Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays
BackgroundThe reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm.ResultsThe optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z’ factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei.ConclusionThe results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.
Background<!><!>Background<!>Materials<!>G6PD expression and purification<!>Optimization of enzymatic assay using WST-8<!>NADH and NADPH standard curve<!>Accuracy and precision determination<!>Enzyme activity assay<!>Determination of steady-state kinetic parameters for GDH and G6PD<!>Applications of the WST-8 assay<!>Spike recovery<!>Assay interference of WST-8/1-mPMS system<!>Enzyme activity assay<!>NADH and NADPH standard curve<!>Determination of accuracy and precision<!>Measurement of G6PD activity in biological samples<!>Statistical analysis<!><!>Accuracy and precision<!><!>Dehydrogenase activity assay<!><!>Discussion<!>Conclusions<!>
<p>Nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) are important biological molecules serving as cofactors in various enzymatic reactions essential for cellular metabolism, mitochondrial functions, protection against oxidative stress, signal transduction, and cell death [1–3]. Various methods are used to assay NAD(P)H [4–6]. Both NADH and NADPH can absorb light at 340 nm and have intrinsic fluorescence. Therefore, the activity of enzymes producing or consuming NAD(P)H (dehydrogenases and oxidoreductases) is commonly determined by measuring the absorbance of NAD(P)H at 340 nm, or monitoring fluorescence [7–9]. Though the detection of NAD(P)H absorption or fluorescence is useful for characterizing enzymes, occasionally, the method is not sufficiently sensitive and/or specific for measuring low concentrations of NAD(P)H. While electrochemical methods for detecting NAD(P)H, such as modified graphene and glassy carbon electrodes, are very attractive, their application in dehydrogenase activity assay remains limited [10, 11]. Electrochemical methods are expensive and require experienced laboratory technicians. In addition, particular substances could be absorbed on the electrode surface and interfere with the assay. Direct detection by oxidation of NADH usually results in a high potential at the platinum or carbon bare electrode. The oxidized form (NAD+) shows strong absorption on the electrode, causing fouling, and leading to reduced sensitivity, reproducibility, and stability [12]. Moreover, redox active species, such as ascorbic acid, uric acid, and glucose, reportedly interfere with the detection of NADH [13]. The bioluminescent assay is very sensitive for NAD(P)H detection but is quite expensive [14].</p><p>A colorimetric method using tetrazolium salts for the measurement of NAD(P)H concentration has been reported [15, 16]. Although tetrazolium salts have been used widely in biological and clinical studies, their application is limited by the low solubility of the formazan product [17–20]. Recently, several water-soluble tetrazolium salts (WST), which include WST-1, WST-3, WST-4, WST-5, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], and WST-9, have been synthesized [21, 22]. In the presence of an electron mediator, such as 1-methoxy-5-methylphenazium methylsulfate (1-mPMS), WST is readily reduced by NAD(P)H to produce a formazan product, which can be determined by monitoring absorbance in the range 430–550 nm. The absorbance of formazan is proportional to the NAD(P)H concentration, so that the WST-based colorimetric assay [21] shows potential for various qualitative and quantitative applications.</p><!><p>Schematic representation of the reaction for detection of dehydrogenase activity using colorimetric WST-8 assay</p><!