Properties and N-terminal sequence of allophycocyanin from the unicellular rhodophyte Cyanidium caldarium

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Abstract
Allophycocyanin from the unicellular rhodophyte C. 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 .**GRAPHIC**. at 650 nm of purified allophycocyanin was 6.35 in 0.05 M-potassium phosphate buffer, pH 7.0. 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 340 nm. Computer analysis of the circular-dichroic spectrum of allophycocyanin from 207-243 nm indicated 42% .alpha.-helix and 58% .beta.-form. The estimated MW of purified allophycocyanin on calibrated Sephadex G-200 columns at pH 7.0 was 196,000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulfate/polyacrylamide gels revealed a single band with apparent MW 16,000. The presence of 2 polypeptide subunits, with nearly the same MW, 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 8 M-urea (pH 3.0) 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, pH 7.0. Automated sequence analysis of the N-terminus of allophycocyanin showed that the sequences of the 2 subunits were different from one another and were different from the subunits of phycocyanin from the same alga, the subunits occurred in a molar ratio of 1:1 and the sequence homology at the N-terminus among .alpha.- and .beta.-subunits of allophycocyanin from blue-green and red algae approached 90%.