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.

Phycocyanin was isolated and purified from the unicellular alga, Cyanidium caldarium. Subunits were prepared on a Bio-Rex-70 column developed stepwise with urea solutions (pH 1.9). The alpha subunit eluted in 8 M urea and the beta subunit eluted in 9 M urea. The alpha and beta subunits displayed absorption maxima at 660, 354, and 277 nm in 8 M and 9 M urea. The alpha:beta ratio of total absorbance under the 660-nm peak was 0.56 suggesting an alpha:beta phycocyanobilin chromophore ration of 1:2. On calibrated sodium dodecyl sulfate gels, the alphs subunit had an estimated molecular weight of 15,500 plus or minus 1100 and the beta subunit has an estimated molecular weight of 18,300 plus or minus 300. Minimum molecular weights based on one histidine residue per subunit were 16,300 for the alpha subunit and 18,750 for the beta subunit. Phycocyanin displayed a single visible absorption maximum at 625 nm and two positive circular dichroic bands at 632 and 610 nm. The alpha and beta subunits displayed single visible absorption maxima at 618 and 600 nm and single positive circular dichroic peaks at 620 and 585 nm, respectively. Two-dimensional maps of tryptic digests of the alpha and beta subunits revealed distinct patterns of peptides each of which was consistent with the lysine and arginine composition of these polypeptides. Maps of tryptic digests of phycocyanin contained 25 major peptides (a total of 27 lysine and arginine residues). Automated sequence analysis of separated subunits revealed a 70% homology within the first 27 residues at the amino terminus of the alpha and beta subunits of C. caldarium phycocyanin.

The unicellular alga Cyanidium caldarium evolves carbon monoxide during the syntheis of the bile pigment, phycocyanobilin. Carbon monoxide and phycocyanobilin were produced in stoichiometric amounts at comparable rates. Therefore, the mechanism of bile pigment formation in this plant parallels that in mammals.

Allophycocyanin from the unicellular rhodophyte Cyanidium caldarium was purified by (NH4)2SO4 fractionation and ion-exchange chromatography on brushite (calcium phosphate) columns and on DEAE-Sephadex A-25 columns. The specific absorption coefficient (A0.1%1cm) at 650nm of purified allophycocyanin was 6.35 in 0.05M-potassium phosphate buffer, pH7.0. Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350 and 275 nm. Circular-dichroic spectra displayed positive-ellipticity bands at 658 and 630 nm and a major negative-ellipticity band at 340nm. Computer analysis of the circular-dichroic spectrum of allophycocyanin from 207 to 243 nm indicated 42% alpha-helix and 58% beta-form. The estimated molecular weight of purified allophycocyanin on calibrated Sephadex G-200 columns at pH7.0. was 196000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulphate/polyacrylamide gels revealed a single band with apparent mol.wt. 16000. The presence of two polypeptide subunits, with nearly the same molecular weight, was revealed on polyacrylamide gels by using a modified electrophoresis buffer. Spectral analysis of the allophycocyanin subunits resolved by ion-exchange chromatography on Bio-Rex 70 columns indicated that a single phycocyanobilin chromophore was present on each polypeptide chain. Treatment of allophycocyanin with 8M-urea (pH3.0) and subsequent removal of urea by dialysis against water yielded a derivative phycobiliprotein with spectroscopic characteristics similar to those of phycocyanin. The original allophycocyanin spectrum was regenerated after incubation in phosphate buffer, pH7.0. Automated sequences analysis of the N-terminus of allophycocyanin showed that (a) the sequences of the two subunits were different from one another and were different from the subunits of phycocyanin from the same alga, (b) the subunits occurred in a molar ratio of 1:1 and (c) the sequences homology at the N-terminus among alpha- and beta-subunits of allophycocyanin from blue-green and red algae approached 90%.

No abstract