PE fluorophore, also known as R-phycoerythrin or PE, is a fluorescent protein pigment found in red algae. It is widely used in biotechnology applications as a tracer due to its bright orange-red fluorescence when excited by blue or green light.
PE belongs to the phycobiliprotein family of light-harvesting pigments along with other proteins like allophycocyanin and phycocyanin. These pigments play a key role in photosynthesis in red algae and cyanobacteria by absorbing light energy and transferring it to chlorophyll where photosynthesis takes place.
Some key properties of PE fluorophore:
- Fluoresces brightly when excited by 488 nm blue laser light or 532 nm green laser light
- Has a peak excitation of 565 nm and peak emission of 578 nm
- High extinction coefficient allowing detection of very low quantities
- Very large Stokes shift (separation between excitation and emission peaks) allowing easy discrimination from excitation light
- High quantum yield of 0.98 (very efficient at fluorescence)
- Stable over a wide pH range from 6-8
These useful properties have led to PE becoming one of the most commonly used fluorophores in flow cytometry, immunology, cell biology research and clinical diagnostics.
Structure
The PE fluorophore consists of a complex of phycobiliproteins that form a hexamer structure made up of α and β subunits. Each subunit contains a brilliantly fluorescent chromophore called phycoerythrobilin (PEB).
PEB is a linear tetrapyrrole structure covalently attached to conserved cysteine residues on the α and β subunits. There are typically 1-2 PEB molecules on each α subunit and 2-3 on each β subunit. The specific arrangement of the subunits brings the PEB molecules in close proximity allowing efficient energy transfer between them.
In total, each PE hexamer contains 18-30 PEB fluorophores giving an exceptionally high fluorescence quantum yield. The complex quaternary structure and assembly of chromophores is what gives PE its unique fluorescent properties compared to single organic dye molecules.
Applications
The bright orange-red fluorescence of PE makes it extremely useful as a tracer across many different applications:
Flow Cytometry
PE is one of the most commonly used fluorophores in multicolor flow cytometry along with fluorophores like FITC and APC. It is used to label antibodies targeted against specific cell surface markers. When passed through the flow cytometer, cells positive for that marker will glow bright orange-red allowing counting and sorting of cell populations. Using 3-4 different colored fluorophores allows simultaneous analysis of multiple markers.
Immunochemistry
In microscopy, PE can be used to visualize the location of antigens and biomarkers within cells and tissues. PE-labeled primary antibodies bind to targets and illuminate their localization when visualized under a fluorescence microscope.
Nucleic acid detection
PE can be coupled to oligonucleotide probes to detect specific DNA/RNA sequences using techniques like fluorescence in situ hybridization (FISH). PE’s brightness allows detection of scarce nucleic acid targets.
Cell labeling and tracing
PE fluorescent dye can be loaded into cells or attached to their surface to then allow tracking and visualization of those labeled cells over time as they move, divide, interact with other cells etc.
Biomolecular labeling
PE can be chemically conjugated to proteins, antibodies, nucleic acids or beads to act as a detectable label or tag in biomolecular research experiments. Assays and techniques like ELISA, pull-down assays, protein labeling, immunoprecipitation all utilize PE for sensitive fluorescent detection of biomolecules.
Clinical diagnostics
PE is used to diagnose diseases by identifying abnormal cells using flow cytometry or microscopic examination of patient samples stained with PE fluorophore biomarkers. Some examples are leukemia diagnosis by abnormal white blood cell staining patterns and HIV quantification by detecting PE stained viral antigens.
Comparison with Organic Fluorophores
While conventional synthetic organic dye molecules like FITC and Alexa Fluor are also widely used, the PE fluorophore has some advantages:
- Brighter fluorescence intensity – PE’s multiple PEB chromophores act cooperatively to give very high brightness.
- Large stokes shift – The ~100 nm separation between excitation and emission peaks reduces background interference.
- Less photobleaching – PE is more resistant to permanent photobleaching than organic dyes during extended illumination.
- More stable pH/solvent resistance – PE maintains fluorescence over a wider range of pH and in different solvents.
However, organic dyes have some benefits like smaller size, the ability to chemically modify their structure, and lower cost at large manufacturing scales. So PE and synthetic dyes each have their own niche uses based on the needs of the application.
Preparation
While PE can be purified from natural sources in red algae, it is more commonly prepared by bioengineering processes to obtain recombinant PE protein:
- The genes encoding the α and β subunits are cloned into bacterial plasmids.
- These are transfected into bacteria like E. coli which then express large quantities of the recombinant subunits.
- The subunits are isolated, purified and combined under conditions that allow hexamer assembly with the PEB chromophores.
- Any excess subunits are removed, leaving just the intact fluorescent PE complex.
The recombinant approach allows large scale production of PE protein tuned to have desired properties like enhanced brightness, solubility or chemical attachment sites. Bioengineered PE typically has higher purity, yield and performance than directly purified PE.
Chemical Modification
While native PE has excellent fluorescence, it can be chemically modified and engineered in several ways to improve its properties and adapt it for different uses:
- Crosslinking – PE subunits can be crosslinked with reagents like glutaraldehyde to stabilize the complex and prevent subunit dissociation.
- Mutagenesis – Site directed mutagenesis of the PE gene sequence can install unique cysteines or lysines to allow specific chemical coupling reactions.
- PEGylation – Attachment of polyethylene glycol polymer chains can improve PE solubility and reduce non-specific binding.
- Bioconjugation – Amine, sulfhydryl, or carboxyl groups are introduced to enable covalent coupling of PE to proteins, antibodies, streptavidin, oligonucleotides, beads etc.
These modifications allow creation of PE reagents optimized for use in immunoassays, nucleic acid hybridization, flow cytometry, cell labeling experiments and other applications.
Spectroscopic Properties
PE has the following key spectroscopic properties that underlie its use as a bright orange-red fluorescent tracer:
| Property | Value |
|---|---|
| Excitation maximum | 565 nm |
| Emission maximum | 578 nm |
| Extinction coefficient | 1.96 x 106 M-1cm−1 |
| Quantum yield | 0.98 |
| Brightness | 1.92 x 106 M-1cm−1 |
| Stokes Shift | ~13 nm |
These optical properties allow PE fluorophore to be efficiently excited and visualized when used in fluorescence-based experiments and instruments. The high extinction coefficient and quantum yield give PE extremely bright orange fluorescence that can be readily detected even at very low concentrations.
Conclusion
In summary, PE fluorophore is an exceptionally bright, stable and useful fluorescent tag widely used in many biotechnology techniques and clinical diagnostics applications. Its multiple, cooperating PEB chromophore structure provides advantages over conventional synthetic organic dyes for techniques like flow cytometry, cell tracing, immunoassays, and nucleic acid detection. Continuing protein engineering efforts are further enhancing PE brightness, photostability and ease of chemical modification to expand its capabilities and applications. PE’s unique Structure and properties will ensure it continues to be a highly valued fluorescent tool for biological research and clinical analysis.