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Quantitation of b-galactosidase (b-gal) enzymatic activity is a commonly used determination in cellular and molecular biology. A colorimetric assay using o-nitrophenol-b-D-galactoside (ONPG) as the substrate for b-galactosidase is described. Introduction Transient expression of cloned DNA sequences into mammalian cells is a commonly used tool in molecular biology for the investigation of transcriptional activity of eukaryotic promoters. However, in most cases the natural product of the promoter cannot be assayed in a quantitative fashion. In these instances, the promoter is joined to a reporter gene, which encodes for a protein with a unique enzymatic activity that can be assayed easily. The level of gene expression is then monitored by determination of that unique enzymatic activity. The LacZ gene from Escherichia coli is frequently used as a reporter gene for detecting the expression of recombinant fusion gene studies and for monitoring the efficiency with transient or stable transfections of mammalian, yeast and bacterial cells. A translational in-frame fusion of the gene of interest and the LacZ gene puts lacZ expression under control of the promoter of interest. The activity of the promoter can then be assayed by measuring b-galactosidase activity. Because b-galactosidase has a high turnover rate and is absent in mammalian cells it serves as a very useful and sensitive reporting tool for gene expression. The enzymatic product of the LacZ gene, b-galactosidase, catalyses the hydrolysis of b-D-galactosides, such as lactose, into their component sugars by hydrolysis of the terminal non-reducing b-D-galactose residues (Figure 1A). Fortunately the substrate specificity of the enzyme is such that a variety of different substrates, each with a b-D-galactopyranoside moiety, can be acted upon. Investigators have taken advantage of this by synthesizing compounds which when hydrolyzed by b-galactosidase result in a colored product. One such compound is o-nitrophenol-b-D-galactoside (ONPG), which when hydrolyzed forms galactose and o-nitrophenol (ONP) (Figure 1B). The compound ONP absorbs light at 420 nm whereas the precursor molecule ONPG does not. Therefore, the increase in light absorbance at 420 nm can be used to monitor b-galactosidase when ONPG is used as a substrate.

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Figure 1. Enzymatic hydrolysis of b-D-galactosides by b-galactosidase. (A) b-Galactosidase catalyses the hydrolysis of terminal non-reducing b-D-galactopyranoside residues from disaccharides such as lactose. (B) Colorless substrate ONPG is hydrolyzed enzymatically by b-gal into galactose and yellow colored product o-nitrophenol (ONP) which absorbs at 420 nm. Other compounds that possess a conformation similar to ONPG or lactose (i.e., have a b-D-galactose moiety), can be used in combination with b-galactosidase. There are several compounds, such as 5-bromo-4-chloro-3-indolyl b-D-galactosidase (X-gal), that form colored insoluble products after hydrolysis. These compounds are quite useful in screening for bacterial plasmid recombinants (1). Bacterial cells containing the LacZ gene (LacZ+) would hydrolyze X-gal and turn blue, while LacZ- cells, unable to hydrolyze the compound would remain white. Alternatively, compounds that cannot be hydrolyzed, but retain a similar structure would be expected to act as a competitive inhibitor. Phenylethyl b-D-thiogalacto-pyranoside (PETG) has a thiol group substituted for the hydroxyl linkage present in compounds that b-galactosidase can normally hydrolyze. PETG when present in a reaction would be expected to occupy the enzyme and thus inhibit its action on hydrolyzable substrates such as ONPG. Here we describe a colorimetric assay for b-galactosidase activity in solution. This assay can be extended to be used for the determination of b-gal activity in cell (prokaryotic or eukaryote) cell extracts. Materials and Methods o-Nitrophenyl b-D-galactopyranoside (ONPG), catalogue number N-8431, was purchased from Molecular Probes (Eugene, Oregon). The 96 well microplates, catalogue number 3635, were purchased from Costar (Cambridge, Massachusetts). b-Galactosidase enzyme (cat. # G-6008), sodium phosphate, magnesium chloride and 2-mercaptoethanol were obtained from Sigma Chemical Company (St. Louis, MO). The b-galactosidase assay was performed according to Sanbrook et al. (1); 100 ml of sample or standard in distilled water were placed in each well of a 96-well microplate. The assay was initiated by the addition of 100 ml of 2X assay buffer. Assay buffer was prepared previously as a 2X stock solution and stored frozen at -20°C. Assay buffer (1X) consists of 100 mM sodium phosphate, pH 7.0; 1 mM MgCl2; 50 mM b-mercaptoethanol; and 0.665 mg/ml ONPG in distilled water. Lyophilized b-galactosidase enzyme was reconstituted with distilled water to stock concentration of 500 U/ml. Enzyme dilutions were made fresh daily and stored on ice until assayed. A series of enzyme dilutions ranging from 0 to 5 U/ml of b-galactosidase (b-Gal) were then made using distilled water as the diluent, where one unit of enzyme will hydrolyze 1.0 mmole of ONPG to ONP and galactose per min. Absorbance determinations at 420 nm were made using a PowerWave 200 Scanning microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT) with the reader controlled by an external PC running KC4 data reduction software (Bio-Tek Instruments, Winooski, VT).

