Allophycocyanin from the filamentous cyanophyte, Phormidium luridum.
Allophycocyanin from the filamentous cyanophyte, Phormidium luridum, was purified by ammonium sulfate fractionation and ion exchange chromatography on brushite columns. The specific absorption coefficient (E 0.1% 1cm) of purified allophycocyanin was 6.1 in distilled water and 7.3 in 0.05 M potassium phosphate buffer (pH 7). Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350, and 275 nm. Circular dichroic spectra displayed positive ellipticity bands at 655 and 625 nm, and a major negative ellipticity band at 340 nm. Computer analysis of the circular dichroic spectrum of allophycocyanin from 207 to 243 nm indicated that the secondary structure contained 60% alpha helix and 40% beta form. The estimated molecular weight of allophycocyanin on Sephadex G-200 columns at pH 7.0 was 155,000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulfate polyacrylamide gels revealed two subunits, alpha and beta, with apparent molecular weights of 17,300 and 19,000, respectively. Densitometric analysis of unstained gels at 600 nm indicated that one phycocyanobilin chromophore was associated with each subunit. Treatment of allophycocyanin with 12% formic acid or 8 M urea and subsequent removal of the denaturant yielded a derivative with spectroscopic characteristics similar to phycocyanin. Subsequent incubation in phosphate buffer (pH 7), but not in acetate buffer (pH 5) or in water, was accompanied by a progressive reappearance of absorption maxima at 650 and 618 nm (shoulder), and positive ellipticity bands at 655 and 617 nm. Automated sequence analysis of allophycocyanin (a) showed that the sequence of amino acids at the amino terminus of the alpha and beta subunits is different, (b) showed that the subunits occur in a ratio of 1:1, and (c) demonstrated sequence homology at the amino terminus of allophycocyanin, phycocyanin, and phycoerythrin.
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008433:Mathematics; D008970:Molecular Weight; D010798:Phycocyanin; D010860:Pigments, Biological / Q000302:isolation & purification; D010940:Plant Proteins / Q000302:isolation & purification; D011487:Protein Conformation; D013045:Species Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D046911:Macromolecular Substances; D010860:Pigments, Biological; D010940:Plant Proteins; D010798:Phycocyanin