Support information for
Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis
Daoyong Yu*, Guihong Huang, Fengxi Xu, Mengfei Wang, Shuang Liu, Fang Huang*
State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 266580, Qingdao, China
Corresponding authors. D. Yu: Tel.: +86 532 8698 1135; fax: +86 532 8698 1135.
F. Huang: Tel.: +86 532 8698 1560; fax: +86 532 8698 1560.
E-mail: (D. Yu); (F. Huang).
Native PAGE analysis
PSI-DDM and PSI-TX particles were dispersed to final concentration of 100 mg Chl/mL in 4 °C MES buffer with 1% (w/v) DDM respectively. Incubate the samples for 30 min to solubilize PSI complexes. Non-solubilized PSI particles were separated away by centrifugation of 10,000´g for 5 min. The Native-PAGE analysis was performed under reducing conditions using Novex® NativePAGE™ Bis-Tris mini gels (Invitrogen), following the manufacturer’s instructions. Set constant voltage 150 V and run 100 min.
(A) (B)
Fig. S1 – Native-PAGE of PSI-TX (A) and PSI-DDM (B) solubilized by 1% (w/v) DDM for 30 min .
Table S1 – Typical price for Triton X-100
Brands / Part# (SKU) / Description / Unit size / Your price (USD)Vetec / V900502 / reagent grade / 100 mL / China RMB 74.72
500 mL / China RMB137.38
Amresco / 0694 / reagent grade / 1 L / 26.80
4 L / 89.33
Teknova / T1100 / reagent grade / 100 g / 33.76
Promega / H5142 / molecular biology grade / 100 mL / 29.00
H5141 / molecular biology grade / 500 mL / 97.00
Sigma-Aldrich / X100 / laboratory grade / 100 mL / 32.90
500 mL / 41.50
1 L / 60.00
1 gallon / 159.50
T9284 / BioXtra / 100 mL / 56.20
500 mL / 78.90
1 L / 116.00
10 L / 806.00
Sigma / T8787 / for molecular biology / 50 mL / 22.80
100 mL / 33.70
250 mL / 67.00
Bio-Rad / 161-0407 / Triton X-100 / 500 mL / 33.00
Anatrace / APX100 / Anapoe-X-100 / 5´10 mL / 63.00
50´1 mL / 75.00
10´10 mL / 113.00
500 mL / 454.00
T1001 / TRITON X-100 / 500 mL / 66.00
1 gallon / 285.00
Data were obtained from the Web on January 9, 2014.
Table S2 – Typical price for n-Dodecyl β-D-maltoside
Sigma / D4641 / ≥98% (GC) / 500 mg / 98.60
1 g / 143.50
5 g / 492.00
25 g / 1,960.00
D5172 / BioXtra, ≥98% (GC) / 250 mg / 57.60
1 g / 143.00
Anatrace / D310S / Sol-Grade, >98% (HPLC); alpha isomer <5% / 1 g / 59.00
5 g / 222.00
25 g / 835.00
P212121 / RP-D12000 / >99.0% / 1 g / 95.00
5 g / 275.00
Bioworld / 40430017-1 / ≥99.0% / 1 g / 52.26
40430017-2 / ≥99.0% / 5 g / 197.42
40430017-3 / ≥99.0% / 10 g / 365.81
40430017-4 / ≥99.0% / 25 g / 740.31
Data were obtained from the Web on January 9, 2014.
Pigment composition of PSI complex
To determine the pigment composition of PSI, the concentrations for chlorophyll a (chla), chlorophyll b (chlb) and the sum of carotenoids were calculated with the following equations given in reference (Lichtenthaler and Buschmann 2001) for acetone with 20%(v/v) water solvent.
The concentrations of chla, chlb, and carotenoids, ca, cb, and c(x+c):
ca (mg/ml)=12.25 A663.2 – 2.79 A646.8
cb (mg/ml)=21.50 A646.8 – 5.10 A663.2
c(x+c) (mg/ml)=(1000 A470 – 1.82 ca – 85.02 cb )/198
The relative absorbance at specified wavelengths could be obtained from Fig. 2B. That is, A663.2=1.000, A646.8=0.244/0.245, and A470=0.346/0.344 were obtained to calculate the relative concentrations of pigments.
