Phycobiliproteins are water-soluble proteins present in cyanobacteria and certain algae (rhodophytes, cryptomonads, glaucocystophytes). They capture light energy, which is then passed on to chlorophylls during photosynthesis. Phycobiliproteins are formed of a complex between proteins and covalently bound phycobilins that act as chromophores (the light-capturing part). They are most important constituents of the phycobilisomes.
YouTube Encyclopedic
-
1/3Views:10 0754 254710
-
Structural Studies of Phycobiliproteins from Spirulina - Amrita University
-
What Is Phycocyanin
-
Noam Adir-"Aspects of Phycobilisome Architecture"
Transcription
Structural Studies of Phycobiliproteins from Spirulina Spirulina is considered to be an excellent source of proteins. The structural composition of spirulina includes phycobiliproteins that contain a covalently attached chromophore called phycocyanobilin, which is in a planar conformation when the protein is in native form. When the protein is denatured, this chromophore undergoes a conformational change, leading to a change in the absorption spectrum which is recorded at 625nm using a UV spectrophotometer. This experiment examines the structural changes occurring in phycobiliproteins upon denaturation with potential denaturants like urea and potassium thiocyanate, employing UV-Vis spectroscopy Materials Required Weighing balance Spirullina tablets Watch glass Spatula. 100 ml beaker Sonication chamber Ammonium Sulphate powder Magnetic stirrer Procedure Arrange the required materials on the Lab bench. Preparation of Phycobiliproteins from spirullina. Place the weighing dish over the weighing balance and tare the balance to zero. Take four spirullina tablets open them and empty the contents into a watch glass. Using a spatula transfer the spirullina powder from the watch glass to the watch glass kept over the weighing balance and note the weight, which should be approximately 2g. Transfer the powder into a 100 ml beaker with a spatula. Using a 100ml measuring jar measure 50 ml of 0.1 Molar potassium phosphate buffer at pH 7. Pour this into the beaker containing the spirullina powder. Mix the solution using a glass rod. The colour of the solution changes to deep blue colour. The spirulina solution is then sonicated to break the cells and release the proteins. Sonication is done 5-6 times for 1 minute at intervals of 5 minutes. The solution is then centrifuged to remove the cell debris. For this, the solution is transferred into the centrifuge tubes. Centrifugation is done for 20 minutes at 24,000 RPM at 4 degree Celsius. After 20 minutes take out the tubes from the centrifuge and you will notice blue pellets in each tube. The supernatant is transferred into a 250 ml beaker. Take 50 ml of 0.1 Molar Potassium Phosphate Buffer at pH 7 in a measuring jar. Make the volume of the spirullina solution to 100 ml by adding 0.1 Molar Potassium Phosphate Buffer at pH 7 from the measuring jar. Weigh 29.00g of ammonium sulphate powder and add it to the spirullina solution. Mix the solution using a magnetic stirrer for 5 minutes. After stirring, the solution is again centrifuged to isolate the proteins. Centrifugation is done for 10 minutes at 16000 rpm at 40C. The supernatant is transferred into a 250ml beaker and discarded, whereas the pellets remain in each tube. Measure 25 ml buffer in a measuring jar and add it to the pellet in each tube. Dissolve the pellet in the added buffer by shaking the contents in the tubes with the hand. Transfer the solution into a conical flask. Protein Denaturation Studies. Transfer 3mL of Potassium Phosphate buffer into a cuvette using a pipette to be used as the blank. To a second cuvette, add 3 ml of Potassium Phosphate buffer using a pipette. Add 100 痞 protein solutions into this cuvette and mix it well using the pipette. Then place the cuvettes in the UV spectrometer slots and record the spectrum at 625nm. This solution is discarded and the cuvette is washed and dried. Now 1.5 ml potassium phosphate buffer is taken in the cuvette. Then add the denaturant 1.50 ml of the denaturant 8M potassium thiocyanate to the cuvette. And finally add 100 痞 protein solution and mix well using a pipette. Then place the cuvette in the spectrophotometer slot and record the spectrum at 625nm. This solution is discarded and the cuvette is washed well and dried. Taken 2.25ml of potassium phosphate buffer in a cuvette . Then add 750ul of the second denaturant 8M urea to the cuvette. Finally transfer 100 ul of protein solution to this cuvette. Mix well using a pipette. Place the cuvette in the spectrophotometer slot and record the spectrum at 625nm. After the measurements are completed, both the cuvettes are removed from the UV spectrometer
Major phycobiliproteins
| Phycobiliprotein | MW (kDa) | Ex (nm) / Em (nm) | Quantum yield | Molar Extinction Coefficient (M−1cm−1) | Comment | Image |
|---|---|---|---|---|---|---|
| R-Phycoerythrin (R-PE) | 240 | 498.546.566 nm / 576 nm | 0,84 | 1.53 106 | Can be excited by Kr/Ar laser Applications for R-Phycoerythrin Many applications and instruments were developed specifically for R-phycoerythrin. It is commonly used in immunoassays such as FACS, flow cytometry, multimer/tetramer applications. Structural Characteristics R-phycoerythrin is also produced by certain red algae. The protein is made up of at least three different subunits and varies according to the species of algae that produces it. The subunit structure of the most common R-PE is (αβ)6γ. The α subunit has two phycoerythrobilins (PEB), the β subunit has 2 or 3 PEBs and one phycourobilin (PUB), while the different gamma subunits are reported to have 3 PEB and 2 PUB (γ1) or 1 or 2 PEB and 1 PUB (γ2). |