><p>In this study, the WST-8 colorimetric method was applied to quantify the concentration of NAD(P)H and the enzymatic activity of NAD(P)+-dependent dehydrogenases. GDH and G6PD were studied as models to evaluate the performance of the WST-8-based enzymatic assay. The assay conditions, which include the effect of pH, the concentrations of WST-8, cofactor and substrate, and reaction-time course, were optimized. The optimized conditions were used to measure the dehydrogenase activity of purified enzymes, as well as dehydrogenase activity in biological samples. The efficiency of the method developed was compared with that of the conventional UV-spectrophotometric method, where absorbance at 340 nm of NAD(P)H was measured. Furthermore, the assay was applied in substrate screening of an uncharacterized short-chain dehydrogenase/oxidoreductase (SDR) from Burkholderia pseudomallei. It was demonstrated that the WST-8 assay is rapid and sensitive, and can be used for dehydrogenases high-throughput screening.</p><!><p>A Cell Counting kit-8 containing 5 mM WST-8 and 0.2 mM 1-mPMS was obtained from Sigma-Aldrich (St. Louis, MO, USA). NAD(P)+, NAD(P) H, D-(+)-glucose, and glucose-6-phosphate (G6P) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Merck (Darmstadt, Germany). Bacillus GDH was obtained from a GDH activity colorimetric assay kit (Biovision, Mountain View, USA).</p><!><p>The human G6PD enzyme was expressed and purified as previously described [32]. In brief, the enzyme was expressed in E. coli BL21 (DE3). The cells were grown at 37 °C until OD600 reached 0.8; protein expression was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG). The cells were grown at 20 °C for an additional 20 h and harvested by centrifugation. The cell pellet was resuspended in lysis buffer (20 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole) and lysed by sonication. After centrifugation at 20,000 x g for 1 h, the supernatant was incubated with cobalt TALON Metal Affinity Resin (BD Biosciences). Unbound proteins were removed by washing buffer (20 mM sodium phosphate pH 7.4, 300 mM NaCl, 20 mM imidazole) and G6PD protein eluted with increasing imidazole concentrations from 40 to 400 mM in 20 mM sodium phosphate pH 7.4, 300 mM NaCl. The G6PD protein was dialyzed against 20 mM Tris-HCl pH 8.0 containing 10% glycerol (v/v) and stored at − 20 °C. The purity of the protein was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the protein concentration was determined by Bradford assay [33].</p><!><p>The WST-8 assay was performed in a 96-well plate (Costar, Corning, NY, USA) with a total volume of 150 μL. The reaction conditions—buffer pH, incubation time, and the concentrations of WST-8, substrate and NAD(P)+—were optimized for determining GDH and G6PD activities. The assays were performed at 37 °C and 25 °C for GDH and G6PD, respectively. Absorbance at 450 nm was measured with the reference wavelength at 600 nm using a microplate reader (Sunrise, Tecan, Männedorf, Switzerland). Absorbance at 450 nm of a reaction mixture set up in the absence of substrate was used for background subtraction.</p><p>For the effect of pH, the activities of Bacillus GDH and human G6PD were determined in a buffer mixture containing 20 mM of each of the following buffers: MES [2-(N-morpholino) ethanesulfonic acid], MOPS [3-(N-morpholino) propanesulfonic acid], HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], Tris-HCl, and CAPSO [3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid], at pH range 6.0–9.5. Incubation time was varied from 0 to 120 min for GDH and 0 to 15 min for G6PD. The concentration of WST-8 was varied between 0 and 300 μM. For the GDH assay, the concentrations of glucose substrate and NAD+ were 0–500 mM and 0–1 mM, respectively. For the G6PD assay, the concentrations of the NADP+ substrate and G6P were varied from 0 to 400 μM and 0–1 mM, respectively.</p><!><p>The linear response curve for formazan absorbance (450 nm) at varying NAD(P)H concentrations was constructed. NADH and NADPH were treated with WST-8. The total volume of 150 μL contained 20 mM Tris-HCl (pH 9.0 for NADH and pH 8.0 for NADPH), 200 μM WST-8/8 μM 1-mPMS, and varying amounts of NAD(P)H (0–33 nmole). Formazan absorbance at 450 nm was plotted versus the amount of NAD(P)H and the linear range with a correlation coefficient > 0.99 was selected [34].