**3. Structure of phycocyanin**

The biological and pharmacological properties of *Spirulina* were attributed mainly to Cphycocyanin (CPC). CPC is a major light-harvesting or photosynthetic pigment protein present in the antenna rods of *Spirulina platensis*. *S. platensis* also contains allophycocyanin (APC) as a minor component present at the core of the antenna rods. CPC and APC including phycoerythrin (PE) are the principal classes of phycobiliproteins which form supramolecular complexes known as phycobilisomes assemblies in cyanobacteria (Figure. 2a). In phycobiliproteins, a linear tetrapyrrole (bilin) as the chromophore is covalently attached to the apoprotein by thioether bonds to cysteine residues. CPC molecule is composed of two kinds of subunits, α- and β-subunits to form trimeric aggregation α3β3 (Figures. 2b and 2c). Padyana et al. (2001) solved the crystal structure of CPC by molecular replacement technique (Figures. 2d and 2e). The α- and β-subunit polypeptides exhibit high affinity for one another and associate into (αβ)-monomers, which in turn aggregate into (αβ)3-trimers and (αβ)6-hexamers (Figure. 2f).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 123

A phycobilisome (a) has six antenna rods with a three-cylinder core of allophycocyanin, APC (circles), two of the core cylinders lie on the thylakoid membrane, while the third one does not. Each rod has four hexameric disk-like structures, two of phycoerythrin, PE (red), and two of phycocyanin, CPC (blue) (MacColl, 2004). CPC consists of αand β-subunit polypeptides to form trimeric aggregation α3β3 and nine phycocyanobilin moieties as a chromophore shown in closed circles (b). c shows chemical structure of phycocyanobilin (Li et al., 2006). d and e show ribbon representation of CPC α-subunit and CPC β-subunit, respectively, with chromophores shown in ball and stick representation (Padyana et al., 2001). f shows coil representation of the two (αβ)6-hexamers in the crystal asymmetric unit, and the box drawn at the center highlights the close proximity of phycocyanobilins at the position 155 on each β-

**Figure 2.** Schematic representation of one type of phycobilisome (a), and various representations of C-

**Preparation of phycocyanin solution in the experiments** Phycocyanin was extracted from spray-dried *Spirulina platensis* with 50 mM sodium-phosphate buffer (pH 6.0). The crude extract was partially purified by DE-52 ion-exchange chromatography. The eluate was dialyzed against distilled water and lyophilized. Phycocyanin contents of the resultant powder were over 80%, and the recovery from the crude extract was approximately 6%. The

subunit in the region between the adjacent hexamers (Padyana et al., 2001).

d. e.

phycocyanin (b - f).

f.

The medicinal and pharmacological properties of CPC have been reported earlier (Romay et al., 1998). Recent studies have demonstrated the antioxidant (Vadiraja et al., 1998; Wu and Annie Ho, 2008), anti-inflammatory (Reddy et al., 2000), and hepatoprotective properties (Vadiraja et al., 1998), in addition to anticancer, anti-allergic, immune-enhancing (Hayashi et al., 2008), blood-vessel-relaxing and blood-lipid-lowering effects of CPC (Gershwin and Belay, 2008).

(αβ)3-trimers and (αβ)6-hexamers (Figure. 2f).

a.

b. c.

Belay, 2008).

The biological and pharmacological properties of *Spirulina* were attributed mainly to Cphycocyanin (CPC). CPC is a major light-harvesting or photosynthetic pigment protein present in the antenna rods of *Spirulina platensis*. *S. platensis* also contains allophycocyanin (APC) as a minor component present at the core of the antenna rods. CPC and APC including phycoerythrin (PE) are the principal classes of phycobiliproteins which form supramolecular complexes known as phycobilisomes assemblies in cyanobacteria (Figure. 2a). In phycobiliproteins, a linear tetrapyrrole (bilin) as the chromophore is covalently attached to the apoprotein by thioether bonds to cysteine residues. CPC molecule is composed of two kinds of subunits, α- and β-subunits to form trimeric aggregation α3β3 (Figures. 2b and 2c). Padyana et al. (2001) solved the crystal structure of CPC by molecular replacement technique (Figures. 2d and 2e). The α- and β-subunit polypeptides exhibit high affinity for one another and associate into (αβ)-monomers, which in turn aggregate into

The medicinal and pharmacological properties of CPC have been reported earlier (Romay et al., 1998). Recent studies have demonstrated the antioxidant (Vadiraja et al., 1998; Wu and Annie Ho, 2008), anti-inflammatory (Reddy et al., 2000), and hepatoprotective properties (Vadiraja et al., 1998), in addition to anticancer, anti-allergic, immune-enhancing (Hayashi et al., 2008), blood-vessel-relaxing and blood-lipid-lowering effects of CPC (Gershwin and

Rod Cor Rod

**3. Structure of phycocyanin** 

A phycobilisome (a) has six antenna rods with a three-cylinder core of allophycocyanin, APC (circles), two of the core cylinders lie on the thylakoid membrane, while the third one does not. Each rod has four hexameric disk-like structures, two of phycoerythrin, PE (red), and two of phycocyanin, CPC (blue) (MacColl, 2004). CPC consists of αand β-subunit polypeptides to form trimeric aggregation α3β3 and nine phycocyanobilin moieties as a chromophore shown in closed circles (b). c shows chemical structure of phycocyanobilin (Li et al., 2006). d and e show ribbon representation of CPC α-subunit and CPC β-subunit, respectively, with chromophores shown in ball and stick representation (Padyana et al., 2001). f shows coil representation of the two (αβ)6-hexamers in the crystal asymmetric unit, and the box drawn at the center highlights the close proximity of phycocyanobilins at the position 155 on each βsubunit in the region between the adjacent hexamers (Padyana et al., 2001).

