doi:10.1016/j.pep.2008.09.013
Copyright © 2008 Published by Elsevier Inc.
Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica
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Li Suna, Shumei Wangb, 
, 
, Xueqin Gonga, Mingri Zhaoa, Xuejun Fua and Lu Wanga
aCollege of Chemistry Engineering and Biology Sciences, Yantai University, No. 30 Qingquan Road, Yantai 264005, PR China
bCollege of Photo-electronic Information Science and Technology, Yantai University, Yantai 264005, PR China
Received 21 August 2008;
revised 16 September 2008.
Available online 27 September 2008.
Abstract
R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A565 to A280 of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.
Keywords: Phycobiliprotein; R-phycoerythrin; Heterosiphonia japonica; Chromatography; Gel electrophoresis; Isoelectric focusing
Fig. 1. The R-PE isolation from the R-PE extract by the gel filtration with a Sepharose CL-4B column (50 × 460 mm). The filtration was developed at a flow rate of 60 ml/h with: (a) the 50 mM phosphate buffer (pH 7.0) containing 4 mM NaN3 and 2 mM EDTA; (b) the 50 mM phosphate buffer (pH 7.0) containing 0.4% (v/v) Triton X-100. The elution was monitored by the absorption at 280 nm.
Fig. 3. The absorption spectra of the R-PE samples in pH 7.0 phosphate buffer: (a) the R-PE extract; (b) the salting-out R-PE extract; (c) the R-PE fraction from the Sepharose CL-4B gel filtration of the R-PE extract; (d) the R-PE fraction from the Sepharose CL-4B gel filtration of the salting-out R-PE extract; (e) the R-PE fraction from the Sephadex G-200 gel filtration of the salting-out R-PE extract; (f) the R-PE fraction from the Sephadex G-200 gel filtration following the Sepharose CL-4B gel filtration developed with no Triton buffer; (g) the R-PE fraction from the ion exchange chromatography on DEAE Sepharose FF. The fluorescence emission spectrum (h) of the purified R-PE was recorded in pH 7.0 phosphate buffer on the excitation at 495 nm.
Fig. 2. The R-PE isolation by the gel filtration with a Sephadex G-200 column (50 × 540 mm) from three R-PE samples: (a) the R-PE extract; (b) the salting-out R-PE extract; (c) the salting-out R-PE extract filtered with a sieve of 1000–1200 mesh before being loaded on the column (50 × 610 mm). The gel filtrations were developed with the 50 mM phosphate buffer (pH 7.0) containing 4 mM NaN3 and 2 mM EDTA at a flow rate of 60 ml/h and monitored by the absorption at 280 nm.
Fig. 4. The Sephadex G-200 gel filtration of the R-PE fraction from the Sepharose CL-4B chromatography developed with the 50 mM phosphate buffer (pH 7.0) containing 0.4% (v/v) Triton X-100 (a), and that of the 0.4% (v/v) Triton X-100 in the 50 mM phosphate buffer (pH 7.0) containing Blue Dextran 2000 and p-aminobenzene sulfonic acid (b). The Sephadex G-200 column (50 × 610 mm) was eluted with the 50 mM phosphate buffer (pH 7.0) at a flow rate of 60 ml/h and the elution was monitored by the absorption at 280 nm.
Fig. 5. The purification of the R-PE from the fractions of the gel filtrations by the DEAE Sepharose FF column (26 × 100 mm) chromatography developed at a flow rate of 60 ml/h with a linear ionic strength gradient of NaCl from 100 to 400 mM in 50 mM phosphate buffer (pH 7.0) of 400 ml. The elution was monitored by the absorption at 280 nm.
Fig. 6. The purity of the R-PE prepared by the DEAE Sepharose FF chromatography was examined by the native PAGE performed in the neutral (lanes A and B) and alkaline (lanes C and D) buffer systems. For the PAGE in the neutral buffer system, the resolving gel of 6.5% (w/v) polyacrylamide was in the pH 7.5 Tris–HCl buffer and the electrode buffer was the pH 7.0 Tris–diethylbarbituric acid buffer; for the PAGE in the alkaline buffer system, the resolving gel of 8.0% (w/v) polyacrylamide was in pH 8.8 Tris–HCl buffer and the pH 8.3 Tris–Glycine buffer was used as the electrode buffer. After the electrophoresis, the protein band was observed under ultraviolet light at 365 nm (lanes A and C) and then after Coomassie Blue G-250 staining (lanes B and D).
Fig. 7. The polypeptide analysis of the R-PE from the DEAE Sepharose FF chromatography by the SDS–PAGE with the separating gel of 13% (w/v) polyacrylamide. (M) Coomassie Blue G-250 staining bands of marker proteins; (A) polypeptide bands of the R-PE showed by Coomassie Blue G-250 staining; (B) fluorescent bands of the R-PE chromophore-carrying subunits exhibited under ultraviolet light at 365 nm after the slab gel was stained with 0.2 M ZnSO4.
Fig. 8. The isoelectric focusing of the R-PE prepared by the DEAE Sepharose FF chromatography. The IEF was performed in both of the native (lanes A–C) and denaturing (lanes D–F) conditions from pH 4.0 to 6.5. The bands showed in the native IEF were: (A) yellow fluorescence under ultraviolet light at 365 nm; (B) native red; and (C) Coomassie Blue G-250 staining. The polypeptide bands exhibited in the denaturing IEF were: (D) orange red fluorescence under ultraviolet light at 365 nm; (E) native red; and (F) Coomassie Blue G-250 staining.
Fig. 9. Chromatographic isolation on Sephacryl S-300 HS column (20 × 500 mm) for the R-PE from the salting-out R-PE extract and the R-PE fraction obtained by the Sepharose CL-4B gel filtration developed with the 50 mM phosphate buffer (pH 7.0) containing 0.4% (v/v) Triton X-100: (a) solid line is for the salting-out R-PE extract, and short dash line is for the purified R-PE; (b) solid line is for the R-PE fraction from the Sepharose CL-4B gel filtration, and short dash line is for the 0.4% (v/v) Triton X-100 in the 50 mM phosphate buffer (pH 7.0) containing Blue Dextran 2000 and p-aminobenzene sulfonic acid. The column was eluted with 150 mM NaCl in 50 mM phosphate buffer (pH 7.0) at a flow rate of 20 ml/h and the elution was monitored by the absorption at 280 nm.
Table 1.
Absorbance of six R-PE samples at 565 and 280 nm and their ratio of absorbance at 565 nm to that at 280 nm in pH 7.0 phosphate buffer
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