TITLE: 2,4-dinitrophenylhydrazine alpha-ketoglutarate detection assay for Prolyl Hydroxylase Domain (PHD) proteins
AUTHORS: sjwong  

[DESCRIPTION]
The 2,4-dinitrophenylhydrazine (2,4-DNPH) alpha-ketoglutarate detection assay was developed to support the study of prolyl hydroxylase domain (PHD) proteins in a substrate-independent manner. This protocol was extensively optimized for the PHD protein reaction, and is applicable to the study of enzyme kinetics or to high-throughput screening. 

[GUIDELINES]
The following materials must be prepared fresh: 

1 mM iron (II) sulfate: Prepare 500 mM in 20 mM HCl, then dilute to 1 mM in water just before use (Hewitson, K. S., Schofield, C. J., & Ratcliffe, P. J. (2007). Hypoxia-inducible factor prolyl-hydroxylase: purification and assays of PHD2. Methods in enzymology, 435, 25–42. https://doi.org/10.1016/S0076-6879(07)35002-7)
PHD enzyme source: frozen stocks may be use, but fresh enzyme is best, especially for PHD3 due to its lability.
2,4-dinitrophenylhydrazine (2,4-DNPH): Dissolve in 0.5 M phosphoric acid, let stand for 30-60 mins, then add water to intended concentration. Filter through 0.45 µM filter to remove precipitate.

[STEPS]
SECTION: In vitro hydroxylation assay
2. 
Prepare 5 Eppendorf tubes containing 50 µl of 10% TCA.
Label tubes: 0 min, 1 min, 2 min, 5 min, 15 min.


SECTION: In vitro hydroxylation assay
3. Prepare cofactor solution containing HEPES/MES, catalase, DTT, ascorbic acid, FeSO4, a-ketoglutarate, and peptide in a 150 µl volume in an Eppendorf tube (using the working concentrations).
SECTION: In vitro hydroxylation assay
4. Add 150 µl of 20 µM PHD enzyme into the cofactor solution.
SECTION: In vitro hydroxylation assay
5. Vortex briefly.
SECTION: In vitro hydroxylation assay
6. Place into a 37 oC tabletop shaking incubator and start the timer (counting up).
This step equilibrates the temperature of the reaction to 37 oC
SECTION: In vitro hydroxylation assay
7. At T = 1 min on the timer, withdraw 50 µl of the reaction solution and quench in the “0 min” tube containing 10% TCA, and replace the reaction tube in the incubator.
SECTION: In vitro hydroxylation assay
8. Repeat this for the other time points.
At T = 2 min, withdraw 50 µl of the reaction solution and quench in the “1 min” tube
At T = 3 min, withdraw 50 µl of the reaction solution and quench in the “2 min” tube
At T = 6 min, withdraw 50 µl of the reaction solution and quench in the “5 min” tube
At T = 16 min, withdraw 50 µl of the reaction solution and quench in the “15 min” tube
SECTION: In vitro hydroxylation assay
9. Briefly vortex the quenched reactions.
SECTION: In vitro hydroxylation assay
10. Keep the quenched reactions at 4 oC until ready for downstream processing.
Reactions have been stored up to 3 days with no loss of signal.
SECTION: Color development with 2,4-DNPH
11. Centrifuge the quenched reactions at 13,000 rpm for 15 minutes.
SECTION: Color development with 2,4-DNPH
12. Meanwhile, add 10 µl of 0.5 M sodium phosphate to 5 wells of a 96-well plate.
SECTION: Color development with 2,4-DNPH
13. Transfer 90 µl of the supernatant of the quenched reaction to a well containing 10 µl of 0.5 M sodium phosphate (VT = 100 µl).
Do the same for the other 4 quenched supernatants.
SECTION: Color development with 2,4-DNPH
14. Using a multi-channel pipette, add 100 µl of 50 mM 2,4-DNPH to the wells (VT = 200 µl). Pipette up and down gently to mix.
SECTION: Color development with 2,4-DNPH
15. Leave at room temperature for 20 minutes.
SECTION: Color development with 2,4-DNPH
16. Using a multi-channel pipette, add 50 µl of 6 M NaOH to the wells (VT = 250 µl). Pipette up and down gently to mix.
SECTION: Color development with 2,4-DNPH
17. Leave at room temperature for 5 minutes.
SECTION: Color development with 2,4-DNPH
18. Read at 425 nm on a spectrophotometer.
SECTION: Data handling
19. Calculate the amount of a-ketoglutarate consumed from a corresponding standard curve processed in exactly the same way as the samples.
SECTION: Data handling
20. Plot the amount of a-ketoglutarate consumed against time, to obtain a curve that looks like this:


  

SECTION: Data handling
21. The initial rate should be taken as the linear portion of the curve. In this case, from T = 0 to 2 mins.
SECTION: Overview of assay schematic
1.