**Figure 2.** Schematic representation of one type of phycobilisome (a), and various representations of Cphycocyanin (b - f).

**Preparation of phycocyanin solution in the experiments** Phycocyanin was extracted from spray-dried *Spirulina platensis* with 50 mM sodium-phosphate buffer (pH 6.0). The crude extract was partially purified by DE-52 ion-exchange chromatography. The eluate was dialyzed against distilled water and lyophilized. Phycocyanin contents of the resultant powder were over 80%, and the recovery from the crude extract was approximately 6%. The

phycocyanin powder was dissolved in distilled water to a concentration of 0.05%, centrifuged in a refrigerated machine for 10 min at 1,500 g, and the supernatant was sterilized by filtration through a 0.20-μm-pore filter (Hayashi et al., 2006).

Proliferation and Differentiation of Hematopoietic Cells and Preservation of Immune Functions 125

The figure illustrates the arrangement of chromophores at various locations within the hexamers. The box drawn at the center highlights the close proximity of phycocyanobilins at the position 155 on each β-subunit in the region between

**Figure 5.** Coil representation of the two hexamers in the crystal asymmetric unit, a view through the

**4. Enhancement of proliferation and differentiation of bone marrow cells** 

Immunomodulation properties of *Spirulina* have been widely studied in chickens, prawns and fish, other animals, and humans. Generally, *Spirulina* and its extracts, such as hot-water extracts and phycocyanin, tended to enhance immune functions including mucosal or innate immunity through macrophage and secretions of the related cytokines (Belay, 2002; Hirahashi et al., 2002; Nemoto-Kawamura et al., 2004). Mao et al. (Mao et al., 2000) demonstrated that *Spirulina* stimulated the secretion of IL-1ß and IFN-γ in human peripheral blood mononuclear cells (PBMC) examined to nearly 2.0 and 3.3 times basal levels, respectively, and suggested that *Spirulina* helped balance the production of Th1 and Th2 cytokine stimulation. Phycocyanin, a characteristic photosynthesis pigment protein and an antioxidant in *Spirulina*, has been known to promote the growth of a human myeloid cell line, RPMI 8226 (Shinohara et al., 1988). Liu et al. (2000) reported that phycocyanin inhibited growth of human leukemia K562 cells and enhanced the arrest of the cell growth at G1

We have reported that *Spirulina* and its extracts enhanced immune responses in mice, mainly through increased production of interleukin-1 (IL-1) in macrophages (Hayashi et al., 1994; Hayashi et al., 1998). In the mice which ingested phycocyanin for 6 weeks, a marked increase of OVA antigen-specific IgA, as well as total IgA level was observed in the Peyer's patches, mesenteric lymph nodes and intestinal mucosa, as well as in the spleen cells

the adjacent hexamers (Padyana et al., 2001).

approximate central axis of hexamers.

**stimulated with** *Spirulina* **and its extracts** 

phase, suggesting enhancement of differentiation of the cells.

a. CPC molecule composed of two kinds of subunits(α- and β-subunits) to form α3β3 and nine phycocyanobilin moieties as a chromophore (closed circles). b. Chemical structure of phycocyanobilin. Phycocyanobilin was covalently bound to polypeptide chain of CPC by ways of thioetherlinkage to cysteine residues, one on the α-chain and two on the β-chain (Li et al., 2006).

**Figure 3.** Structure of CPC (αβ)3-trimer and phycocyanobilin

**Figure 4.** Ribbon representation of (a) CPC α-subunit (b) CPC β-subunit. Chromophores are shown in ball and stick representation (Padyana et al., 2001).

the β-chain (Li et al., 2006).

**Figure 3.** Structure of CPC (αβ)3-trimer and phycocyanobilin

ball and stick representation (Padyana et al., 2001).

phycocyanin powder was dissolved in distilled water to a concentration of 0.05%, centrifuged in a refrigerated machine for 10 min at 1,500 g, and the supernatant was

a. CPC molecule composed of two kinds of subunits(α- and β-subunits) to form α3β3 and nine phycocyanobilin moieties as a chromophore (closed circles). b. Chemical structure of phycocyanobilin. Phycocyanobilin was covalently bound to polypeptide chain of CPC by ways of thioetherlinkage to cysteine residues, one on the α-chain and two on

**Figure 4.** Ribbon representation of (a) CPC α-subunit (b) CPC β-subunit. Chromophores are shown in

sterilized by filtration through a 0.20-μm-pore filter (Hayashi et al., 2006).

The figure illustrates the arrangement of chromophores at various locations within the hexamers. The box drawn at the center highlights the close proximity of phycocyanobilins at the position 155 on each β-subunit in the region between the adjacent hexamers (Padyana et al., 2001).

**Figure 5.** Coil representation of the two hexamers in the crystal asymmetric unit, a view through the approximate central axis of hexamers.
