PowerWaveX Select and KC4 : A Multifunctional System for Today s Laboratory Environment

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Morgan Norris
6 years ago
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The recent introduction of 384-well plates offers four times the throughput of conventional 96-well microplates.

1 PowerWaveX Select and KC4 : A Multifunctional System for Today s Laboratory Environment Figure 1. PowerWaveX Select Microplate Spectrophotometer Introduction With today's requirements for high throughput, microplate instrumentation has become more commonly used in the laboratory. The recent introduction of 384-well plates offers four times the throughput of conventional 96-well microplates. However, they also present many challenges to providing the same degree of functionality that has become standard with 96-well plates. Microplate readers determine the absorbance of solutions in microplates following the completion of a colorimetric assay. Alternatively, kinetic assays can be monitored over a period of time using sequential periodic measurements but, in order to maintain appropriate assay conditions, temperature regulation must be provided. Regardless of the reading mode, the rapid and accurate determination of absorbance measurements is most desirable. The role of the data reduction software is to control reader functions (such as wavelength(s) selection for endpoint determinations, reading interval for kinetic reactions and wavelength range increment for spectral scanning), perform data reduction, and store the data on a PC's hard drive or network. To meet these needs as well as others, BioTek Instruments has developed the PowerWaveX Select Scanning Microplate Spectrophotometer and KC4 v3.0 with PowerReports data reduction software for the reading and data analysis of both 96-well and 384-well microplates. Enzyme kinetics is an area where microplate readers with high throughput capabilities, in conjunction with powerful data reduction software, can provide high value to the investigator. Most biological processes require an enzyme to act as a catalyst in order for the reaction to take place. With a few exceptions, most reactions catalyzed by enzymes can be described BioTek Instruments, Inc., P.O. Box 998, Highland Park, Winooski, Vermont USA COPYRIGHT 2006 TEL: FAX: Outside the USA: customercare@biotek.com

satisfactorily by the Michaelis-Menten Equation (Eq. 1), where v = reaction rate, [S] = substrate concentration, Vmax = maximal velocity, and Km is the Michaelis constant.

2 satisfactorily by the Michaelis-Menten Equation (Eq. 1), where v = reaction rate, [S] = substrate concentration, Vmax = maximal velocity, and Km is the Michaelis constant. Each enzyme has physical characteristics in regard to substrate specificity, reaction velocity or required cofactors that affect the Michaelis-Menten constants. The determination of these constants involves the performance of kinetic enzymatic activity measurements. Eq. 1 Because Vmax is frequently difficult to determine experimentally, the Michaelis-Menten equation has been mathematically transformed in order to make estimations of Michaelis-Menten values. These transformations include the Eadie-Hofstee and the Lineweaver-Burk plots. The Eadie- Hofstee transformation plots velocity (v) against the velocity divided by the substrate concentration (v/[s]), while the Lineweaver-Burk or double reciprocal transformation plots the reciprocal of velocity (1/v) against the reciprocal of substrate concentration (1/[S]). These types of plots, which generate linear regressions, allow the investigator to more easily determine Km and Vmax. The PowerWaveX Select Microplate Spectrophotometer is a multichannel absorbance plate reader that has the capability of measuring absorbance from the UV to the near IR range in 96- and 384-well microplates (Figure 1 on page 1). This monochromator-based instrument, which has a wavelength range of nm, requires no absorbance filters and can perform spectral scans of substances in increments as small as 1 nm. Powered by a xenon-flash lamp, the optics of the PowerWaveX Select is depicted in Figure 2. Elevated temperatures are regulated by a four-zone natural convection design that ensures superior temperature uniformity. By utilizing the near IR capability of the instrument, pathlength can be corrected automatically in liquid samples. This allows for the direct quantitation of unknown sample concentrations from absorbance measurements using extinction coefficients of the analyte rather than a standard curve. The reader can read 96- and 384-well microplates with an absorbance range of 0-4 OD. Additionally, with a compact footprint and RS232 connection, the PowerWaveX Select is compatible with many of the commonly preferred robotics systems. Figure 2. Schematic Diagram of the PowerWaveX Select Optics. Xenon-Flash lamp is used to illuminate a high-precision diffraction-grating monochromator. Monochromatic light is then split into 8 experimental and 1 reference channels. The experimental channels are then focused onto the microplate well samples in a staggered array pattern, which eliminates crosstalk. Unabsorbed light is then focused onto silicon diode detectors.

