METHYLENETETRAHYDROFOLATE REDUCTASE RADIOASSAY (reductive direction)

ASSAY PRINCIPLES

Summary
14CH2-THF (prepared from THF and a two-fold molar excess of H14CHO) is incubated with NAD(P)H and enzyme in the presence of a recycling system for NAD(P)H. The recycling system prevents NAD(P)+ formed in the reaction (or by NAD(P)H oxidase in crude extracts) from mediating 14CH2-THF oxidation via the CH2-THF dehydrogenase activity present in crude extracts. At the end of the reaction, the remaining 14CH2-THF is decomposed to H14CHO by adding a large excess of unlabelled HCHO, and the reaction product (14CH3-THF), which is strongly cationic, is bound to Dowex 50 resin and counted while bound to the resin (14CH3-THF can be eluted from the resin with 6N NH4OH, but the volume required is inconvenient (>12 vol/ vol resin).
 
1. Preparation of the substrate
14CH2-THF substrate is prepared by incubation of tetrahydrofolate (THF, dissolved in 40 mM b-mercaptoethanol in triethanolamine buffer) with a twofold molar excess of H14CHO, at room temperature. The reaction proceeds according to the scheme:
THF + H14CHO --> 14CH2-THF
The reaction is allowed to reach equilibrium for 5 minutes (~100% conversion of THF substrate into CH2-THF, see the assay procedure section).

 

The substrate should always be prepared fresh, immediately before use.

2. The assay reaction scheme and NAD(P)H regeneration system
Conversion of CH2-THF into CH3-THF in the assay proceeds according to the scheme:
 
14CH2-THF + NAD(P)H --> 14CH3-THF + NAD(P)+
 
The NAD(P)+ formed is recycled to NAD(P)H by addition of glucose-6-phosphate dehydrogenase (G6PDH) and glucose-6-phosphate to the assay. G6PDH catalyzes the reaction:
glucose-6-phosphate + NAD(P)+  --> 6-phosphoglucono-d-lactone + NAD(P)H
The equilibrium lies far to the right.
3. Termination of the assay
At the end of incubation period, the assay is stopped by adding a large excess of 100 mM HCHO. This in effect destroys the remaining 14CH2-THF by exchange of the label with unlabelled HCHO.
The 14CH3-THF product formed in the assay is bound to Dowex AG-50 (H+ form) resin, washed three times with 100 mM HCHO, and counted while still bound to the resin.
The assay should be done in conical 2-mL screw-top tubes (NOT 1.5-ml), Fisher catalog #05-664-33
 
ASSAY REAGENT STOCKS

1M K-Phosphate buffer pH 7.2

0.5 M Na2EDTA pH 8.0

10X K-Phosphate (pH 7.2) – EDTA mix

(1 M K-Phos containing 3 mM Na2EDTA)

Mix 1 mL K-Phos pH 7.2 with 6 mL EDTA 0.5 M

THF (6R,S)-5,6,7,8-Tetrahydrofolic acid trihydrochloride

MW = 554.8

Supplier: Schircks laboratories

Catalog # 16.209-100, 3X 100 mg ampules
 

Note: The ampules should be stored at –80°C. After an ampule is opened, the contents should be transferred into a glass screw-top vial with a teflon-lined cap, and stored desiccated at –80°C.

The compound is a racemic mixture, 50% is in the biologically active S-form.

TESH (40 mM b-Mercaptoethanol in triethanolamine-HCl)

0.25 M Triethanolamine-HCl pH 7.0 is prepared in advance and stored frozen. b-Mercaptoethanol (2.8 mL/mL) should be added to it just before use.


4 mM NADH
MW = 709.4 , 2.84 mg/mL

 
4 mM NADPH
MW = 833.4, 3.33 mg/mL
 
0.5 mM FAD
MW = 829.5 + 1 H2O/mol, 8.5 mg/20 mL H2O, store 1-mL aliquots at -20°C
20 mM glucose-6-phosphate
MW = 282.1, 5.64 mg/mL stock stored at -20°C

Glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides

Supplier: Sigma, Catalog # G8529, active with either NAD+ or NADP+
2 manufacturer’s units/µL stock solution in a buffer (100 mM K-Phos, pH 7.2, 1mM Na2EDTA, 10% glycerol) can be prepared in advance and stored at +4°C for several weeks. It is then diluted appropriately before use (see below).
The activity of G6PDH in the buffer used for MTHFR assay (100 mM K-Phos, pH 7.2) is lower than its activity in the buffer recommended by manufacturer. Therefore, the activity of G6PDH in 100 mM K-Phos, pH 7.2, should be determined spectrophotometrically before the enzyme is used in the MTHFR assay. The method is given below. Our measurements indicate that the initial activity of G6PDH in 100 mM K-Phos 7.2, using NAD+ as a cofactor, is about 25 % of its activity under the assay conditions recommended by manufacturer (i.e. 1 manufacturer’s unit = 0.25 measured units).
The activity of G6PDH stored in solution at +4°C will slowly decrease with time. Therefore, the enzyme should be periodically re-assayed before use (at least once/week) to determine its activity. The amount of G6PDH in the MTHFR assay should be adjusted according to this activity measurement. The quantity of G6PDH used in the MTHFR assay should be 0.06 measured units/assay.
 