</p><!><p>To determine the accuracy and precision, known NAD(P)H amounts within the linear range were measured on five consecutive days in triplicate. NADH amounts were 4.3, 8.5, and 12.8 nmole, and NADPH amounts were 4.3, 8.6, and 12.9 nmole. The accuracy of the assay was expressed as the percent relative error (% RE), where % RE = 100% × (measured amount - prepared amount)/prepared amount. Both within- and between-run precisions were assessed as percent coefficient of variation (%CV).</p><!><p>GDH and G6PD activities were determined according to the optimal conditions obtained. The assays were performed at 37 °C and 25 °C for GDH and G6PD, respectively. For the GDH assay, the standard reaction mixture contained 20 mM Tris-HCl pH 9.0, 250 mM glucose, 1 mM NAD+, 200 μM WST-8/8 μM 1-mPMS and 1 μg GDH enzyme (Biovision, Mountain View, USA). For the G6PD assay, the standard reaction mixture contained 20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 500 μM G6P, 200 μM NADP+, 200 μM tetrazolium salt and 0.1 μg G6PD enzyme. The amount of NAD(P)H generated was determined using the standard curve described above. Enzyme activity was expressed as nanomole of NAD(P)H produced per minute per microgram of protein (nmole/min/μg).</p><!><p>To further confirm the performance and accuracy of the dehydrogenase assay using WST-8, the kinetic parameters for GDH and G6PD were determined and compared with those obtained from the UV-spectrophotometric standard method, which measures the absorbance of NAD(P)H at 340 nm [32, 35–37]. The assays were performed at 37 °C and 25 °C for GDH and G6PD, respectively. For the GDH enzyme, to determine the KM for glucose, the assay was performed by fixing the concentration of NAD+ at 1 mM and varying the concentrations of glucose from 0 to 500 mM, while the KM for NAD+ was determined by fixing the concentration of glucose at 250 mM and varying the concentrations of NAD+ from 0 to 1 mM. For the G6PD enzyme, to determine the KM for the G6P substrate, the G6P concentration was varied from 0 to 500 μM, while fixing the concentration of NADP+ at 200 μM. To determine KM for NADP+, the concentration of NADP+ was varied from 0 to 200 μM, while fixing the concentration of G6P at 500 μM. The rate of NAD(P)H product formation was calculated and expressed as micromolar NAD(P)H produced per minute (μM/min). The kinetic parameters were determined by fitting the data to the Michaelis–Menten equation using GraphPad Prism (GraphPad Software).</p><!><p>The WST-8 assay was applied to measure the dehydrogenase activity of the biological samples. G6PD activity in E. coli crude extract was measured at 25 °C. Crude extracts (20 μg) of E. coli harboring empty pET28a and recombinant pET28a-G6PD plasmids were assayed in the standard reaction mixture. The absorbance obtained from E. coli crude extracts harboring recombinant pET28a-G6PD plasmid was subtracted with that of E. coli crude extract harboring empty pET28a plasmid. Enzyme activity was determined using NADPH standard curve and was expressed as nanomole of NADPH produced per minute per microgram of protein (nmole/min/μg).</p><p>The WST-8 assay was also used to screen for the substrate of an uncharacterized SDR from B. pseudomallei. Crude lysate (150 μg) of E. coli expressing recombinant SDR was screened for dehydrogenase activity using various substrates, including sugars, alcohols, and aldehydes. Reactions were performed at 37 °C in the standard reaction mixtures containing 20 mM Tris-HCl pH 8.0, 200 μM tetrazolium salt, 500 μM NAD(P)+ and substrate (concentration ranges from 50 mM to 250 mM). The enzyme activity of E. coli lysate expressing recombinant SDR was subtracted with E. coli lysate without SDR. The activity of SDR towards different substrates was expressed as nanomolar of NAD(P)H produced per minute (nM/min).</p><!