8 Analytical Protocols in Phycobiliproteins Analysis
181
grade of APC (Yan et al. 2011) whereas the commonly accepted criterion for PE
purity is when A 565 /A 280 ratio reaches 3.2 (Galland-Irmouli et al. 2000). An additional approach for evaluation of PBPs purity is determination of the ratios between
absorption maxima of individual PBPs. Ratio A 650 /A 620 < 0.3 means that PC preparation contains negligible contamination from APC, while the ratio A 650 /A 620 > 1.5
indicates that APC is pure relatively to PC (McGregor et al. 2008). The purity of RPE is also estimated by means of following indexes: A 565 /A 498 ≤ 1.5 and A 565 /A 620
≤ 0.005, indicating minimal contamination by B-PE and PC, respectively (Niu et al.
2006). However, these absorbance ratios could sometimes give false interpretations.
The pure APC in trimeric form has a peak at 652 nm, but monomers strongly absorb
at 620 nm, preventing discrimination of PC from APC by visible absorption measurements (MacColl et al. 2003). In these cases, additional analytical methods, such as
SDS-PAGE, are needed to obtain reliable results regarding PBPs purity. Induction of
fluorescence after incubation of SDS-PAGE gels with Zn
2+ ions specifically marks
PBPs bands (Berkelman and Lagarias 1986), giving the possibility to make a distinction between PBP and other proteins by comparing electrophoretic gels after Zn
2+ and
CBB staining. The molecular weights of PBPs are determined by gel filtration and
electrophoretic techniques (SDS-PAGE and PAGE), as well as by mass spectroscopy
(MS) (Chen et al. 2006).
Isolation of tetrapyrrole chromophores requires cleavage of thioether bond
between apoprotein and chromophore by acid hydrolysis, enzymatic cleavage, or
extensive refluxing in alcohols. Although the most common procedure for the chromophore cleavage is still conventional reflux in methanol (Fu et al. 1979), it has been
shown that performing ethanolysis in the sealed vessel at 120 °C decreases reaction
time to 30 min and obtained PCB has higher purity in comparison to conventional
reflux method (Roda-Serrat et al. 2018). Purification of released tetrapyrroles is
usually performed by reversed-phase HPLC (Roda-Serrat et al. 2018).
Although recombinant production of PBPs is demanding, as a complete synthesis
of PBPs depends on co-expression of α- and β-chains, in parallel with the synthesis of
chromophores and their covalent attachment to protein, recombinant APC/PC were
successfully produced in E. coli (Chen et al. 2015a; Cherdkiatikul and Suwanwong
2014). Tetrapyrrole chromophores could also be produced recombinantly. PCB was
produced in mammalian cells by metabolic engineering introducing genes for heme
oxygenase-1 and PCB:ferredoxin oxidoreductase with simultaneous knock-down of
biliverdin reductase A to prevent PCB reduction to phycocyanorubin (Muller et al.
2013).
8.3 PBPs Structure and Physicochemical Properties
Evaluation
Early studies based on PBPs enzymatic or CNBr hydrolysis and products detection
after Edman degradation of chromopeptides by MS, revealed that the side chain (at
position 2) of tetrapyrrole ring A is covalently bound to the cysteine residues of
181
grade of APC (Yan et al. 2011) whereas the commonly accepted criterion for PE
purity is when A 565 /A 280 ratio reaches 3.2 (Galland-Irmouli et al. 2000). An additional approach for evaluation of PBPs purity is determination of the ratios between
absorption maxima of individual PBPs. Ratio A 650 /A 620 < 0.3 means that PC preparation contains negligible contamination from APC, while the ratio A 650 /A 620 > 1.5
indicates that APC is pure relatively to PC (McGregor et al. 2008). The purity of RPE is also estimated by means of following indexes: A 565 /A 498 ≤ 1.5 and A 565 /A 620
≤ 0.005, indicating minimal contamination by B-PE and PC, respectively (Niu et al.
2006). However, these absorbance ratios could sometimes give false interpretations.
The pure APC in trimeric form has a peak at 652 nm, but monomers strongly absorb
at 620 nm, preventing discrimination of PC from APC by visible absorption measurements (MacColl et al. 2003). In these cases, additional analytical methods, such as
SDS-PAGE, are needed to obtain reliable results regarding PBPs purity. Induction of
fluorescence after incubation of SDS-PAGE gels with Zn
2+ ions specifically marks
PBPs bands (Berkelman and Lagarias 1986), giving the possibility to make a distinction between PBP and other proteins by comparing electrophoretic gels after Zn
2+ and
CBB staining. The molecular weights of PBPs are determined by gel filtration and
electrophoretic techniques (SDS-PAGE and PAGE), as well as by mass spectroscopy
(MS) (Chen et al. 2006).
Isolation of tetrapyrrole chromophores requires cleavage of thioether bond
between apoprotein and chromophore by acid hydrolysis, enzymatic cleavage, or
extensive refluxing in alcohols. Although the most common procedure for the chromophore cleavage is still conventional reflux in methanol (Fu et al. 1979), it has been
shown that performing ethanolysis in the sealed vessel at 120 °C decreases reaction
time to 30 min and obtained PCB has higher purity in comparison to conventional
reflux method (Roda-Serrat et al. 2018). Purification of released tetrapyrroles is
usually performed by reversed-phase HPLC (Roda-Serrat et al. 2018).
Although recombinant production of PBPs is demanding, as a complete synthesis
of PBPs depends on co-expression of α- and β-chains, in parallel with the synthesis of
chromophores and their covalent attachment to protein, recombinant APC/PC were
successfully produced in E. coli (Chen et al. 2015a; Cherdkiatikul and Suwanwong
2014). Tetrapyrrole chromophores could also be produced recombinantly. PCB was
produced in mammalian cells by metabolic engineering introducing genes for heme
oxygenase-1 and PCB:ferredoxin oxidoreductase with simultaneous knock-down of
biliverdin reductase A to prevent PCB reduction to phycocyanorubin (Muller et al.
2013).
8.3 PBPs Structure and Physicochemical Properties
Evaluation
Early studies based on PBPs enzymatic or CNBr hydrolysis and products detection
after Edman degradation of chromopeptides by MS, revealed that the side chain (at
position 2) of tetrapyrrole ring A is covalently bound to the cysteine residues of