Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Summary

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

Abstract

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Introduction

fPhycobiliproteins are water-soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria (Cyanophyta) and several eukaryotic taxa (Glaucophyta, Rhodophyta, and Cryptophyta)¹. They occur mainly as supramolecular complexes called phycobilisomes and they are typically attached to the surface of the photosynthetic membranes on the stromal side, with the exception of Cryptophyta, where the phycobiliproteins are localized in the thylakoid lumen². Four types of phycobiliproteins have been identified up to date: the c....

Protocol

1. Cyanobacteria Cultivation

1. Cultivate Synechocystis cells in Erlenmeyer flasks or in photobioreactors^10,19 in buffered BG11 medium²⁰ to maintain a pH of < 10 (e.g., using 17 mM HEPES¹⁰).
   NOTE: Standard cultivation conditions require a controlled temperature (typically, 30 °C, the optimal temperature is 35 °C)²¹, illumination (typically, a white light of an intensity up to 800 µmol[photons]/[m²·s])²¹, and a CO₂ supply (in the 400-mL flat-panel photobio....

Results

For the initial method tests, Synechocystis was cultivated as batch cultures in Erlenmeyer flasks on a shaker in BG11 cultivation medium²⁰ (supplemented with 17 mM HEPES) at 25 °C, under a warm white light of an intensity of 50 µmol(photons)/(m²·s) and with 1% CO₂ in the culturing atmosphere. During the cultivation, the cultures were sampled to safe-lock tubes and centrifuged (15,000 x g at laboratory temperature for 5 min), .......

Discussion

This protocol describes a simple, fast, and reproducible method for the quantification of phycobiliprotein content in the model cyanobacterium Synechocystis. Several methods of cell homogenization, protein extraction, and phycocyanin and allophycocyanin quantification are compared, and the final protocol represents a combination of the optimal steps of every single procedure. As representative data, the content of phycobiliproteins was quantified in Synechocystis cells under increasing light intensity. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Synechocystis sp. PCC 6803	Institut Pasteur, Paris, France	6803	Cyanobacterium strain
Roti-CELL PBS	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	9143.1	Phosphate-Buffered Saline (PBS) solution, pH 7.4
Eppendorf safe-lock tubes	Eppendorf, Hamburk, Germany	30120086	Safe-lock tubes 1.5 ml
VWR 80-Place Storage System	VWR International, Radnor, Pennsylvania, USA	30128-282	Holder for safe-lock tubes
RAININ 100 µl -1000 µl	Mettler-Toledo, Columbus, Ohio, USA	17014382	Pipette
GP-LTS-A-1000µL-/F-768/8	Mettler-Toledo, Columbus, Ohio, USA	30389272	Pipette tips
Rotina 420R	Hettich, Kirchlengern, Germany	4701	Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes
LCexv 4010	Liebherr, Bulle, Switzerland	9005382197172	Refrigerator and freezer -20 °C
Revco ExF -86°C Upright Ultra-Low Temperature Freezer	Thermo Fisher Scientific, Waltham, Massachusetts, USA	EXF24086V	Freezer -80 °C
CoolSafe	LaboGene, Lillerød, Denmark	7.001.000.615	Freeze dryer
UV-2600	Shimadzu, Kyoto, Japan	UV-2600	Spectrophotometer
Hellma absorption cuvettes, semi Micro	Sigma-Aldrich, St. Louis, Missouri, USA	Z600288	VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm
Silamat S6	Ivoclar Vivadent, Schaan, Liechtenstein	602286WU	Homogenizer
Solid-glass beads	Sigma-Aldrich, St. Louis, Missouri, USA	Z273627	Glass bead of the diameter 2 mm
CPA225D-0CE	Sartorius AG, Göttingen, Germany	SECURA225D-1OBR	Analytical balances
C-Phycocyanin from Spirulina sp.	Sigma-Aldrich, St. Louis, Missouri, USA	P2172	Phycocyanin standard
Allophycocyanin	Sigma-Aldrich, St. Louis, Missouri, USA	A7472	Allophycocyanin standard
Bicinchoninic Acid Kit	Sigma-Aldrich, St. Louis, Missouri, USA	BCA1, B9643	Complete kit for total proteins determination
AlgaeTron	Photon System Instruments Ltd., Drásov, Czech Republic	AG 130-ECO	Cultivation chamber for E. flasks, with controllable light and atmosphere
Photobioreactor	Photon System Instruments Ltd., Drásov, Czech Republic	FMT-150	Cultivation equipment for cyanobacteria and algae with completely controllable environment
Cellometer	Nexcelom Bioscience, Lawrence, Massachusetts, USA	Auto M10	Cell counter
Corning 15 mL centrifuge tubes	Sigma-Aldrich, St. Louis, Missouri, USA	CLS430791	15 ml Centrifuge tube for dry weigth sampling
Herasafe KS	Thermo Fisher Scientific, Waltham, Massachusetts, USA	51024579	Laminar flow hood