><p>One microgram of GDH or 0.2 μg of G6PD was spiked into 1.5 μL of fetal bovine serum (FBS). The dehydrogenase activity of the spiked samples was monitored at 450 nm in triplicate. The rate of NAD(P)H production measured from the spiked samples was subtracted with that from the FBS. The %recovery = 100% x (measured rate/expected rate).</p><!><p>To determine assay interference in the absence of NAD(P)H, 150 μL reaction mixtures containing 20 mM Tris-HCl pH 8.0 and 200 μM WST-8 were mixed with various chemicals, including mono- and di-saccharides (250 mM), phosphorylated sugars (1 mM), alcohols (100 mM), aldehydes (50 mM), NAD(P)+ (1 mM), detergents (0–1%) and reducing agents (0–10 mM). Absorbance at 450 nm was followed at 37 °C to evaluate assay interference.</p><!><p>To validate the performance of the WST-8 assay, the conventional UV-spectrophotometric method (measurement of NAD(P)H absorption at 340 nm) was used as the standard method for dehydrogenase assay. The assays were performed at 37 °C and 25 °C for GDH and G6PD, respectively. For the GDH assay, the 1 mL reaction mixture contained 20 mM Tris-HCl pH 9.0, 250 mM glucose, 1 mM NAD+, and 5 μg GDH enzyme. For the G6PD assay, the reaction mixture contained 20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 500 μM G6P, 200 μM NADP+, and 0.5 μg G6PD enzyme. The reaction was initiated with the addition of the enzyme and the rate of reaction was monitored at 340 nm for 30 and 10 min for GDH and G6PD, respectively. Blank reactions were carried out in the absence of glucose substrate. The reactions were monitored using a UV-2700 UV-VIS spectrophotometer (Shimadzu). The amount of NAD(P)H generated was calculated using molar extinction coefficient 6220 M− 1 cm− 1 for NAD(P)H. Enzyme activity was expressed as nanomole of NAD(P)H produced per minute per microgram of protein (nmole/min/μg).</p><!><p>To determine the limit of detection of the conventional UV-spectrophotometric method, the NAD(P)H standard curve was constructed. The total volume of 1 mL contained 20 mM Tris-HCl (pH 9.0 for NADH and 8.0 for NADPH), and various amounts of NAD(P)H (0–860 nmole). Absorbance at 340 nm was monitored.</p><!><p>The amounts of NADH were 102, 212.5, and 340 nmole; and the amounts of NADPH were 103.2, 215, and 344 nmole. The within- and between-run accuracy and precision of the assay were calculated as described above.</p><!><p>G6PD activity in E. coli crude extract was determined by assaying crude extract (100 μg) of E. coli harboring recombinant pET28a-G6PD plasmid with the reaction mixture mentioned above. Enzyme activity was determined using molar extinction coefficient 6220 M− 1 cm− 1 and was expressed as nanomole of NADPH produced per minute per microgram of protein (nmole/min/μg).</p><!><p>All experiments were performed in triplicate. All analyses were performed with GraphPad Prism software and the results presented as mean ± standard deviation.</p><!><p>Optimization of GDH assay conditions using colorimetric WST-8. a The effect of pH on enzymatic reaction. b Reaction time course of the assay. c The effect of WST-8/1-mPMS on the absorbance. d The effect of NAD+ concentration on the enzymatic assay. e The effect of glucose concentration on the enzymatic assay</p><p>Optimization of G6PD assay conditions using colorimetric WST-8. a The effect of pH on enzymatic reaction. b Reaction time course of the assay. c The effect of NADP+ concentration on the enzymatic assay. d The effect of G6P concentration on the enzymatic assay</p><p>NADH and NADPH standard curves. a NADH was measured using colorimetric WST-8. b NADPH was measured using colorimetric WST-8. c NADH was measured at 340 nm using UV-spectrophotometry. d NADPH was measured at 340 nm using UV-spectrophotometry. Insets demonstrate assay linearity over the range of concentrations 0 to 19 nmole of NAD(P) measured by colorimetric WST-8 assay and 0 to 400 nmole of NAD(P)H measured by UV-spectrophotometry</p><p>The limit of NAD(P)H detection of colorimetric WST-8 and UV spectrophotometric methods</p><p>The relationship between absorbance and NAD(P)H concentration. NAD(P)H was measured using colorimetric WST-8 at 450 nm (dashed line) and UV-spectrophotometry at 340 nm (solid line). Measurement of NAD(P)H at 340 nm using UV-spectrophotometry was performed in a 1 mL cuvette or b 96-well plate</p><p>Accuracy and precision for measurement of NAD(P)H by colorimetric WST-8 and UV spectrophotometry methods</p><!