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Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

Wang L, Wang S, Fu X, Sun L - PLoS ONE (2015)

Bottom Line: Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6.The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1.These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ').

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Yantai University, Yantai, Shandong, P. R. China.

ABSTRACT
An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ').

No MeSH data available.


The polypeptide analysis of the purified R-PE by SDS-PAGE.The SDS-PAGE was performed with a gradient separating gel of 13%-17% (w/v) in pH 9.2 Tris-HCl buffer and 4% (w/v) stacking gel in pH 6.8 Tris-HCl buffer. Lane 1 and 3 showed the polypeptide bands after Coomassie Blue G-250 staining. Lane 2 and 4 showed fluorescent bands of the subunits under UV-light at 365 nm after Zn(SO4)2 staining. Lane M showed marker proteins. The right part was the profile curve corresponding to band density and area of the Coomassie Blue G-250 stained pattern of the SDS-PAGE.
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pone.0120333.g006: The polypeptide analysis of the purified R-PE by SDS-PAGE.The SDS-PAGE was performed with a gradient separating gel of 13%-17% (w/v) in pH 9.2 Tris-HCl buffer and 4% (w/v) stacking gel in pH 6.8 Tris-HCl buffer. Lane 1 and 3 showed the polypeptide bands after Coomassie Blue G-250 staining. Lane 2 and 4 showed fluorescent bands of the subunits under UV-light at 365 nm after Zn(SO4)2 staining. Lane M showed marker proteins. The right part was the profile curve corresponding to band density and area of the Coomassie Blue G-250 stained pattern of the SDS-PAGE.

Mentions: As shown in Fig. 6, after the SDS-PAGE electrophoresis, the polypeptides were first visualized under 365 nm after Zn(SO4)2 staining (Fig. 6 lane 2 and 4) and then observed after CB G-250 staining (Fig. 6 lane 1 and 3). The results showed that the two staining methods gave the same polypeptide band patterns (Fig. 6). This demonstrates that the R-PE is merely composed of chromophore-carrying subunits and contains no colorless liner, for the Zn(SO4)2 staining specifically visualizes chromophore-containing subunits based on Zn+2 enhancing subunit fluorescence emission by coordinating with subunit chromophores. The subunit bands located at 18.2 kD, 20.6 kD, 34.6 kD and 31.6 kD correspond to α, β, γ and γ′ subunits of the R-PE, respectively. Besides the four subunits, other two weak fluorescent bands located at higher molecular mass were proved to be the subunit complexes which are not completely dissociated into subunits.


Characteristics of an R-phycoerythrin with two γ subunits prepared from red macroalga Polysiphonia urceolata.

Wang L, Wang S, Fu X, Sun L - PLoS ONE (2015)

The polypeptide analysis of the purified R-PE by SDS-PAGE.The SDS-PAGE was performed with a gradient separating gel of 13%-17% (w/v) in pH 9.2 Tris-HCl buffer and 4% (w/v) stacking gel in pH 6.8 Tris-HCl buffer. Lane 1 and 3 showed the polypeptide bands after Coomassie Blue G-250 staining. Lane 2 and 4 showed fluorescent bands of the subunits under UV-light at 365 nm after Zn(SO4)2 staining. Lane M showed marker proteins. The right part was the profile curve corresponding to band density and area of the Coomassie Blue G-250 stained pattern of the SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4364535&req=5

pone.0120333.g006: The polypeptide analysis of the purified R-PE by SDS-PAGE.The SDS-PAGE was performed with a gradient separating gel of 13%-17% (w/v) in pH 9.2 Tris-HCl buffer and 4% (w/v) stacking gel in pH 6.8 Tris-HCl buffer. Lane 1 and 3 showed the polypeptide bands after Coomassie Blue G-250 staining. Lane 2 and 4 showed fluorescent bands of the subunits under UV-light at 365 nm after Zn(SO4)2 staining. Lane M showed marker proteins. The right part was the profile curve corresponding to band density and area of the Coomassie Blue G-250 stained pattern of the SDS-PAGE.
Mentions: As shown in Fig. 6, after the SDS-PAGE electrophoresis, the polypeptides were first visualized under 365 nm after Zn(SO4)2 staining (Fig. 6 lane 2 and 4) and then observed after CB G-250 staining (Fig. 6 lane 1 and 3). The results showed that the two staining methods gave the same polypeptide band patterns (Fig. 6). This demonstrates that the R-PE is merely composed of chromophore-carrying subunits and contains no colorless liner, for the Zn(SO4)2 staining specifically visualizes chromophore-containing subunits based on Zn+2 enhancing subunit fluorescence emission by coordinating with subunit chromophores. The subunit bands located at 18.2 kD, 20.6 kD, 34.6 kD and 31.6 kD correspond to α, β, γ and γ′ subunits of the R-PE, respectively. Besides the four subunits, other two weak fluorescent bands located at higher molecular mass were proved to be the subunit complexes which are not completely dissociated into subunits.

Bottom Line: Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6.The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1.These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ').

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Yantai University, Yantai, Shandong, P. R. China.

ABSTRACT
An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α18.2, β20.6, γ31.6 (γ'), γ34.6 (γ), and no colorless polypeptide; its subunit composition was 6α18.2:6β20.6:1 γ31.6:2γ34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)3 γ (αβ)3γ' and γ (αβ)3 γ'(αβ)3 γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)3 face-to-face with the aid of one γ subunit (γ or γ').

No MeSH data available.