3 KC4 v3.0 with PowerReports, the latest version of KC4, has added several new features to the already powerful KC4 program. Using true Object Linking and Embedding (OLE) technology, KC4 with PowerReports can interact directly with Microsoft Excel and Microsoft Word programs, providing total control over all report formatting using data objects created by KC4. This allows the user to create custom, publication-quality reports of microplate applications. In addition to the reporting features, the latest version of KC4 offers real-time monitoring of specific wells during kinetic assays, as well as enhanced well-area scanning functionality. Here we utilize several different absorbance-based methods to demonstrate the capabilities of the PowerWaveX Select in conjunction with KC4 version 3.0 to perform routine analysis of enzyme kinetics in microplates. Materials and Methods o-nitrophenyl ß-D-galactopyranoside (ONPG), catalogue number N-8431, and phenylethyl ß-Dthiogalactopyranoside (PETG), catalogue number P-1692, were purchased from Molecular Probes (Eugene, Oregon). The 96-well microplates, catalogue number 3635, were purchased from Costar (Cambridge, Massachusetts). ß-galactosidase enzyme (cat. # G-6008), sodium phosphate, magnesium chloride, potassium dichromate, and 2-mercaptoethanol were obtained from Sigma Chemical Company (St. Louis, Missouri). Purified herring sperm DNA, catalogue number D1816, was procured from Promega (Madison, WI). Several series of dilutions of purified herring sperm DNA were made using TE buffer (10 mm Tris, 1 mm EDTA ph 8.0) as the diluent. Following dilution, 100-µl aliquots were pipetted into microplate wells and absorbance measurements were made using a PowerWaveX Select. Samples were blanked using the mean absorbance of water blanks in the same microplate. Determination of the concentration for unknown samples in microplates was carried out by measuring absorbance at 260 nm, followed by correction for pathlength. The ß-galactosidase assay was performed as follows. Briefly, 100 µl of sample or standard in distilled water were placed in wells of a 96-well microplate. The assay was initiated by the addition of 100 µl of 2X assay buffer. Assay buffer (1X) consists of 100 mm sodium phosphate, ph 7.0; 1 mm MgCl2; 50 mm ß-mercaptoethanol; and mg/ml ONPG in distilled water. Assay buffer was prepared previously as a 2X stock solution and stored frozen at -20 C. Lyophilized ß-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 ß-galactosidase (ß-gal) was then made using distilled water as the diluent. All absorbance determinations were made at 420 nm using a PowerWaveX Select Scanning Microplate Spectrophotometer with the reader controlled by an external PC running KC4 data reduction software. After the assay was initiated by the addition of the 2X assay buffer, kinetic readings commenced immediately with absorbance determinations made every 30 seconds for a total of 60 minutes at ambient temperature.

4 Figure 3. Pathlength Correction in 384-well Microplates. Volumes ranging from 10 to 100 µl of DNA samples (100 µg/ml) were aliquoted into 384-well microplates in triplicate. The absorbance at 260 nm was determined with and without pathlength correction and the average absorbance was plotted against sample volume. Table 1. Sample Number Expected Conc. (mg/ml) Observed Conc. (mg/ml) ± ± ± ± ± ± ± ± ± ±0.04 Table 1. Comparison of Expected and Experimentally Determined DNA Concentrations. Serial Dilutions of DNA were made using distilled water as the diluent. Aliquotes (100 µl) of each dilution were placed in a 384-well microplate in triplicate and the absorbance measured at 260 nm. Absorbance values were blanked with a water-only control, corrected to a 1-cm pathlength, and the DNA concentration calculated using the conversion factor of 1 OD is equivalent to a 50-µg/ml solution. Observed concentrations are represented by the mean of the three replicates ± the standard deviation of the means. Potassium dichromate solutions (1 mm) were prepared at the required ph levels in 500 mm Tris buffer. For each ph level tested, 100-µl aliquots were pipetted in replicates of eight into Costar 3535 UV-transparent microplates. Buffer-only blanks for each ph tested were also included. Spectral scans from 200 nm to 500 nm in 1-nm increments were performed using a PowerWavex Select controlled by KC4 running on an external PC. The average absorbance at each wavelength of eight determinations was calculated and the buffer blank subtracted at each corresponding wavelength.

