples.) Prepare a few dilutions of this sample. What will be the diluent?
4. Add The Samples and Control To Z Buffer. Add 10-50 µl of the diluted sample(s) to 1 ml of Z buffer. As always, remember to write down how much sample you used.
5. Prepare a control tube by adding a volume of buffer that is equal to the volume of sample used in step 4 (10-50 µl) to a tube containing 1 ml of Z buffer. This functions as a control for the spontaneous hydrolysis of your substrate, ONPG, and will serve as your calibration blank for the spectrophotometer.
6. Equilibrate. Equilibrate the samples to be assayed to 37°C. Make sure the ONPG (4 mg/ml in 0.1 M sodium phosphate buffer pH 7.5) is also at 37°C. Prepare fresh ONPG solution each day.
7. Begin The Reaction. Add 0.2 ml of ONPG to each tube and record the time.
8. Incubate. Incubate the samples at 37°C until yellow color appears. The color change should take at least 10 minutes but may take longer to result in an absorbance of 0.3 to 0.9. A very rapid color change (1-5 minutes) indicates that there is too much activity to measure accurately. Such samples should be repeated at a higher dilution. Samples containing low concentrations of ß-galactosidase may take significantly longer than 10 minutes to become yellow. (Why is a rapid color change (0.5 O.D. in 2 minutes) not accurate?)
9. Stop The Reaction. Add 0.5 ml of 1 M NaC0₃ (Stop Solution) to terminate the reaction and record the time.
10. Read The Samples. Transfer the samples to plastic cuvettes and read the A₄₂₀ against the blank.
11. Determine The Enzyme Activity: Calculate the enzyme activity present in your sample using the formula shown below:

Units of ß-galactosidase	=	A420 x 0.380
		---------------------
		minutes at 37° C



One unit is the amount of enzyme that will hydrolyze 10^-6 moles/minute of ONPG at 37°C and 0.380 in the above equation is the constant used to convert the A₄₂₀ reading into these units. This constant is a function of the molar extinction coefficient for ONP+ that, under these conditions, is 4500 M^-1cm^-1. What is a molar extinction coefficient? (Refer to Seidman and Moore, chapters 19-21.)

Contact Us:

Lisa Seidman
lseidman@matcmadison.edu
(608) 246-6204

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jmowery@matcmadison.edu
(608) 243-4307