Simply Sensitive: Phosphate Detection Made Easy.

Kevin R. Kupcho, Caroline M. Wessely, Steve M. Riddle, and Kevin L. Vedvik, Life Technologies, 501 Charmany Dr., Madison, WI, USA, 53719

ABSTRACT RESULTS Figure 7. Phosphodiesterase Assay Schematic. Figure 12. PDE-5 Titration – Kinetic Mode. 30000 4 ng PDE5 Figure 3. Phosphatase Assay Schematics. Numerous enzymatic reactions release inorganic phosphate (Pi) as a result of 25000 2 ng PDE5 their activity. For example, phosphatases liberate phosphate from proteins as a Phosphate Sensor 1 ng PDE5 20000 Phosphatase Phosphate 0.5 ng PDE5 way of regulating the activity of their targets, ATPase/GTPases hydrolyze NTP Sensor into NDP + Pi in order to generate energy to pump ions across a membrane, and 15000 0.25 ng PDE5 Phosphate helicases use ATP to open double stranded DNA. We describe here a simple tool Phosphorylated-Peptide Substrate Peptide Substrate Pi Sensor-Pi 0.125 ng PDE5 10000 that can be used to interrogate the activity of phosphate releasing enzymes. BIOMOL GreenTM 0.063 ng PDE5 BioMol Green Phosphatase 0.031 ng PDE5 This tool is based on a bacterially-derived phosphate binding protein that has Reagent 5000

Fluorescence (Em450) 0.016 ng PDE5 been modified with a fluorophore such that upon the binding of phosphate an BioMol Phosphorylated-Peptide Substrate Peptide Substrate Pi Green -Pi 0 increase in fluorescence is detected. Not only is the reagent simple to use, it can 0 1000 2000 3000 4000 be used in conjunction with a variety of enzyme systems to give screening ProFluor® Tyrosine Phosphatase Assay time (sec) Stabilizer Protease Phosphatase Solution Human Phosphodiesterase-5 reaction was carried out as described previously except that the 10 µL of 1 µM Phosphate quality data, and is orders of magnitude more sensitive than the commonly used Sensor was added immediately and read every minute over a 60 minute incubation period.

Rhodamine reagent, Malachite Green. Phosphorylated-Peptide Peptide Substrate- The ability of the Phosphate Sensor to gather kinetic data is unique for the measurement of inorganic phosphate. Pi Rhodamine 110 110 Substrate-Rhodamine 110 Rhodamine 110

The Phosphate Sensor is a simple two step assay with a fluorescent readout. Figure 13. GTPase Assay Schematic. INTRODUCTION The BIOMOL Green™ Reagent is simple to use but lacks sensitivity and is an absorbance measurement. The ProFluor® Tyrosine Phosphatase Assay requires the use of a protease which could be inhibited by test compounds Drug candidate screening typically requires complex assay systems that employ (indicated by red X), adding complexity to the detection method. multiple enzymatic reactions to measure a response or need to have a different Figure 5. PTP1B titration - Phosphate reagent for each target class. Along with this many of these systems suffer from Figure 4. PTP1B titration - Phosphate The Phosphate Sensor can be configured to detect phosphodiesterase activity through coupling with a hydrolase enzyme like alkaline Sensor and BIOMOL Green™ Reagent. Sensor and ProFluor® Assay. phosphatase. lack of sensitivity which results in increased reagent usage. To this end we have The PDE-Glo™ Phosphodiesterase Assay measures phosphodiesterase activity by coupling though PKA and Luciferase, adding 30000 0.40 developed the Phosphate Sensor (PV4406). This simple, versatile, and easy to 110 complexity to the method. Test compounds could inhibit PDE-5, PKA, or luciferase as indicated by the red X’s. 0.35 use tool enables direct measurement of inorganic phosphate formation in 25000 0.30 90 picomolar quantities. This reagent is based on a bacterially-derived phosphate 20000

0.25 A Figure 8. Alkaline Phosphatase Figure 9. Human PDE5 titration. binding protein modified with a fluorophore such that upon binding of phosphate 620 70 15000 0.20 titration. an increase in fluorescence is detected (figure 1) (1). The observed 6-10 fold 0.15 50 55000 10000 50000 increase in fluorescence can be used to interrogate a variety of enzymatic 0.10 60000 45000 The Phosphate Sensor can be configured to measure the activity of GTPase enzymes such as RhoA. As RhoA is stimulated by its 5000 30 40000 reactions that release phosphate. This system is orders of magnitude more 0.05 Normalized 50000 associated GTPase Activating Protein (GAP), hydrolysis of GTP to GDP and inorganic phosphate increases. The Phosphate Sensor

Fluorescence (EmFluorescence 450) 35000 can be used to measure this hydrolysis event. sensitive than the commonly used phosphate detection system Malachite Green 0 0.00 30000 -3 -2 -1 0 1 2 3 4 5 6 10 40000

