Allophycocyanin
Allophycocyanin (from Greek: ἄλλος (allos) meaning "other", φύκος (phykos) meaning “alga”, and κυανός (kyanos) meaning "blue") is a protein from the light-harvesting phycobiliprotein family, along with phycocyanin, phycoerythrin and phycoerythrocyanin. It is an accessory pigment to chlorophyll. All phycobiliproteins are water-soluble and therefore cannot exist within the membrane like carotenoids, but aggregate forming clusters that adhere to the membrane called phycobilisomes. Allophycocyanin absorbs and emits red light (650 & 660 nm max, respectively), and is readily found in Cyanobacteria (also called blue-green algae), and red algae. Phycobilin pigments have fluorescent properties that are used in immunoassay kits. In flow cytometry, it is often abbreviated APC. To be effectively used in applications such as FACS, High-Throughput Screening (HTS) and microscopy, APC needs to be chemically cross-linked.
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. The molecular weight of APC is 105,000 Daltons.
Spectral characteristics
| Absorption maximum | 652 nm |
| Additional Absorption peak | 625 nm |
| Emission maximum | 657.5 nm |
| Stokes Shift | 5.5 nm |
| Extinction Coefficient | 700,000 M−1cm−1 |
| Quantum Yield | 0.68 |
| Brightness | 1.6 x 105 M−1cm−1 |
Cross-linked APC
As mentioned above, in order for APC to be useful in immunoassays it must first be chemically cross-linked to prevent it from dissociating into its component subunits when in common physiological buffers.[1] The conventional method for accomplishing this is through a destructive process wherein the treated APC trimer is chemically disrupted in 8M urea and then allowed to re-associate through in a physiological buffer.[2] An alternative method can be used which preserves the structural integrity of the APC trimer and allows for a brighter, more stable end-product.[3]
Applications
Many applications and instruments were developed specifically for Allophycocyanin. It is commonly used in immunoassays such as FACS, flow cytometry, and High Throughput Screening as an acceptor for Europium via Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) assays.
References
- ↑ George C. Papageorgiou, Thoula Lagoyanni. Effects of chaotropic electrolytes on the structure and electronic excitation coupling of glutaraldehyde- and diimido ester-cross-linked phycobilisomes. Biochimica et Biophysica Acta (BBA) - Bioenergetics Volume 724, Issue 3, 30 September 1983, Pages 323–332
- ↑ Yeh SW, Ong LJ, Clark JH, Glazer AN. Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer. Cytometry. 1987 Jan; 8(1):91-5.
- ↑ United States Patent 7256050; High fluorescent intensity cross-linked allophycocyanin. Assigned to Columbia Biosciences Corp.