><p>Furthermore, the robustness of colorimetric WST-8 for high-throughput screening was evaluated by determining the Z' factor. The means and standard deviations of the negative control were used to calculate the Z' factor according to the equation developed by Zhang and colleagues [42]. Z' factor is defined as Z' = 1 – [(3σpos + 3σneg)/|μpos - μneg|]. It is calculated based on the scale of the assay and the distribution of the positive and negative signals, and has a value between 0 and 1. An assay with a wide separation, i.e. close to 1, would be a good assay. WST-8 exhibited excellent reproducibility, with Z' values ranging between 0.9 to 0.99 for NAD(P)H measurement, which are comparable to UV-spectrophotometry (0.96–0.98). The Z' factor of WST-8 was greater than the analytical acceptance criteria (0.7), indicating that the WST-8 assay is suitable for high-throughput screening.</p><!><p>The effect of various chemicals on the reduction of WST-8. Background absorbance in the absence of NAD(P)H of a sugars, b alcohols, c aldehydes, d NAD(P)+, e detergents, f reducing agents and g dose-response curve of reducing agents</p><p>GDH and G6PD activities measured by colorimetric WST-8 and UV spectrophotometry methods</p><!><p>The performance of WST-8 for measuring dehydrogenase activity in biological samples was assessed using E. coli crude extract. The recombinant human G6PD activity of 0.112 ± 0.001 nmole/min/μg was obtained, comparable to that of UV-spectrophotometry, 0.145 ± 0.003 nmole/min/μg. This indicates that WST-8 is a sensitive assay for the detection of dehydrogenase enzyme activity. It also suggests that the colorimetric WST-8 assay can measure dehydrogenase activity in biological samples that usually contain several other proteins and enzymes.</p><!><p>Spike recoveries of GDH and G6PD in FBS using colorimetric WST-8 assay</p><p>Steady-state kinetics assay determined by colorimetric WST-8 method. Enzyme kinetic plots of GDH for a glucose and b NAD+. Enzyme kinetic plots of G6PD for c G6P and d NADP+</p><p>Steady-state kinetic parameters of GDH and G6PD determined by colorimetric WST-8 assay compared with previous studies using UV-spectrophotometry</p><p>Substrate screening of crude extract of E. coli harboring SDR from B. pseudomallei</p><p>na no activity</p><!><p>In this study, we described the application of tetrazolium salt WST-8 to determine dehydrogenase activity using GDH and G6PD as model enzymes. The reaction catalyzed by GDH generates NADH, while that catalyzed by G6PD produces NADPH. In the presence of 1-mPMS, an electron mediator, NAD(P)H reacts with WST-8 to yield a formazan product, which can be determined by measuring absorbance at 450 nm. The assay is applicable to the quantitative measurement of NAD(P)H produced from dehydrogenase reactions. Moreover, the WST-8 assay developed was applied to screening the substrate of an uncharacterized SDR from B. pseudomallei. The results indicated that the colorimetric WST-8 method has several advantages for measuring NAD(P)H and dehydrogenase activity. The method shows great sensitivity in the detection of NAD(P)H, where as low as 0.3 nmole can be detected. It is 5-fold more sensitive than conventional UV-spectrophotometry, which measures NAD(P)H absorption at 340 nm. The greater sensitivity of the WST-8 assay when compared to UV-spectrophotometry can be explained by a higher extinction coefficient of formazan product (30,700 M− 1·cm− 1), which is about 5-fold greater than that of NAD(P)H (6,220 M− 1·cm− 1) [21]. Although UV-spectrophotometry is able to measure small amounts of NAD(P)H accurately (i.e. as low as 1.7 nmole), the technique is slow and is, therefore, not suitable for high-throughput dehydrogenase-activity screening. On the other hand, the colorimetric WST-8 assay can be performed in a 96-well plate with a final volume of 150 μl and the volume can further be minimized for the purposes of economy. Though NAD(P)H absorption at 340 nm can also be measured in a 96-well plate, the lower extinction coefficient of NAD(P)H and small reaction volume usually give low absorbance. This makes conventional UV-spectrometry less sensitive than colorimetric WST-8 assay.