Figure 1. Chromogenic reactions in the assay kit

Materials and Reagents

EnzChek Pyrophosphate Assay kit (Molecular Probes, catalog number: E-6645 )
1. The contents of the components A~E are shown below.
2. 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG)
3. Purine nucleoside phosphorylase (PNP)
4. 20x reaction buffer: 1.0 mM Tris-HCl, 20 mM MgCl₂ (pH 7.5)
5. Pyrophosphate standard solution: 50 mM Na₄P₂O₇
6. Inorganic pyrophosphatase
1 M MgCl₂
100 mM substrates (our examples in Table 1)
Enzyme preparation (with or without imidazole, see note 5 below)

Equipment

Block incubator (ASTEC, model: BI-516S )
Spectrophotometer (Shimadzu, model: UV-2600 )
Electronic cooling and heating cuvette holder (Shimadzu, model: S-1700 )

Quarts cuvette with a lid

Table 1. An example of substrate reagents

Substrate	Company	Catalog number
Adenosine triphosphate (ATP)	Sigma-Aldrich	A-2383
Guanosine triphosphate (GTP)	Sigma-Aldrich	G-8877
Cytidine triphosphate (CTP)	Sigma-Aldrich	C-1506
Uridine triphosphate (UTP)	Sigma-Aldrich	U-6625
Deoxythymidine triphosphate (dTTP)	Sigma-Aldrich	T-0251
Glucose-1-phosphate (Glc-1P)	Sigma-Aldrich	G-9380
N-acetylglucosamine-1-phosphate (GlcNAc-1P)	Sigma-Aldrich	A-2142
Galactose-1-phosphate (Gal-1P)	Sigma-Aldrich	G-0380
Mannose-1-phosphate (Man-1P)	Santa Cruz Biotechnology	SC-284868

Procedure

Measurement of enzyme activity
1. Set up the spectrophotometer and electronic cooling and heating cuvette holder. Hold a cuvette in the holder to keep the temperature (usually 37 °C).
2. Prepare the assay kit solutions as written in the product manual and keep them on ice. The instruction states component A should be used within 4 h when melted.
3. Prepare reaction mixtures according to Table 2.
4. Preincubate the mixture at 22 °C for 20 min to react the contaminated pyrophosphate and phosphate in the block incubator. We usually incubate the mixture for another 10 min at 37 °C (the measuring temperature) to warm up the mixture. The original instruction recommends the preincubation for 10 min at 22 °C.
5. Put the reaction mixture into the cuvette in the spectrophotometer and record the absorbance at 360 nm in a time-scan mode for 5 ~ 10 min. Usually, the linear time course of the color development was obtained often initial lag time of 10 ~ 20 sec. Therefore, it may be sufficient to record time points every 30 ~ 60 sec.
Data analysis
1. Measure the rate of absorbance change (∆A₃₆₀/sec).
2. Convert the reaction rate (∆A₃₆₀/sec) to the velocity (molar concentration/sec) by the difference of final A₃₆₀ between the positive control and the negative control 1 (see the formula below).
  
  V (M / sec) =
  ∆A₃₆₀ (cm^-1) / sec - ∆A₃₆₀ (cm^-1) / sec (negative control)
  ε (M^-1 cm^-1)
  
  We experimentally determine the molar extinction coefficient of ~ 11,000 (M^-1 cm^-1) at 360 nm every time based on the final absorption of the positive control as follows.
  
  ε (M^-1 cm^-1) =
  final A₃₆₀ (cm^-1, positive control) - final A₃₆₀ (cm^-1, negative control 1)
  pyrophosphate concentration (M, positive control)
  
  The kit provides standard solution of 50 mM pyrophosphate (component D). We obtained fairy reproducible value for ε in our hands although the instruction does not mention about the known ε value, the original literature (Upson et al., 1996) states ε (M^-1 cm^-1) = 11,000 at pH 7.6.
  
  Table 2. Preparation of reaction mixtures
  
  The asterisk represent the kit components A~E. I~IV: The order of addition for preparation of reaction mixtures.

Representative data

Here are representative data of cyanobacterial UDP-glucose pyrophosphorylase expressed and purified from E. coli cells (Maeda et al., 2014). The initial slope of the graph was used for calculation. We usually use enzymes at 50 ~ 200 nM, and substrates [(d)NTP and sugar-1P] at 50 µM ~ 2 mM.

Table 3. Preparation of reaction mixtures in our example

Figure 2. An example of measurements for polyhistidine-tagged cyanobacterial UDP-glucose pyrophosphorylase, which was purified by Ni-affinity chromatography

Notes

Before measurement, it is essential to check the reaction capacity of kit. Add 50~75 µM sodium pyrophosphate to the kit to confirm the maximum activity of pyrophosphorylase and purine nucleoside phosphorylase in the kit. We cannot measure NTP transferase activities higher than the maximum activity of the kit (Figure 2).
To check the quality of the assay kit, we must measure the positive control first.
Negative control 2 gives the level of contamination of phosphate and pyrophosphate. Negative control 3 and 4 give background enzyme activities to degrade (d)NTP and sugar-1P, respectively. Phosphate and pyrophosphate in the sample solutions or substrate reagents increase the background absorbance at 360 nm. We experienced some reagents such as mannose-1-phosphate contained too much phosphate. Such impurities may depend on the lot of the reagent or product manufacturer.
A simple representative example for an enzyme sample is shown in Figure 2. The slope of the sample should be between the pyrophosphate positive control and the negative control. The initial slope of the graph was used for calculation according to the Data analysis.
The imidazole (up to final 100 mM in the assay mixture) has little effect on the assay kit activity. This means that the kit accepts usual preparations of His-tagged enzymes, which are (partially) purified by Ni-affinity chromatography.
Manganese ion (Mn²⁺) inhibits the assay kit activity at final 5 mM or even lower concentration.
Reproducibility of the measurements is pretty high, though the maximum activity of the kit depends on the freshness of the kit.
According to the kit instructions, the reagents of the kit are stable for six months to one year at < -20 °C. Reconstituted MESG (component A) may be stored at < -20 °C for at least one month. Reconstituted purine nucleoside phosphorylase (component B) may be stored at 4 °C for at least one month. Diluted inorganic pyrophosphatase (component E) may be stored at 4 °C for at least one week.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from MEXT and PRESTO from JST (to R. N.) and by Grants-in-Aid for Scientific Research and the GCOE program “From the Earth to Earths” from MEXT, and CREST from JST (to M. I.).

References

Enomoto, G., Nomura, R., Shimada, T., Ni Ni, W., Narikawa, R., and Ikeuchi, M. (2014). Cyanobacteriochrome SesA is a diguanylate cyclase that induces cell aggregation in Thermosynechococcus. J Biol Chem 289(36): 24801-9.
Kawano, Y., Sekine, M. and Ihara, M. (2014). Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120. J Biosci Bioeng 117(5): 531-538.
Maeda, K., Narikawa, R., and Ikeuchi, M. (2014). CugP is a novel ubiquitous non-GalU-type bacterial UDP-glucose pyrophosphorylase found in cyanobacteria. J Bacteriol 196 (13): 2348-54.
Upson, R. H., Haugland, R. P., Malekzadeh, M. N. and Haugland, R. P. (1996). A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal Biochem 243(1): 41-45.