Molecular characterization of a water-soluble chlorophyll protein from main veins of Japanese radish

A water-soluble chlorophyll (Chl) protein (WSCP) from the main veins of the Japanese radish, Raphanus sativus L. var. hortensis, was purified and characterized. We sequenced 26 N-terminal residues of the Raphanus-WSCP and found a complete signature motif of Künitz proteinase inhibitors, as in WSCPs from other Brassicaceae plants. The Raphanus-WSCP sequence shared 92% identity with that of a Lepidium-WSCP; however, the antibody against recombinant Lepidium-WSCP did not recognize the Raphanus-WSCP, indicating that the N-terminal region may not be a major antigenic epitope. On the other hand, while the absorption spectrum of the red band of the Raphanus-WSCP was distinct from that of the Lepidium-WSCP, it was similar to that of Brassica-WSCP. Spectrophotometric and spectrofluorometric analyses of Raphanus-WSCP from the main veins revealed that the red band was comprised of four Chl a and one Chl b forms. In addition, this WSCP contained an intense absorption component with a peak at 698 nm, which is not prominent in WSCP from Raphanus leaflets. As an initial step in determining its molecular structure, the Raphanus-WSCP was crystallized. A green, parallelepiped, single crystal was obtained. This is the first characterization and crystallization of WSCP from the genus Raphanus, which is the third documented genus possessing WSCP in Brassicaceae plants.