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Products from a b-galactosidase assay were scanned from 200 nm to 550 nm in 1 nm increments using a PowerWave 200 scanning microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT). Samples containing either distilled water, 1X reaction buffer, or 1X reaction buffer and 0.5 U/ml of b-galactosidase enzyme were scanned at 5, 30 and 60 minutes following the addition of reaction buffer. Data was collected using KC4 software (Bio-Tek Instruments, Winooski, VT) on a separate PC controlling reader function and data capture. Corrected data was generated by exporting the raw data from KC4 to Excel (Microsoft Redmond, WA), and subtracting the background absorbance at each wavelength of the substrate only mixture (B) for each time point. Results b-Galactosidase activity was investigated using several methods under a variety of conditions. A comparison of the spectral scans from 200 nm to 800 nm of the reaction products to the substrate demonstrates the generation of absorbance at wavelengths ranging from 350 nm to 500 nm (Figure 2). Although the wavelength with maximal absorbance was found to be 363 nm, it was found that the reaction substrate mix contributes to the absorbance in this wavelength region and therefore, higher wavelengths were used to ascertain enzyme activity. The wavelength 420 nm was determined to be close to maximal in terms of end-product absorbance, with little interference from the enzyme substrates (Figure 2). As demonstrated in Figure 3, when the background absorbance of the substrate reaction is subtracted from the products, although the peak maxima is at 400 nm, the absorbance at 420 nm returns values very close to maximal. Figure 3 also demonstrates that the absorbance increases with time. The small change in peak amplitude from 20 minutes to 60 minutes is the result of the exhaustion of ONPG substrate. This is further corroborated by data depicted in Figure 4, where increasing enzyme concentration does not necessarily result in a corresponding increase in absorbance. Endpoint absorbance determinations were made for b-galactosidase enzyme concentrations ranging from 0.0 to 1.0 U/ml (Figure 4). When endpoint reactions were measured 5 minutes after initiation of the reaction the response was virtually linear for all of the enzyme concentrations tested, reaching a maximal absorbance mean of 1.669. Increasing the reaction time of the assay to 10 minutes resulted in greater absorbance values for all enzyme concentrations. For example, the absorbance at 10 minutes for the 1 U/ml sample increased to 2.342. However, the endpoint response was no longer a straight line, thus necessitating the use of a 4-parameter logistic best fit of the data to describe the data.

A B

C

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Figure 2. Spectral scans of reaction products from a b-galactosidase assay. Reaction A, contained only distilled water; reaction B, contained reaction buffer, but distilled water was added instead of b-galactosidase enzyme to initiate the reaction; and reaction C, contained complete reaction mix (reaction buffer and 0.5 U/ml b-galactosidase enzyme). Spectral scans were made 20 minutes after the initiation of the reaction. Dashed line indicates the wavelength used for subsequent endpoint determinations.