 |
| B-Phycoerythrin (B-PE) | 240 | 546.566 nm / 576 nm | 0,98 | (545 nm) 2.4 106
(563 nm) 2.33 106 |
Applications for B-Phycoerythrin
Because of its high quantum yield, B-PE is considered the world's brightest fluorophore. It is compatible with commonly available lasers and gives exceptional results in flow cytometry, Luminex and immunofluorescent staining. B-PE is also less "sticky" than common synthetic fluorophores and therefore gives less background interference. Structural Characteristics B-phycoerythrin (B-PE) is produced by certain red algae such as Rhodella sp. The specific spectral characteristics are a result of the composition of its subunits. B-PE is composed of at least three subunits and sometimes more. The chromophore distribution is as follows: α subunit with 2 phycoerythrobilins (PEB), β subunit with 3 PEB, and the γ subunit with 2 PEB and 2 phycourobilins (PUB). The quaternary structure is reported as (αβ)6γ. |
 |
| C-Phycocyanin (CPC) | 232 | 620 nm / 642 nm | 0,81 | 1.54 106 | Accepts the fluorescence for R-PE; Its red fluorescence can be transmitted to Allophycocyanin | |
| Allophycocyanin (APC) | 105 | 651 nm / 662 nm | 0,68 | 7.3 105 | Excited by He/Ne laser; double labeling with Sulfo-Rhodamine 101 or any other equivalent fluorochrome. Applications for Allophycocyanin Many applications and instruments were developed specifically for allophycocyanin. It is commonly used in immunoassays such as flow cytometry and high-throughput screening. It is also a common acceptor dye for FRET assays. Structural Characteristics Allophycocyanin can be isolated from various species of red or blue-green algae, each producing slightly different forms of the molecule. It is composed of two different subunits (α and β) in which each subunit has one phycocyanobilin (PCB) chromophore. The subunit structure for APC has been determined as (αβ)3. |
 |
| ↑ = FluoProbes PhycoBiliProteins data | ||||||
Characteristics
Phycobiliproteins demonstrate superior fluorescent properties compared to small organic fluorophores, especially when high sensitivity or multicolor detection required :
- Broad and high absorption of light suits many light sources
- Very intense emission of light: 10-20 times brighter than small organic fluorophores
- Relative large Stokes shift gives low background, and allows multicolor detections.
- Excitation and emission spectra do not overlap compared to conventional organic dyes.
- Can be used in tandem (simultaneous use by FRET) with conventional chromophores (i.e. PE and FITC, or APC and SR101 with the same light source).
- Longer fluorescence retention period.
- High water solubility
Applications
Phycobiliproteins allow very high detection sensitivity, and can be used in various fluorescence based techniques fluorimetric microplate assays Archived 2018-03-18 at the Wayback Machine,[6][7][8] FISH and multicolor detection.
They are under development for use in artificial photosynthesis, limited by the relatively low conversion efficiency of 4-5%.[9]
References
- ^ Contreras-Martel, C.; Legrand, P.; Piras, C.; Vernede, X.; et al. (2000-05-09). "Crystal structure of R-phycoerythrin at 2.2 angstroms". RCSB Protein Data Bank (PDB). doi:10.2210/pdb1eyx/pdb. PDB ID: 1EYX. Retrieved 11 October 2012.
{{cite journal}}: Cite journal requires|journal=(help) - ^ Contreras-Martel C, Martinez-Oyanedel J, Bunster M, Legrand P, Piras C, Vernede X, Fontecilla-Camps JC (January 2001). "Crystallization and 2.2 A resolution structure of R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning". Acta Crystallographica D. 57 (Pt 1): 52–60. doi:10.1107/S0907444900015274. PMID 11134927. S2CID 216930. PDB ID: 1EYX.
- ^ a b Image created with RasTop (Molecular Visualization Software).
- ^ Camara-Artigas, A. (2011-12-16). "Crystal Structure of the B-phycoerythrin from the red algae Porphyridium cruentum at pH8". RCSB Protein Data Bank (PDB). doi:10.2210/pdb3v57/pdb. PDB ID: 3V57. Retrieved 12 October 2012.
{{cite journal}}: Cite journal requires|journal=(help) - ^ Camara-Artigas A, Bacarizo J, Andujar-Sanchez M, Ortiz-Salmeron E, Mesa-Valle C, Cuadri C, Martin-Garcia JM, Martinez-Rodriguez S, Mazzuca-Sobczuk T, Ibañez MJ, Allen JP (October 2012). "pH-dependent structural conformations of B-phycoerythrin from Porphyridium cruentum". The FEBS Journal. 279 (19): 3680–3691. doi:10.1111/j.1742-4658.2012.08730.x. PMID 22863205. S2CID 31253970. PDB ID: 3V57.
- ^ "MicroPlate Detection comparison between SureLight P-3L, other fluorophores and enzymatic detection Table 1: Comparison of honeypot with other detection methods". Columbia Biosciences. 2010. doi:10.7717/peerj-cs.350/table-1.
- ^ "Flow Cytometry" (PDF). Archived from the original (PDF) on 2018-03-18. Retrieved 2014-06-07.
- ^ Telford, William G; Moss, Mark W; Morseman, John P; Allnutt, F.C.Thomas (August 2001). "Cyanobacterial stabilized phycobilisomes as fluorochromes for extracellular antigen detection by flow cytometry". Journal of Immunological Methods. 254 (1–2): 13–30. doi:10.1016/s0022-1759(01)00367-2. ISSN 0022-1759. PMID 11406150.
- ^ Lavars, Nick (2021-10-19). "Encasing algae triples the efficiency of artificial photosynthesis". New Atlas. Retrieved 2021-10-24.
This page was last edited on 4 October 2023, at 17:29