Biliprotein

Biliproteins found in plants and algae serve as a system of pigments whose purpose is to detect and absorb light needed for photosynthesis.  The absorption spectra of biliproteins complements that of other photosynthetic pigments such as chlorophyll or carotene.[3]  The pigments detect and absorb energy from sunlight; the energy later being transferred to chlorophyll via internal energy transfer.[4] According to a 2002 article written by Takashi Hirata et al., the chromophores of certain phycobiliproteins are responsible for antioxidant activities in these biliproteins, and phycocyanin also possesses anti-inflammatory qualities due to its inhibitory apoprotein. When induced by both collagen and adenosine triphosphate (ADP), the chromophore phycocyanobilin suppresses platelet aggregation in phycocyanin, its corresponding phycobiliprotein.[5]

In insects, biliprotein lipocalins generally function to facilitate the changing of colours during camouflage, but other roles of biliproteins in insects have also been found. Functions such as preventing cellular damage, regulating guanylyl cyclase with biliverdin, among other roles associated with metabolic maintenance, have been hypothesised but yet to be proven. In the tobacco hornworm, the biliprotein insecticyanin (INS) was found to play a crucial part in embryonic development, as the absorption of INS into the moth eggs was observed.[6]

Phycobiliproteins are found in cyanobacteria (also known as blue-green algae) and algae groups such as rhodophyta (red algae) and cryptophytes.[10] Major phycobiliproteins include variations of phycocyanin (blue-pigment), variations of phycoerythrin (red pigment), and allophycocyanin (light-blue pigment); each of them possessing different spectral properties.  These water-soluble biliproteins are not essential for the functioning of cells. Some special qualities of phycobiliproteins include antioxidant properties and high fluorescence, and it is their chromophores that give these proteins their strong pigment.[5][11] Phycobiliproteins are classified into two categories based on their amino-terminal sequences: "α-type" and "β-type" sequences. In biliproteins where the number of bilins on the two subunits is unequal, the subunit with more bilins has a β-type amino sequence.[12]

Phycochromes are a subclass of phycobiliprotein that was initially recognised only as light sensory pigments in cyanobacteria. They are now deemed to constitute of all possible photoreversibly photochromic pigments, regardless of function. They are also found in red algae.[10][13] In a series of journal articles written by G.S. and L.O. Björn, it was reported that phycochromes a, b, c and d were discovered by scientists who fractionated samples of blue-green algae using electrofocusing. The fractions with isoelectric points at or around 4.6 seemed analogous to phytochromes in that they possessed photochromic properties, yet were sensitive to light of shorter wavelengths. All four phycochromes except phycochrome c were extracted from the blue-green algae Tolypothrix distorta; whereas phycochrome a was also found in Phormidium luridum, Nostoc muscorum 1453/12 and Anacystis nidulans; and phycochrome c was extracted from Nostoc muscorum A and Tolypothrix tenuis.[14][15]

Phytochromes (also known as phys) were initially discovered in green plants in 1945. The photoreversible pigment was later found in fungi, mosses, and other algae groups due to the development of whole-genome sequencing, as explained in Peter H. Quail's 2010 journal article Phytochromes.[16]  As described in Hugo Scheer's 1981 journal article Biliproteins, phytochromes function as a sensor of light intensity in ‘high-energy’ reactions, i.e. in higher plants (e.g. underground seedlings), during transformation of heterotrophic blanching growth to autotrophic photosynthetic growth.[10] They carry out this function by monitoring the various parameters of light signals (such as presence/absence, colour, intensity and photoperiodicity). This information is then transduced via intracellular signaling pathways that trigger responses specific to the organism and its development state on both cellular and molecular levels, as explained by Quail. Phytochromes are also responsible for regulating many aspects of a plant's growth, development and reproduction throughout its lifecycle.[16]

The large white butterfly (Pieris brassicae), from which the biliprotein known as 'bilin-binding protein' was extracted.

The lipocalins that have been identified as biliproteins have been found in a wide variety of insects, but mainly in the order Lepidoptera. Scientists have discovered them in the large white butterfly and a number of moth and silkmoth species, including the ailanthus and domestic silkmoths, giant silkworm moth, tobacco hawk moth, honeycomb moth, and the puss moth.[6][8] The biliproteins associated with these insect species are the bilin-binding proteins, biliverdin-binding proteins, bombyrin, lipocalins 1 and 4, insecticyanin, gallerin and CV-bilin respectively.[6][7] The biliproteins found in the tobacco hawk moth and pussmoth make up a major part of the insects’ haemolymph fluids.

The biliproteins that have been found in other insect orders apart from Lepidoptera still have unknown sequences, and so their lipocalin nature is still open.[6]

Fluorescent proteins have had a substantial impact on bioimaging, which is why biliproteins have made suitable candidates for the application, due to their properties of fluorescence, light-harvesting, light-sensitivity and photoswitching (the latter occurring only in phytochromes). Phycobiliproteins, which are highly fluorescent, have been used in external applications of bioimaging since the early 1980s. That application requires the bilin chromophore to be synthesised from haem, after which a lyase is needed to covalently bond the bilin to its corresponding apoprotein. An alternative method of uses phytochromes instead; some phytochromes only require one enzyme, haem oxygenase, for synthesising chromophores. Another benefit of using phytochromes is that they bind to their bilins autocatalytically. While there are photochromic pigments with poor fluorescence, this problem has been alleviated by engineering protein variants that reduce photochemistry and enhance fluorescence.[17]

Properties of phycobiliproteins, such as their natural antioxidant, anti-inflammatory, food colourant, strong pigment and anti-aging activities, have given them considerable potential for use in food, cosmetics and medicinal applications. They have also proven to be therapeutic in treating diseases such as Alzheimer's disease and cancer. Given their large range of applications and potential uses, researchers have been trying to find and develop ways to produce and purify phycobiliproteins to meet the growing demand for them.[18] One such phycobiliprotein is C-phycocyanin (C-PC), which is found in spirulina. A limiting factor of C-PC's usage in these applications is its protein stability, given that in its natural form, C-PC is highly sensitive to light and heat when in aqueous solution, due to its photosensitive phycocyanobilin (PCB) chromophore, which also makes it prone to free-radical oxidation. Like other natural food colourants, C-PC is also sensitive to acidic conditions and oxidant exposure. This has prompted studies to develop methods of stabilising C-PC/PCB and expand their applications to other food systems.[19]

More details on the applications of phycocyanin in food and medicine can be found here.

The fluorescence signals emitted from phycoerythrin and phycocyanin have made them suitable for use as indicators to detect cyanotoxins such as microcystins in drinking water. A study examined the nature of the biliproteins' fluorescence signals regarding their real-time character, sensitivity and the biliproteins' behaviour in different treatment stages (of water) in comparison to microcystins. The fluorescence signals' real-time character was confirmed by fluorescence measurements, as they can be carried out without having to pre-concentrate the biliproteins. If the ratio of biliprotein to microcystin is above 1, the fluorescence signals can estimate very low concentrations of microcystins. A test conducted in 2009 compared the behaviour of both biliproteins and selected microcystins MC-LR and MC-RR during water treatment. The test results showed that the biliproteins have an early warning function against microcystins in conventional treatment stages that use pre-oxidation with permanganate, activated carbon and chlorination. However, the early warning function does not occur when chlorine dioxide is used as a pre-oxidant or final disinfectant. It is important for the biliprotein/toxin ratio of raw water to be known in order to use the biliproteins for control measurements in drinking water treatment.[20]