</p><p>It is worth mentioning that NAD(P)H detection method could be chosen according to the quantity of the NAD(P)H. Although WST-8 is highly sensitive and uses a low detection amount of NAD(P)H, its linear range is much smaller than other NAD(P)H detection methods. Therefore, serial dilutions are necessary when a large amount of NAD(P)H is present, to ensure that the detection value is within the linearity range of the WST-8 method (0.4–19 nmole). In contrast, the UV-spectrophotometric method is more appropriate to measure a high amount of NAD(P)H (i.e. > 1.72 nmole). The physiological concentration of NAD(P)H varies among cell types. The concentration of NADH is approximately 100 μM (15 nmole in 150 μL reaction) and 60 μM (10 nmole in 150 μL reaction) in breast and brain tissues, respectively [48, 49]. In bacterial cells, the NADH concentration is 4–11.7 μmole/g [50]. The WST-8 method could be used to measure the amount of NAD(P)H in all of these biological samples, because the concentrations present in the samples are within the detection limit of the assay.</p><p>Dehydrogenase activity measurement using colorimetric WST-8 is an end-point assay and could be more time-consuming than conventional UV-spectrophotometry, because the WST-8 assay usually requires a long incubation time. However, the required incubation time in the WST-8 assay depends upon enzyme activity. In this study, we monitored GDH activity at 60 min and G6PD activity at 10 min to ensure maximum sensitivity. In fact, the enzyme activity of GDH and G6PD can be determined using a shorter incubation time; any time points that are in the linear absorbance range can be used. Nevertheless, the shortest incubation time should give measurable absorbance and this value should be greater than the detection limit of a spectrophotometer. Our results indicate that dehydrogenase activity can be measured at 5 min for GDH and 1 min for G6PD, which is similar to those monitored by UV-spectrophotometry.</p><p>In terms of accuracy, precision and reproducibility, the performance of the colorimetric WST-8 assay is indistinguishable from UV-spectrometric method that measures the absorbance of NAD(P)H at 340 nm. The WST-8 method shows high accuracy and precision for measuring NAD(P)H. For accuracy, the method exhibited %RE of 0.7–0.25. For precision, %CV for within-run and between-run ranged between 0.6–4.5. These values were very small and also within the statistical acceptance criteria of 15%. Excellent reproducibility, with Z' values of 0.9–0.99, was observed for the WST-8/1-mPMS detection system. Altogether, this suggests that the WST-8 assay is suitable for high-throughput screening in clinical and research applications. In addition, it was demonstrated here that the WST-8 assay is effective for measuring the dehydrogenase activities of purified enzymes as well as those of biological samples. Dehydrogenase activity measured using WST-8 was comparable to that obtained from UV-spectrophotometry. Though enzyme activity measured by WST-8 was 10–25% lower than that measured by UV spectrophotometry, it was not statistically significant by Mann-Whitney analysis. p-values of 0.0765, 0.1 and 0.0765 were obtained for activity measurement of purified GDH, purified G6PD, and crude G6PD assay, respectively. This indicates that the WST-8 method is accurate and also compatible with dehydrogenase activity measurement in the presence of other contaminated proteins. Even though WST-8 is quite stable and is not light-sensitive, the reagent should be kept in the dark and exposure to the air