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).
G6PD expression and purification
The human G6PD enzyme was expressed and purified as previously described . 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 .
Optimization of enzymatic assay using WST-8
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.
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.
NADH and NADPH standard curve
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 .
Accuracy and precision determination
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).
Enzyme activity assay
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).
Determination of steady-state kinetic parameters for GDH and G6PD
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, 35,36,37]. 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).
Applications of the WST-8 assay
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).
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).
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).
Assay interference of WST-8/1-mPMS system
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.
Enzyme activity assay
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).
NADH and NADPH standard curve
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.
Determination of accuracy and precision
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.
Measurement of G6PD activity in biological samples
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).
All experiments were performed in triplicate. All analyses were performed with GraphPad Prism software and the results presented as mean ± standard deviation.