Soybean trypsin inhibitor (STI) was immobilized on the agarose gel modified with spiropyran compound (spiropyran gel), and photocontrolled binding and releasing oftrypsin was examined. The STI-spiropyran gel showed reverse photochromism. Trypsin was bound on the STI-spiropyran gel in the dark and released with visible light irradiation. The optimum conditions for photocontrolled binding and releasing of trypsin were pH 6.6 and the buffer concentration of 0.05 M. Approximately 60-80% of bound trypsin was released with visible light irradiation. The activity of released trypsin was the same as that of native trypsin. Approximately 21-fold purification of trypsin was performed with the STIspiropyran gel column.
Selective isolation and purification of biologically important substances such as enzymes and coenzymes by affinity chromatography exploits the unique biological property of these substances to bind ligands specifically and reversibly (1). Bound substances can be released with a large amount of eluent including a linear salt gradient. If releasing of a substance is controlled with external energy such as light and electric field, this technique become more convenient for separation process.
Substances that undergo reversible color formation with light irradiation are called photochromic compounds. The polarity of spiropyran compound drastically changes with light irradiation. Photosensitive enzymes were prepared by modifying enzymes with the photochromic spiropyran compound (2,3). Furthermore, the activity of enzymes immobilized in photosensitive collagen membrane was also controlled with light irradiation (4,5). In this case, the change of microenvironmental properties around enzymes induced the activity change of immobilized enzymes. Agarose gel modified with the spiropyran compound was used as a ligand of cytochrome c binding (6). Cytochrome c bound on immobilized spiropyran under visible light was released from the gel in the dark. However, as other proteins also bound on the immobilized spiropyran under