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Figure 3. Spectral scans from 300 to 550 nm of reaction products at various times after the initiation of the reaction. Spectral scans of a b-galactosidase assay reaction at indicated times. Data was corrected by subtracting the absorbance of a reaction containing no enzyme at each wavelength for each time point. Similarly, endpoint determinations at 30 and 60 minutes result in concentration curves that plateau. Likewise, these curves can be described using a 4-parameter logistic best fit of the data. In terms of detecting small quantities of enzyme, this colorimetric assay can detect as little as 0.0005 U/ml b-gal enzyme activity with statistical significance (p < 0.0002), after a 60 minute incubation (data not shown). As expected, allowing the reaction to continue for a total of 120 minutes increases the detection limit. With a two hour incubation at ambient temperature, 0.0001 U/ml can be distinguished from the 0.0 U/ml blank (p < 0.004). When corrected for the volume of sample in each well, as little as 1 X 10-5 Units of enzyme per well can be distinguished from a reaction buffer-only blank.

Figure 4. b-Galactosidase concentration curves at different endpoint times. The absorbance at 420 nm for increasing concentrations of b-galactosidase were determined at 5, 10, 30, and 60 minutes. KC4 data reduction package was used to plot absorbance against enzyme concentration. Equations describing the concentration curve used a 4-parameter logistic best fit of the data. Discussion

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The ability to quantitate b-galactosidase activity is of particular importance in molecular biology. Because the LacZ gene is not normally present in mammalian cells, it can be used as a reporter for gene expression. Initial studies demonstrated that the hydrolysis of ONPG catalyzed by b-galactosidase results in a colored product with a broad peak in absorbance centered approximately at 400 nm. The utilization of absorbance at 420 nm, which is near the maxima, allows an accurate assessment of hydrolyzed product without any background absorbance from the non-hydrolyzed substrate. The small change in peak amplitude seen in Figure 3 between 20 minutes and 60 minutes is due to the depletion of ONPG substrate. Maintaining an appropriate relationship between the catalytic enzyme concentration, substrates, and incubation time is paramount in obtaining appropriate linearity in the assay. For example, the plateau of absorbance seen with either the 30 or 60 minute endpoint reactions at higher enzyme concentrations indicates that either shorter incubation times (e.g. 5 or 10 minutes) would provide superior linearity for the indicated enzyme concentrations. Similarly, lower enzyme concentrations could be used to maintain linearity for 30 or 60 minutes incubations. Increasing the ONPG substrate is limited by the solubility of the compound in the aqueous reaction buffer and by the microplate’s absorbance limits. The addition of 2M sodium carbonate has been demonstrated to increase the absorbance at 420 nm as well as stop the reaction from proceeding further. Increasing the pH above 8.0 results in the complete deprotonation of o-nitrophenol to the yellow compound o-nitrophenolate, as well as denaturing the b-galactosidase protein (2). This has the utility of allowing the samples to truly be endpoint reactions. While the addition of a stop solution is vital when the reactions are being read in cuvettes on a spectrophotometer, which is by its nature a slow process, the rapidity of the PowerWave precludes the need to stop the reaction. The addition of stop solutions would be expected to increase the absorbance signal if a significant portion of the end-product was in the protonated form, as would occur if the reaction was performed at a pH less than 6.0. The increase in signal when 2M sodium carbonate was added using the reaction buffer described in this report was approximately 10%. However, the addition of sodium carbonate does allow the plate to be read later without the reactions continuing to develop. In the past such b-gal determinations have been performed using a conventional spectrophotometer. This method usually entailed using a pair or at most a set of four matched cuvettes to perform the analysis resulting in a very low throughput. The ability to use the PowerWaveÔ 200 scanning microplate reader to perform this analysis allows this routine procedure to be performed on 96 samples in a matter of seconds leading to a tremendous increase in productivity and throughput. References (1) Sanbrook, J., E.F. Fritsch, and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2) Maloy, S.R., J.E. Cronan Jr. and D. Freifelder (1994), Microbial Genetics 2nd ed. Jones and Bartlett, Boston, MA. Paul G. Held Ph.D. Senior Scientist & Applications Lab Manager

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