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Protocol for in vitro assay of dihydroflavonol 4-reductase enzyme activity

This protocol was developed from the assay described by Stafford and Lester (1982). While their protocol made use of native proteins extracted from fresh flower tissue, the method presented here uses enzymes overexpressed in E. coli. We have successfully made DFR enzymes using Invitrogen's pDEST17 and pDEST14 vectors, though other workers have had success with a variety of bacterial and yeast overexpression vectors; we use the E. coli line BL-21 Star (DE3) from Invitrogen. The assay works well for the DFR substrates naringenin, eriodyctiol, dihydrokaempferol, dihydroquercetin and dihydromyricetin, though these substrates may have different optimal assay conditions that we have not yet determined. The assay protocol presented below is for 1 mL reactions; due to the cost of the reagents, we generally do 500 µL reactions. Adjust both assays contents and subsequent extraction steps accordingly.

Bacterial Overexpression

1. Start 5 mL overnight cultures from single colony picks. Grow in shaking incubator at 37ºC in LB+Amp100.

2. Autoclave 200 mL LB and a 250mL Erhlenmeyer flask for each clone. Add ampicillin to LB at a concentration of 100 mg/L. Leave the LB in the non-shaking incubator overnight to get the broth warm.

3. The next morning, draw 2µL of overnight culture and run a PCR to check for the presence of the insert. Use T7 primer and an internal reverse GSP. (Alternatively, do this at the end of the 3-hour grow-out)

4. Dilute overnight into 200mL LB+Amp100 and grow at 37ºC in shaking incubator at 200rpm until OD600 reaches ~0.5 (2-3 hrs from room temp LB).

5. Add 500uL 200 mM IPTG solution to the 200 mL culture.

6. After three hours, aliquot samples into labeled 50 mL tubes and spin down in swing-rotor at 4ºC for 20 minutes.

7. Pour off LB and store at -80ºC.

Preparing Bulk Protein for Enzyme Assays

1. The DFR assay works well working off protein extracted from the pellets of 100 mL of culture grown for 3 hours.

2. Thaw pelleted cells on ice. Add 500µL Lysis buffer (to make 10 mL of buffer, add to 7.85 mL H20: 500µL 1M HEPES, 87 mg NaCl, 1mL 200 mM PMSF, 100µL 100 mM TPCK, 50µL 200 mM TLCK) and 10µL Lysozyme (stock: 100 mg/mL) to one tube of cells from 50 mL of culture. Flick to resuspend pellet.

3. Set swing-rotor temperature to 4ºC.

4. Incubate lysis reactions on ice 30 minutes with occasional mixing.

5. Spin down 20 minutes at 4ºC and transfer supernatant to pre-chilled Eppendorf. DFR is a soluble protein and will remain in the supernatant.

6. Yield is generally ~1.5 mL crude bacterial protein extract from two 50 mL tubes of cells.

DFR Assay

In 1 mL reaction:
1 µmol substrate (= 0.304 mg DHQ, 0.288 mg DHK, 0.32 mg DHM, 0.286 mg eriodyctiol: dissolve in >95% MeOH. For Sigma naringenin, which is racemic, use 2 µmol, 0.54 mg)
1µmol NADPH (= 1 vial ~ 1 mg; I generally dissolve this in the Tris)
6µmol glucose-6-P (= 1.7 mg)
1 U glucose-6-P dehydrogenase (= 0.25 mg)
200-400 µg enzyme (I use 250uL of bacterial extract)
0.1M Tris (pH 7.4) to 1 mL

1. Mix and incubate 3 hr at 30ºC

2. Add 15uL 6N HCl to stop the reaction.

3. Immediately extract three times in 1 mL ethyl acetate (I shake these vigorously and then spin down ca. 30 sec in the centrifuge. EtAc is the top layer and contains the leucoanthocyanidins, which you want).

4. Wash twice with 200 µL H2O (H2O forms a small bubble at the bottom- try to get as much as possible out).

5. Evaporate EtAc under vacuum at 35-45ºC into ~ 100 µL aqueous phase.

6a. For leucoanthocyanidins (DHK, DHQ, DHM as substrate), add 1 mL butanol-HCl reagent (95:5 v/v) and heat at 95ºC for 30 min (in the past, we have used n-butanol but t-butanol is preferable to reduce ether formation at the substrate 3-carbon).

6b. For flavan-4-ols (naringenin, eriodyctiol as substrate), add 1mL 2N HCl and heat at 95ºC for 30 min, then extract into isoamyl to clean, if desired.

7. After formation of anthocyanidins, the products can be visualized using light spectroscopy and compared to purified standards for quantification.

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