In vitro P5C dehydrogenase assay

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In vitro P5C dehydrogenase assay

P5C preparation.

Reaction mix.
100 mg DL-^-pyrroline-5-carboxylic acid (2, 4-dinitrophenyl-hydrochloride double salt)
19.2 ml HCl (0.25 N)
25.3 ml acetophenone

1. Mix with a magnetic stir bar for 5 min/room temperature (fume hood). Transfer to aseparatory funnel. Under acidic conditions the dinitrophenyl group (yellow) is extracted by the acetophenone phase (bottom phase).

2. Remove the bottom layer and discard.

3. Add 10 ml acetophenone to the aqueous phase to remove any remaining yellow color.
Repeat steps 1-3 until yellow no longer leaches into the acetophenone phase.

4. Extract the aqueous phase twice with toluene (top phase).

5. Store at 4oC in 500 ul volumes until use.

Using P5C in P5C dehydrogenase assays.

1. Concentrate two 500 ul P5C stocks to ~ 100 ul in a speed vac and pool (Vt ~ 200ul).

2. Adjust pH to 6.0 with 4 N NaOH (~ 80 ul for each 500 ul stock).

3. Purify via HPLC on a S5-SCX cation-exchange column to remove salts.
Buffer: CaSO4, pH 5.5
Run conditions: 1 ml min -1
Collect in 200 ul volumes

4. Assay peak fractions (usually fractions 5-10) for P5C using the O-AB (O aminobenzaldehyde)

O-AB assay.

1. Dissolve O-AB in the following mixture.

15 mg O-AB
0.5 ml ethanol
0.75 ml dH2O

2. From the above mixture:

0.7 ml O-AB mixture
5.4 ml dH2O
6.0 ml 10% TCA (trichloroacetic acid)

3. Using a microtitre plate, place 100 ul in duplicate for each fraction and dilution of fraction.
For each fraction add 10 ul in duplicate and 10 ul of a 1/10 dilution in duplicate to the microtire plate.

4. Incubate for 20 min at room temperature to allow yellow color to develop.

5. Read at OD450 and correct each reaction for an O-AB mix only control.

mM P5C = [OD450][205 uM][Dilution factor][mM]
--------- -------
[0.5 cm] [1000 uM]

P5C dehydrogenase assay.

Stock solutions.

4X reaction buffer.
0.76 g Tris HCl
4.20 g Tris base
40 ml ethylene glycol
1 ml Tween-20
q.s. 100 ml

B-NAD (10 mM).
6.6 mg ml -1 into 1 ml dH2O.

INT (p-iodo-nitrotetrazolium violet) (3.2 mg ml -1).
160 mg INT into 50 ml dH20.
Heat to 80oC to dissolve.
PMS (phenazine methylsulfate) (0.4 mg ml -1).
4 mg PMS into 10 ml dH2O.

PMS/INT reaction mix
210 ul PMS
960 ul INT
4.83 ml 0.5 M Tris, pH 8.0

P5C stocks (Vt 600 ul).

X Stock (uM) Y mM P5C (ul) dH2O (Z ul) Reaction (uM)
----------------------------------------------------------------------
500 60 540 50
400 48 552 40
300 36 564 30
200 24 576 20
100 12 588 10
50 6 594 5
25 3 597 2.5
- - 600 -
a. The above numbers are based on 5 mM P5C stock from the following equation:

Y ul (5 mM stock P5C) = (uM X stock) (Vt 600 ul)
------------------------
5 mM P5C

Z ul (dH2O) = Vt 600 ul - Y

B-NAD stock (Vt 100 ul)

Stock (mM) 10 mM NAD (ul) dH2O (ul) Reaction (mM)
10 200 - 1
8 160 40 0.8
6 120 80 0.6
4 80 120 0.4
2 40 160 0.2
1 20 180 0.1
0.5 10 190 0.05
- - 200 -

Reaction set up.

1. Prepare stock mixture: 2.50 ml 4X reaction buffer.
2.25 ml dH2O.
2. From this stock add 35 ul to each well of a microtitre plate.
3. Add 5 ul appropriate B-NAD stock/dilution to each well (see plate below).
4. Add 5 ul appropriate P5C stock/dilution to each well.
5. Add 50 ul PMS/INT reaction mix to each well.
6. Start reactions in triplicate (one sample in columns 1-3 and 7-9, and any other in 4-6 and 10-12) by adding 5 ul PutA (diluted to 0.1 ug ul -1) to appropriate wells.
7. Incubate 10 min/37oC.
8. Stop reactions with 100 ul 2 N HCl.
9. Read at OD490 and correct each reaction with the appropriate control.

Specific activity (nmol INTR min -1 mg -1 ) =

OD49 0.1 ml
[ ----------------- ] [ ------------------ ]
6.64 X 10-3 ml -1 (10 min) (5 X 10-4 mg)

Link: http://www.sci.sdsu.edu/~smaloy/Research/pdf%20files/P5C_DH_in_vitro_.doc