PLANT FOODS FOR HUMAN NUTRITION
Phytoferritin association induced by EGCG inhibits protein degradation by proteases
Aidong Wanga, Kai Zhoua, Xin Qib, Guanghua Zhaoa,*
aCAU & ACC Joint-Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University, Key Laboratory of Functional Dairy,Ministry of Education, Beijing 100083, China
b National institute of Metrology, Beijing 100013, China
*Corresponding authors. Address: Guanghua Zhao, College of Food Science and NutritionalEngineering, China Agricultural University, China. E-mail addresses: (G. Zhao). Tel./fax: +86 10 62738737.
Online resource 1: Detailed information about the methods used in the manuscript.
- Chemicals
Ascorbic acid (AA), 2,2-bipyridyl, N,N-bis-methyleneacrylamide, sodium dodecyl sulphate (SDS), trypsin, pepsin, TEMED, tris(hydroxymethyl)aminomethane (TRIS), β-nercaptoethanol, and Coomassie Brilliant Blue R250 were obtained from Sigma-Aldrich Co. (Beijing, China). SDS and native electrophoresis marker were purchased from GE Healthcare Bio-Sciences AB (Beijing, China). 3-(N-Morpholino)propanesulfonic acid (MOPS) was obtained from Amerseco (Beijing, China). All other reagents used were of analytical grade or purer.
2. Protein gel electrophoresis
Determination of the molecular weight of the native rH-2 ferritin was done using a 4-20% polyacrylamide gradient gel run at 4 mA for 24 h at 4 °C and with Tris-HCl (25mM, pH 8.3) as running buffer. Electrophoresis of proteins under denaturing conditions was done in 15% SDS-polyacrylamide gel Gels. All gels were stained with Coomassie Brilliant Blue R-250.
3. Fluorescence quenching titration
Fluorescence titration experiments were performed using the Cary Eclipse spectrophotometer (Varian, Palo Alto, USA). The concentration of the aporH-2 was0.5 μM in 50mM Tris-HCl, 150mM NaCl, pH8.0, at 25 °C. The titrations were conducted by adding 2 μL of EGCG (0.5 mM) to 0.8 mL aporH-2 in 14 increments. Fluorescence spectra were scanned from 290 nm to 450 nm after each addition. The excitation wavelength was set at 280 nm.
4. Circular dichroism (CD) spectra
Protein samples were dissolved in 0.8 mM Mops buffer, pH 7.5. CD spectra were recorded with a Chirascan Plus spectrometer (Applied Photophysics, Leatherhead, UK), using quartz cuvettes of 0.5 mm optical path length at 25 °C. CD spectra were scanned at the far UV range (190-260 nm) with 3 replicates at 50 nm/min, bandwidth = 1 nm. The CD data were expressed in terms of mean residual ellipticity, (θ), in deg cm2 dmol-1. The induced ellipticity was defined as the ellipticity of the EGCG-aporH-2 mixture, minus the ellipticity of EGCG alone under the same experimental conditions. Percentage of secondary structure was calculated using the CDNN secondary structure analysis with Milli-Degrees signal type.
5. Stopped-flow light scattering (SLS) and dynamic light-scattering (DLS)
The SLS measurements were performed with a pneumatic drive Hi-Tech SFA-20 M apparatus in conjunction with a Cary Eclipse spectrofluorimeter (Varian, Polo Alto, USA) as previously described [1]. To observe the binding of rH-2 to EGCG, equal 140 μL volumes of EGCG (1.1-2.2 mM) and buffered rH-2 (0.9 μM) were mixed at 25 °C in the thermostated sample compartment containing a 280 μL quartz stopped-flow cuvette. All quoted concentrations are final concentrations after mixing the two reagents. The DLS measurements were performed at 25 °C using a Viscotek model 802 dynamic light-scattering instrument (Viscotek Europe Ltd.) as recently reported [2]. The hydrodynamic radius (RH) was calculated with the regularization histogram method using the spheres model, from which an apparent molecular mass was estimated according to a standard curve calibrated from known globular proteins. OmniSIZE 2.0 software was used to calculate the size distribution of aggregated protein from the addition of EGCG.
6. SGF and SIF digestion stability experiments
Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion stability experiments were carried out as recently described [3].
SGF consists of 3.2 mg/mL pepsin in 100mM HAC-ACNa buffer, pH 4.0. Aliquots (200 μL) of SGF was placed in 1.5 mL microcentrifuge tubes and incubated in a water bath at 37 °C.40 μL of rH-2 (1 mg/mL in 5 mM Mops buffer and 30 mM NaCl at pH4) in the presence of EGCG (1–100 mg/mL) was added to each of the SGF vials to start the digestion reaction.The ratio of pepsin to rH-2 wasabout 16:1 (w/w). 1 h later, the reaction was immediately stopped by adding 60μL 1M NaOH into the tubes. SDS-PAGE was run to analyze the stability of rH-2.
SIF consists of 10 mg/mL trypsin in 100 mMKH2PO4–NaOH buffer, pH 7.5. Aliquots (200 μL) of SIF was placed in1.5 mL microcentrifuge tubers and incubated in a water bath at 37 °C.40 μL of rH-2 (1 mg/mL in 5 mM Mops buffer and 30 mM NaCl at pH7.5) in the presence of EGCG (1–100 mg/mL) was added to each of the SIFvials to start the digestion reaction. The ratio of trypsin to rH-2 wasabout 50:1 (w/w). 1 h later, the reaction was immediately stopped byplacing the tube in a boiling water bath for 10 min. Then, the sampleswere analyzed by SDS-PAGE.
7. Kinetics of iron release from holo-rH-2
Iron release from holo-rH-2 was investigatedusing the assay procedure as previously described [4]. Holo-rH-2 were produced by adding approximately 400 iron atoms per ferritin in 8 increments at intervals of 20 min in 50 mM Tris-HCl, 150 mM NaCl, at pH 8.0.All concentrations stated were final concentrationsafter mixing the two reagents. Briefly, the assay system (1 mL of total volume) contained 0.5 μM holo-rH-2 (Fe3+/protein = 400/1), 500 μM ferrozine, 0.1 MNaCl, and 400 μM EGCG or AA in 0.1 M Mops buffer, pH 8.0. Reactionswere carried out at 25 °Cagainst reference cuvettes containing allreactants and were initiated by the addition of EGCG. The development of[Fe(ferrozine)3]2+ was measured by recording the increase inabsorbance at 562 nm using a Varian Cary 50 spectrophotometerand the iron released was estimated using ε562 nm = 27,900 M-1 cm-1.
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