5 Figure 4. DNA Concentration Curve. Concentration curve from 0 to 104 µg/ml with a sample volume of 100 µl and subtraction of the water blank. Solid line depicts the result of linear regression analysis of the data, while filled circles represent the mean values of four determinations at each concentration. Linear regression analysis was performed using KC4 data reduction software. Results The PowerWaveX Select Scanning Microplate Spectrophotometer has a number of capabilities that make it suitable for today's molecular biologist. Figure 3 demonstrates the ability of the PowerWaveX Select to correct for sample pathlength. When the absorbance of various volumes of a DNA solution is measured, one observes a direct correlation between sample volume and absorbance. With increased sample volume the well of the microplate is filled to a greater extent, resulting in a greater light absorption pathlength. However, when the pathlength is corrected to 1 cm for all samples, the resultant "corrected" absorbance for each sample is very close to the same value. The corrected absorbance values can then be used in conjunction with reported extinction coefficients to directly calculate sample concentrations. Note that without pathlength correction, calculations based on extinction coefficients will result in aberrant results. The data presented in Table 1 demonstrate the ability of the PowerWaveX Select to determine nucleic acid concentrations. When serial dilutions of DNA with a known concentration are measured using absorbance and the resultant concentration compared to the expected result, very close correlations are observed (Table 1). These data suggest that the pathlength correction provided by the PowerWaveX Select is sufficient to make accurate direct concentration determinations from microplate absorbance measurements. Using KC4 data reduction software to perform data reduction, several different curve fits can be used to best fit the data. As seen in Figure 4, when the absorbance at 260 nm of a serial dilution of DNA is measured, a linear regression analysis can be used to best describe the data. These data also demonstrate the linearity of the optics of the PowerWaveX Select microplate reader. As demonstrated in Figure 5, spectral scans can be performed using the PowerWaveX Select. Potassium dichromate demonstrates a remarkable change in absorbance spectrum with a change in ph from 2 to 10. In an environment with a basic ph (i.e., ph 10), potassium dichromate exhibits peaks in absorbance at 273 nm and 372 nm not seen in an acidic environment. The absorbance of potassium dichromate did not change with ph at wavelengths above 500 nm (data not shown). Kinetic analysis can be performed using the PowerWaveX Select and KC4. Figure 6 demonstrates the effect of PETG on the ß-galactosidase enzyme activity.

Figure 5. Spectral Scan of potassium dichromate (1 mm) in aqueous solution at different ph levels. Spectral scans of 1 mm potassium dichromate at ph 2.0 or 10.

6 Figure 5. Spectral Scan of potassium dichromate (1 mm) in aqueous solution at different ph levels. Spectral scans of 1 mm potassium dichromate at ph 2.0 or 10.0 from 200 to 500 nm in 1-nm increments were performed using a PowerWaveX Select Scanning microplate spectrophotometer. Kinetic measurements of ß-galactosidase reactions containing constant amounts of enzyme and ONPG substrate were made in the presence of increasing amounts of the non-hydrolysable compound PETG. Increasing the amount of PETG resulted in a sigmoidal decrease in the Vmax values for ß-galactosidase (Figure 6). Inhibitor concentrations above 1000 µg/ml result in virtually no enzyme activity, while concentrations below 0.15 µg/ml demonstrate very little inhibition. Using KC4 data reduction software to control reader function and perform data reduction, several different curve fits can be used to best fit the data. In this example, the 4- parameter logistic fit best describes these data. Figure 6. Effect of PETG inhibitor concentration on ß-galactosidase Vmax. Increasing concentrations of PETG were added to samples containing 0.25 U/ml ß-galactosidase enzyme and the kinetic readings at 420 nm were taken every 30 seconds. Vmax values were calculated for each inhibitor concentration and plotted using a 4-parameter logistic fit. Note that the ordinate axis (PETG concentration) is expressed in a log scale. Kinetic analysis can also be used to calculate Michaelis-Menten constants, such as Km and Vmax. Using KC4 with PowerReports to perform the necessary transformations in Microsoft Excel, Lineweaver-Burk plots were generated where inhibitor and substrate concentrations were varied. When no PETG inhibitor was present, Vmax, which can be calculated from the reciprocal of the y-intercept using a Lineweaver-Burk plot, was determined to be mod/min (Figure 7). When similar Lineweaver-Burk plots are made with various concentrations of PETG present, regression lines with different slopes are observed, suggesting that the inhibitor influences the Km of the reaction. The linear regressions all have relatively the same y-intercept point

indicating that all of the reactions have the same Vmax. When the Vmax values are calculated using KC4 from the y-intercepts of the plot values of 29.98, 26.05, and 23.