10 10 10 10 10 10 10 10 10 10 Fluorescence Signal (such as the BIOMOL Green™ Reagent formulation (figure 2) and its single 25000 [PTP1B] (pM) -10 30000 20000 Figure 14. RhoA GTPase Assay. reagent system makes it much simpler than other assay formats. 10-3 10 -2 10-1 100 101 10 2 103 10 4 10 5 106 15000 50 M substrate, Phosphate Sensor 20000 10000 14000 Here, we demonstrate how the Phosphate Sensor can be used with a variety of [PTP1B] (pM) 5000 10 M substrate, Phosphate Sensor Fluorescence (Em 450) 10000 0 50 M substrate, Biomol Green™ Reagent 5 M PTP1B subs, Phosphate Sensor 12000 different target classes. Data gathered with Protein Tyrosine Phosphatase 1B Fluorescence (Em 450) 0.001 0.01 0.1 1 10 100 1000 10000 100000 10 M substrate, Biomol Green™ Reagent 5 M R110 subs, ProFluor® Assay 0 [PDE5] (pg/well) (PTP1B) demonstrates the Phosphate Sensor is more sensitive than both 0.000010.0001 0.001 0.01 0.1 1 10 100 1000 10000 BIOMOL Green™ Reagent and ProFluor® Tyrosine Phosphatase Assay, resulting Protein Tyrosine Phosphatase 1B (PTP1B, Calbiochem #539735) PTP1B was 2-fold serial diluted and incubated with 5 µM Alkaline PPase (mU/mL) no cGMP, no Alk. PPase no cGMP, 10 mU/mL Alk. PPase in the reduction of both PTP1B and substrate used. In the Rho GTPase assay, was 2-fold serial diluted and incubated with either 10 or 50 µM substrate for 60 minutes. The Phosphate Sensor data was no nucleotide 5 M GMP 5 M cGMP 5 M cGMP, no Alk. PPase 5 M cGMP, 10 mU/mL Alk. PPase 8000

PTP1B Substrate (Calbiochem #539737) for 60 minutes. The performed as described earlier using PTP1B substrate. The 10 RFU the Phosphate Sensor can measure increase in activity of Rho GTPase by the Phosphate Sensor reaction was performed in 10 µL and inorganic µL ProFluor® Tyrosine Phosphatase Assay was conducted Alkaline phosphatase (Calbiochem #524545) was 2-fold serial Human Phosphodiesterase-5 (PDE-5 SignalChem #P93-31G) 6000 addition of its associated GTPase Activating Protein (GAP). The Phosphate phosphate was detected with the addition of 10 µL 1 µM using 5 µM PTPase R110 Substrate; this was followed by a 5 µL diluted and incubated with 5 µM GMP, cGMP or no nucleotide was 2-fold serial diluted and incubated in the presence and Phosphate Sensor and read on a Safire2™; excitation 430(10) nm, addition of Protease Solution and a 30 minute incubation. Then for 60 minutes in 10 µL. 10 µL of 1 µM Phosphate Sensor and absence of 5 µM cGMP and alkaline phosphatase for 60 4000 Sensor can also be configured to detect activity of a phosphodiesterase assay, emission 450(10) nm. The BIOMOL Green™ Reagent reaction 5 µL of Stabilizer Solution was added and read on a Safire2™, read immediately after addition as described earlier. minutes in 10 µL. 10 µL of 1 µM Phosphate Sensor was added 2000 illustrated by the Phosphodiesterase-5 titration and inhibitor titration presented. was carried out in 50 µL with a 100 µL addition of BIOMOL excitation 485 (10) nm and emission 530 (10) nm. Signal above background is only observed when GMP is and read as described earlier. Green™ Reagent and the plate was read on a Safire™ in The Phosphate Sensor is orders of magnitude more hydrolyzed into gaunosine and inorganic phosphate. The PDE-5 generates a sigmoidal curve with an EC of 290 50 0 absorbance mode at 620 nm. sensitive than the ProFluor® Assay. EC50 values are 12 pM pg/well. Background signals from the alkaline RhoGAP only (50 HIS-RhoA only (50 HIS-RhoA + HIS-Cdc42 only (50 HIS-Cdc42 + HIS-Rac1 only (50 HIS-Rac1 + HIS-Ras p21 only HIS-Ras p21 + The Phosphate Sensor is more sensitive than BIOMOL and 13 nM respectively. phosphatase or cGMP are negligible. uM GTP) uM GTP) RhoGAP (50 uM uM GTP) RhoGAP (50 uM uM GTP) RhoGAP (50 uM (50 uM GTP) RhoGAP (50 uM GTP) GTP) GTP) GTP) Green™ Reagent with EC50 values of 12 pM and 990 pM respectively. Additionally, improved sensitivity of the GTPase assays were conducted using 4.75 µM GTPase with and without 5.2 µM Rho-GAP in the presence of 50 µM GTP in 10 µL total Phosphate Sensor enables the experiment to be performed Figure 10. Inhibition of Human PDE5 Figure 11. PDE-5 Titration - Phosphate Figure 1. Phosphate Senor Schematic. volume, and were incubated at 37°C for 2 hours. 10 µL of 1 µM Phosphate Sensor with 20 mM EDTA was added and the plate was read as with 1/5th the amount of substrate with no impact to assay with sildenafil citrate. Sensor and PDE-Glo™ described previously. window. Phosphodiesterase Assay. As expected Rho-GAP stimulated His-RhoA and His-Cdc42; marginal activation was observed for His-Rac1 and His-Ras p21. 50000 Figure 6. PTP1B titration – Kinetic mode. 110 110 40000 35000 90 90 CONCLUSIONS Normalized