G6PDH assay (spectrophotometric):
Stocks:
10 mM NAD+  (prepared fresh)
10 mM NADP+  (prepared fresh)
20 mM Glucose-6-phosphate
100 mM K-Phos pH 7.2
Enzyme: dilute the 2 manufacturer’s units/mL stock 100X in 100 mM K-Phos
For an 80- mL assay, mix in an UV microcuvette:
68 mL 100 mM K-Phos, pH 7.2
8 mL NAD+ or NADP+
2 mL enzyme (100X diluted)
Start the reaction by addition of 4 mL of 20 mM glucose-6-phosphate and follow the increase in A340. The activity in the presence of NADP+ is equal to ~ 80% of the activity in the presence of NAD+
Molar extinction coefficient of NAD(P)+ at 340 nm is 6270 M-1cm-1
[14C]Formaldehyde

Supplier: NEN, Catalog# NEC039H, specific activity ~ 50 mCi/mmol (in ampule)

The compound is volatile and the contents of the ampule should be condensed before opening, immediately dissolved in water at 100 nCi/mL, and transferred to a glass screw-top vial with a teflon-lined cap. This stock should be stored at +4°C.

40 mM Formaldehyde

MW = 30

36.6 % w/w soln. = 13.2 M
30.3 µL of 36.6 % in 10 mL H2O, store at +4°C for up to 1 week
100 mM Formaldehyde
760 µL of 36.6 % in 100 mL H2O, store at +4°C for up to 1 week
Dowex 50 resin (Bio Rad AG-50)

Supplier: Bio Rad. Mesh size 200

AG-50 (H+) resin
1:1 mixture with H20 stored at +4°C
ASSAY PROCEDURE (20 mL final volume)
Preparation of 14CH2-THF:
Just before use:

1. Put 1 ml of TESH in a screw-top tube and keep under a stream of N2 for 5 min to reduce oxygen concentration.

2. Working quickly, dissolve 5.5 mg/ml of THF in N2-gassed TESH. The solution will be 10 mM with respect to THF.

3. Mix the 10 mM THF solution at once with 1M K-Phos-EDTA, pH 7.2, 40 mM HCHO, and H14CHO (100 nCi/mL) in ratio 2(THF):2(K-Phos-EDTA):1(HCHO):1(H14CHO).

4. Incubate the mix at room temperature for 5’ to allow 14CH2-THF formation before adding to the other assay constituents.Use 6 mL of the mix per 20-mL assay.

The substrate should always be prepared fresh, immediately before use.

Assay constituents:

Mix on ice:
 
 
Constituent
mL
Final concentration
 
 
 
14CH2-THF mix
6
1 mM THFa
2.1 mM HCHO (100 nCi) 
4 mM b-mercaptoethanol 
4 mM NAD(P)H
1
200 mM
0.5 mM FAD
1
25 mM
20 mM Glucose-6-phosphate 
1
1 mM
0.06 measured units G6PDHb
1
 
Yeast protein extract
 
~10 mg/assay 
H2O
to 20 mL
 
 
 
 

a concentration of (6-S) form (biologically active) = 0.5 mM

b measured in K-Phos pH 7.2 with NAD+ as electron acceptor

Blank: Reaction minus pyridine nucleotide

Recovery determination (spike) : Reaction with unlabelled CH2-THF substrate and a 14CH3-THF spike added at the beginning of the incubation.

Incubation: 30°C, 20 min

Stop:

1. Add 1 ml of 100 mM HCHO to each reaction, vortex briefly, and let the system equilibrate at room temperature for 20 min.
2. Add 200 mL of AG-50 (H+) resin (1:1 mixture with H20) with a cut-off pipette tip (vortex the resin/water mix briefly just before pipetting). Vortex the tubes for 2 min (using a vortex micro tube attachment for multiple tubes, Fisher catalog # 12-812B), microfuge 15 sec to pellet the resin , aspire off and discard the supernatant.
3. Rinse the resin 3 X with 1.5 ml of 100 mM HCHO (vortex 5 min each wash and centrifuge as before to pellet the resin ).
4. Add 1 ml of Beckman Ready Gel scintilation cocktail to the resin, vortex 2 min, place the screw-top tube in a small scintillation vial, count. The counting efficiency of this system is approx. 40 %.

 

Calculation of results

1. Correction for counting efficiency and recovery:

correction factor = cpm found for 14CH3-THF-spiked sample (see above)/dpm of 14CH3-THF added to each assay (typical values are ~ 0.39)

2. Specific activity of the substrate:

specific activity of H14CHO = 100 nCi/42 nmol

max. conversion of label = nmol THF (20)/nmol HCHO (42) x 50% (6-S stereoisomer) x 100 nCi  =  24 nCi x 0.39 = 21 000 cpm.