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R-phycocyanin was purified from two independent isolates of the unicellular red alga Porphyridium cruentum. At pH 7.0 the protein sediments as a single component with s 20,w of 5.98 S (at 2 mg/ml, gamma/2=0.02). Over a protein concentration range of 0.2 to 0.5 mg/ml (gamma/2=0.16), sedimentation equilibrium gave a molecular weight of 103,000 +/- 6,000 with no evidence of heterogeneity. In common with C-phydocyanins, R-phycocyanin consists of alpha and beta subunits of molecular weights of 18,200 and 20,500, determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Isoelectric focusing in polyacrylamide gels resolves two bands, blue (at pI of 5.2), and purple (at pI of 5.3), believed to correspond to the alpha and beta subunits, respectively. The native protein gave a single precipitin band when tested against the homologous antiserum by the Ouchterlony double diffusion technique. No cross-reaction was observed with antiserum to the allophycocyanin from the same organism. The absorption spectrum of native trimeric R-physocyanin at pH 7.0 exhibited epsilomN (555 nm) of 1.51 x 10(5) M(-1) cm(-1), epsilonM (618 nm) 2.55 x 10(5) M(-1) cm(-1), and A 1% 1cm (618 nm) of 70.0. The circular dichroism spectrum of the native protein was characterized by the following molecular ellipticity maxima in deg cm2 per dmol x 10(-5): [theta]311 = -2.36, [theta]343 = -3.27, [theta]552 = 4.67, and [theta]627 = 6.27. All of these values were based on an alphabeta molecular weight of 36,3000, calculated from the amino acid composition. To permit quantitative estimation of the chromophore composition of R-phycocyanin, the absorption properties of Aphanocapsa sp. C-phycoerythrin were determined. At pH 7.0, native C-phycoerythrin exhibited epsilonM (562 nm) of 4.88 x 10(5) M(-1) cm(-1), and A 1% 1cm of 127, based on an alphabeta molecular weight of 38,400 calculated from the amino acid composition. The molar extinction coefficients for polypeptide-bound phycoerythrobilin were calculated from the spectrum of denatured C-phycoerythrin in 8 M urea at pH 1.9, on the assumption that each alphabeta unit contains six such chromophores. The analogous data for phycocyanobilin was available from an earlier study (Glazer, A.N., and Fang, S. (1973) J. Biol. Chem. 248, 659-662). The absorption curve of denatured R-phycocyanin was fitted with high precision by a theoretical curve calculated for a mixture of two phycocyanobilin and one phycoerythrobilin chromophore. The amino acid analyses of R-phycocyanin and of its separated alpha and beta subunits demonstrated a 1:1 stoichiometry for the subunits in the native protein. The absorption spectra of the isolated subunits were consistent with the conclusion that the alpha subunit carries a single phycocyanobilin chromophore, while one phycoerythrobilin and one phycocyanobilin chromophore are bound to the beta subunit... |