250 pM PTP1B signal RLU 30000 70 70 30000 125 pM PTP1B 50 50 The Phosphate Sensor is a simple assay system to directly measure the 20000 25000 62.5 pM PTP1B 30 30

RFU signal amount of inorganic phosphate generated in an enzymatic reaction. Normalized 31.25 pM PTP1B 10000 20000 10 10 15.63 pM PTP1B Fluorescence (Em 450) 0 The Phosphate Sensor is very sensitive; detecting picomolar quantities -5 -4 -3 -2 -1 0 1 2 3 4 5 -10 -10 15000 7.81 pM PTP1B 10 10 10 10 10 10 10 10 10 10 10 0.01 0.1 1 10 100 1000 10000 100000 [sildenafil citrate] (nM) [PDE5] (pg/well) of inorganic phosphate. 10000 3.91 pM PTP1B Sildenafil citrate (Tocris #3784) was 3-fold serial diluted and Phosphate Sensor PDE-Glo™ Assay 1.95 pM PTP1B incubated with 0.8 nM PDE-5 in 7.5 µL for 10 min. 2.5 µL of 20 Human Phosphodiesterase-5 (PDE-5 SignalChem #P93-31G) Protein ribbon diagram of the Phosphate Senor with fluorescent dye indicated in green. Fluorescence 5000 The Phosphate Sensor is versatile and can be used for a variety of target µM cGMP and 40 mU/µL alkaline phosphatase was added to was 2-fold serial diluted and assayed using the Phosphate intensity of the Phosphate Sensor increases 6-10 fold upon binding inorganic phosphate. Fluorescence (Em 450) 0.98 pM PTP1B 0 start the reaction and incubated for 60 minutes. 10 µL of 1 µM Sensor as described earlier and the PDE-Glo™ classes; phosphatases, phosphodiesterases, GTPases, and ATPases. 0 300 600 900 1200 1500 1800 2100 2400 2700 no enzyme control Phosphate Sensor was added and read as described earlier. Phosphodiesterase Assay. The PDE-Glo™ Assay protocol Figure 2. Phosphate Standard Titration. An IC50 value of 2.3 nM was obtained which agrees with the includes the PDE-5 reaction steps, addition of a PDE-Glo™ time (sec) reported value of 3.7 nM (2). Termination Buffer, a PDE-Glo™ Detection Solution, and the REFERENCES and Useful Weblinks (1) Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. Direct, real-time measurement of rapid inorganic phosphate 50000 The PTP1B assay was run as described previously and read in kinetic mode, collecting data every minute for a total of 40 minutes. Kinase-Glo® Reagent. The plate was read on a BMG release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry, 1994 33, 8262- 0.5 The ability of the Phosphate Sensor to directly measure the amount of inorganic phosphate present allows for real time detectionof LabTech PHERAStar Plus in Luminescence mode. The Phosphate Sensor and BIOMOL 8271. 40000 its release. This is a unique feature of the Phosphate Sensor and can be used to determine enzymatic rates. The Phosphate Sensor is about 10 fold more sensitive 0.4 Green™ Reagent were compared using a than the PDE-Glo™ Phosphodiesterase Assay. EC (2) Mitsi A. Blount, Alfreda Beasley, Roya Zoraghi, Konjeti R. Sekhar,Emmanuel P. Bessay, Sharron H. Francis, and Jackie D. two-fold serial dilution of a phosphate 50 Binding of Tritiated Sildenafil, Tadalafil, or Vardenafil to the Phosphodiesterase-5 Catalytic Site Displays Potency, Specificity, 30000 A values are 240 pg/well and 2.6 ng/well respectively.

0.3 620 standard. Heterogeneity, and cGMP Stimulation. Molecular Pharmacology, 2004, 66, 1, 144-152. The Phosphate Sensor is orders of For More information about the Phosphate Sensor, please visit: www.invitrogen.com. 20000 0.2 magnitude more sensitive as compared to BIOMOL Green™ Reagent; EC values 10000 50 TRADEMARKS/LICENSING 0.1 are 9.6 pM and 2200 pM respectively. BIOMOL Green™ is a trademark of Enzo Life Sciences, Inc. Fluorescence (Em 450) 0 0.0 ProFluor® and PDE-Glo™ are trademarks of Promega Corporation. 0.001 0.01 0.1 1 10 100 1000 10000 100000 Safire™ and Safire2™ are trademarks of Tecan Group LTD. phosphate (pmol) Phosphate Sensor Biomol Green™ Reagent (Malachite Green) The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. For Research Use Only. Not intended for animal or human therapeutic or diagnostic use

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