Formation of Hybrid Proteins from the α and β Subunits of Phycocyanins of Unicellular and Filamentous Blue-green Algae

Abstract

Phycocyanins of two unicellular (Synechococcus sp., and Aphanocapsa sp.), and one filamentous (Anabaena sp.) blue-green alga, have been purified and characterized with respect to behavior on polyacrylamide gel electrophoresis in the absence of denaturing agents, isoelectric point, absorption spectra in the visible region, subunit molecular weights, and amino acid composition. The α and β subunits of each of the phycocyanins were separated by chromatography in urea solutions, and characterized in a similar manner. Each phycocyanin was reconstituted from its separated subunits. Hybrid phycocyanins were formed from the α subunits of Synechococcus sp. phycocyanin and the β subunits of Aphanocapsa sp. and Anabaena sp. phycocyanins, and from the β subunit of Synechococcus sp. phycocyanin and the α subunits derived from the phycocyanins of the other two blue-green algae. The reconstituted and hybrid phycocyanins were purified by ion exchange chromatography, and recovered in an over-all yield of 40 to 60% based on the starting weight of subunit protein. The reconstituted and hybrid phycocyanins were characterized in the same manner as the native proteins. The native, reconstituted, and hybrid phycocyanins, all consisted of α and β subunits in a ratio of 1:1, as established by quantitative amino acid analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chromatographic properties, absorption spectra, and behavior on isoelectric focusing, of both the reconstituted and hybrid phycocyanins, were consistent with the possession by these macromolecules of a three-dimensional structure very similar to that of the native proteins. The divergence of unicellular and filamentous blue-green algae occurred over 2.5 billion years ago. The successful hybridization of subunits of phycocyanins from these two types of organisms indicates a conservation of structural features over a span of time considerably greater than had been involved in previous comparative studies of protein structure.