avoided, to minimize any decrease in its activity. More importantly, the developed method was further applied for substrate screening of an uncharacterized SDR from B. pseudomallei, in which the frequently used UV-spectrophotometric method is not precisely responsive. In fact, no dehydrogenase activity was observed by direct NAD(P)H absorbance measurement. On the other hand, dehydrogenase activity was detected for glucose, xylose, ethanol, propanol, butanol, hexanol, and octanol, when assayed using the WST-8 method. This indicates that the WST-8 based assay is essential for measuring low concentrations of NAD(P)H. The WST-8 assay is sensitive and able to measure the dehydrogenase activities of uncharacterized SDR in bacterial crude lysate. Work is currently underway to investigate the true function of uncharacterized SDR from B. pseudomallei.</p><p>It was reported that the UV-spectrophotometric method was not adequately sensitive to detect hydroxysteroid dehydrogenase activity, since it is often present at low levels, especially in biological samples. Therefore, a greater sensitivity (2–3 fold) nitroblue tetrazolium salt was used to detect activity in steroidogenic tissue extracts [51]. In addition to sensitivity, direct measurement of NAD(P)H at 340 nm from lysate samples usually results in low absorbance, of around 0.04–0.05, leading to high noise/signal ratio in the lysate assay [52].</p><p>Compared with other qualitative and quantitative dehydrogenase kits that must be stored at − 20 °C and are stable for only 2–3 months, the WST-8 solution is highly stable and can be stored at 4 °C for up to 1 year (according to the manufacturer). Though measurement using WST-8 assay requires reagent preparation, it is easy and the cost per reaction is much lower ($0.35) than commercial dehydrogenase assay kits ($3.50). According to the results shown here, monosaccharide and disaccharide sugars, alcohols, aldehydes and detergents did not interfere with the current developed method. However, it should be cautioned that the WST-8/1-mPMS detection system is not compatible with a reducing agent in buffer systems, as this can generate false-positive signals. In addition, caution should be taken when working with ketonic monosaccharides, such as fructose. On the other hand, some chemicals may interfere with the measurement of NAD(P)H at 340 nm, including bilirubin and several inorganic compounds, such as phosphorus, nickel, and chromium [53]. Furthermore, hemoglobin also interferes with the spectrophotometric detection of NAD(P)H at 340 nm, causing a shift in the UV spectra [54]. Both bilirubin and hemoglobin are endogenous substances in many biological samples. Therefore, it should be taken into consideration when using the UV method to measure dehydrogenase in biological samples directly.</p><!><p>WST-8 is a sensitive assay for the measurement of NAD(P)H and dehydrogenase activity. The method is specific for NAD(P)H and measurement of dehydrogenase activity. In addition to sensitivity, the lower cost per reaction, high stability and relative ease of preparation and long-term storage life are other advantages of the WST-8 assay. The assay could serve as a high-throughput method for measuring dehydrogenase activity.</p><!><p>Percent coefficient of variation</p><p>Percent relative error</p><p>1-methoxy-5-methylphenazium methylsulfate</p><p>β-mercaptoethanol</p><p>Dithiothreitol</p><p>Glucose-6-phosphate</p><p>Glucose-6-phosphate dehydrogenase</p><p>Glucose dehydrogenase</p><p>Isopropyl β-D-thiogalactoside</p><p>Lower limit of quantitation</p><p>Limit of detection</p><p>Oxidized form of nicotinamide adenine dinucleotide</p><p>Reduced form of nicotinamide adenine dinucleotide</p><p>Reduced form of nicotinamide adenine dinucleotide phosphate</p><p>Oxidized form of nicotinamide adenine dinucleotide phosphate</p><p>Short-chain dehydrogenase/oxidoreductase</p><p>Sodium dodecyl sulfate-polyacrylamide gel electrophoresis</p><p>Upper limit of quantitation</p><p>Water-soluble tetrazolium salts</p>