The molar mass 527.76, average of 21 complete b-carotenes and 1 incomplete b-carotene, was used for carotenoids. The number of Chla molecules is normalized to 96.00, thus the calculated number for b-carotene is 22.17 for PSI-TX and 21.91 for PSI-DDM respectively. The molar ratio of Chl/carotene is 4.36±0.05, which is well consistent with theoretical ratio of 96/22=4.36 , which is well consistent with theoretical ratio of Chl/carotene for the crystal structure of cyanobacterial PSI in which 22 carotenoid molecules have been identified and modeled as 21 complete and 1 incomeplete b-carotene (Jordan et al. 2001).
Table S3 – Pigments and their molar mass
Pigments / Chla / Chlb / Incomplete b-carotene / Complete b-caroteneMolar mass (g/mol) / 893.49 / 907.47 / 336.55 / 536.87
HPLC analysis of PSI pigments
The pigment composition of isolated PSI was determined using Waters HPLC. The LHCII samples were extracted with 80% acetone and loaded onto a C18 reverse phase HPLC column (4.6mm´150mm, 5μm, Waters SunFireTM). The column was first washed with 90% acetonitrile in water for 5 min, followed by a gradient from 0% to 95% ethyl acetate in 35 min. The HPLC system was equipped with a diode-array optical absorption detector, which allowed identification of the peaks in the chromatograms by their absorption spectra.
The absorption spectra and retention times of pigments extracted from PSI-DDM were the same as those of pigments extracted from PSI-TX. In the 480nm traces (Fig.7), small peaks 1-5 exhibiting the absorption spectra of b-carotene (Fig.S2), might be assigned as incomplete b-carotene, 9-cis, 9,9¢-cis, 9,13¢-cis and 13-cis b-carotene respectively, and peak 6 might be all-trans b-carotene according to the PSI structure revealed at 2.5 Å resolution by X-ray crystallography (Jordan et al. 2001). In the 663.9 nm traces (Fig.8), small peaks 1-3 exhibiting the absorption spectra of Chl a (Fig.S3), might be assigned as biosynthetic intermediates of Chl a. Peak 4-7 would be Chl a allomer, Chl a, Chl a¢ and Phe a respectively (Nakamura et al. 2003).
Fig. 7 HPLC traces of pigments extracted from PSI-DDM (A) and PSI-TX (B) detected at 480nm.
Peak 1, incomplete b-carotene; Peaks 2-5, 9-cis, 9,9¢-cis, 9,13¢-cis and 13-cis b-carotene;
Peak 6, all-trans b-carotene
Fig. 8 HPLC traces of pigments extracted from PSI-DDM (A) and PSI-TX (B) detected at 663.9nm.
Peaks 1-3, Chl a GG, Chl a DHGG and Chl a THGG; peak 4, Chl a allomer(s);
peak 5, Chl a; peak 6, Chl a¢; and peak 7, Phe a.
Fig. S2 – Absorption spectra of minor peaks detected in Fig.7 by HPLC
Peak 1, incomplete b-carotene; Peaks 2-5, 9-cis, 9,9¢-cis, 9,13¢-cis and 13-cis b-carotene.
Fig. S3 – Absorption spectra of minor peaks detected in Fig.8 by HPLC
Peaks 1-3, Chl a GG, Chl a DHGG and Chl a THGG; peak 4, Chl a allomer(s); peak 7, Phe a.
References
Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauß N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature 411 (6840):909-917. doi:http://dx.doi.org/doi:10.1038/35082000
Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. In: Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc. doi:http://dx.doi.org/10.1002/0471142913.faf0403s01
Nakamura A, Akai M, Yoshida E, Taki T, Watanabe T (2003) Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. European Journal of Biochemistry 270 (11):2446-2458. doi:http://dx.doi.org/10.1046/j.1432-1033.2003.03616.x
8
S