Glazer A N, Hixson C S
J Biol Chem. 1975 Jul 25;250(14):5487-95.
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...
从单细胞红藻紫球藻（Porphyridium cruentum）的两个独立分离株中纯化出了R-藻蓝蛋白。在pH 7.0时，该蛋白质作为单一成分沉降，其s 20,w为5.98 S（蛋白质浓度为2 mg/ml时，γ/2 = 0.02）。在0.2至0.5 mg/ml的蛋白质浓度范围内（γ/2 = 0.16），沉降平衡给出的分子量为103,000 ± 6,000，没有异质性的迹象。与C-藻蓝蛋白一样，R-藻蓝蛋白由分子量分别为18,200和20,500的α和β亚基组成，这是通过在十二烷基硫酸钠-聚丙烯酰胺凝胶中电泳测定的。在聚丙烯酰胺凝胶中进行等电聚焦可分辨出两条带，蓝色（pI为5.2）和紫色（pI为5.3），据信分别对应于α和β亚基。当通过奥克特洛尼双扩散技术用同源抗血清测试时，天然蛋白质产生单一沉淀带。未观察到与来自同一生物体的别藻蓝蛋白抗血清的交叉反应。pH 7.0时天然三聚体R-藻蓝蛋白的吸收光谱显示，εN（555 nm）为1.51×10⁵ M⁻¹ cm⁻¹，εM（618 nm）为2.55×10⁵ M⁻¹ cm⁻¹，A 1% 1cm（618 nm）为70.0。天然蛋白质的圆二色光谱的特征在于以下每dmol×10⁻⁵的分子椭圆率最大值：[θ]311 = -2.36，[θ]343 = -3.27，[θ]552 = 4.67，[θ]627 = 6.27。所有这些值均基于根据氨基酸组成计算得出的αβ分子量36,3000。为了对R-藻蓝蛋白的发色团组成进行定量估计，测定了集球藻属C-藻红蛋白的吸收特性。在pH 7.0时，天然C-藻红蛋白的εM（562 nm）为4.88×10⁵ M⁻¹ cm⁻¹，基于根据氨基酸组成计算得出的αβ分子量38,400，A 1% 1cm为127。假设每个αβ单元包含六个这样的发色团，从pH 1.9的8 M尿素中变性的C-藻红蛋白的光谱计算出与多肽结合的藻红胆素的摩尔消光系数。藻蓝胆素的类似数据可从早期研究中获得（格拉泽，A.N.，和方，S.（1973年）《生物化学杂志》248，659 - 662）。变性R-藻蓝蛋白的吸收曲线通过为两种藻蓝胆素和一种藻红胆素发色团的混合物计算的理论曲线进行了高精度拟合。R-藻蓝蛋白及其分离的α和β亚基的氨基酸分析表明，天然蛋白质中亚基的化学计量比为1:1。分离亚基的吸收光谱与以下结论一致：α亚基携带单个藻蓝胆素发色团，而一个藻红胆素和一个藻蓝胆素发色团与β亚基结合……