7 indicating that all of the reactions have the same Vmax. When the Vmax values are calculated using KC4 from the y-intercepts of the plot values of 29.98, 26.05, and mod/min are obtained for samples containing 0.167, 1.67, and 16.7 mm PETG, respectively. Figure 7. Lineweaver-Burk plot of b-galactosidase activity in the presence of PETG inhibitor. Reciprocals of experimentally determined reaction velocity (1/v) were plotted against the reciprocal of substrate concentration (1/[S]) and linear regression trend analysis performed. Indicated legend represents experiments with 0 mm, mm, 1.67 mm and 16.7 mm PETG, respectively. Discussion The PowerWaveX Select is a monochromator-based microplate reader, which allows the user to select any wavelength from 200 nm to 999 nm for absorbance determinations. In addition, the PowerWaveX Select has pathlength conversion technology that allows for the correction of absorbance values to a 1-cm pathlength. Corrected absorbance values would be expected to directly compare to values obtained in conventional spectrophotometers. Pathlength correction also provides for the direct quantitation of nucleic acids by measuring absorbance at 260 nm or assessing nucleic acid purity by A260/A280 ratio determination. The use of a monochromator rather than filters for wavelength selections allows the microplate reader to be used as a true spectrophotometer. While the example of measuring potassium dichromate at different ph levels using the scanning function of the PowerWaveX Select scanning microplate spectrophotometer is relatively simplistic, it does demonstrate the utility of the function. This feature is most commonly used for the determination of peak absorbance values of colorimetric determinations; however, fluorescent compounds could also be scanned to find a region, preferably a peak, in the absorption spectra that does not overlap the fluorescent emission spectrum. Spectral scans can be used as a means to check for purity of samples. Additionally, using the KC4 data reduction package to capture the spectral scan data has several features not presented with these examples. The KC4 data reduction software allows the user to automatically determine maximum and minimum values, along with their corresponding wavelengths. After scanning, the wavelength range to be examined can also be narrowed to provide information concerning minor peaks. When analyzing enzyme kinetics, the Michaelis-Menton constants, Vmax and Km, can be determined in several different ways. Prior to the advent of computers, several methods were developed to determine these constants using linear regression because of the difficulty of estimating the Vmax. Two such examples are the Eadie-Hofstee transformation and Lineweaver-Burk plots. By extrapolating the experimentally determined linear regression plots, Vmax and Km can be estimated from intercept points and the slope. Similar results were obtained for Vmax regardless of the method.

8 These data presented also indicate that PETG is a competitive rather than a non-competitive inhibitor of ß-galactosidase. Measurements of the reaction rates at different concentrations of substrate and inhibitor serve to distinguish between competitive and noncompetitive inhibition. Competitive inhibitors compete for enzyme binding sites and a large excess of substrate to inhibitor will result in reaction rates similar to samples that do not contain inhibitor. This would result in Vmax values that are equivalent to that of enzyme without inhibitors present. The discrepancy between the calculated Vmax for the 1.67 mm PETG samples and controls (0 mm PETG) is the result of ONPG not being in sufficient excess. Noncompetitive inhibitors would show different Vmax values regardless of the substrate concentration (3). PETG also has a chemical structure very similar to ONPG, suggesting that it will bind to the active binding site of the ß-galactosidase enzyme. In the past, kinetic determinations have been performed using a conventional spectrophotometer, requiring the use of matched cuvettes to perform the analysis, resulting in a very low throughput. The ability to use the PowerWaveX Select scanning microplate reader to perform this analysis allows this routine procedure to be performed on 96 or 384 samples in a matter of seconds leading to a tremendous increase in productivity and throughput. These data presented in this report also demonstrate the utility of KC4 data reduction software to perform routine enzyme kinetic studies. Automatic determination of values such as Vmax and Km allows the end user flexibility in regard to data reduction of kinetic assays. In this monograph, we have provided several examples of the different capabilities of the PowerWaveX Select in conjunction with KC4 with PowerReports data reduction software. Because the instrument and the software package have been designed for flexibility as well as high throughput, several distinctly different examples have been shown. The already powerful KC4 data reduction package has been vastly enhanced by the addition of PowerReports. This unique feature allows all of the reader control and data reduction capabilities of KC4 to be combined with the printing and page layout features present in Microsoft Word and Microsoft Excel. Using PowerReports technology, the investigator can combine the throughput advantages of microplates to the specific data reduction already being performed manually into an automatically generated report. Paul G. Held, Ph.D. Senior Scientist & Applications Lab Manager Rev. 1/16/01

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