No abstract

Association and dissociation reactions of cytochrome b562 on solid surfaces were investigated by means of surface plasmon resonance spectroscopy. Propionates of side chains in the heme molecule were modified with sulfur moiety as an anchor part to be adsorbed on the gold substrate. This modified heme was firstly reconstituted with apo-protein in solution. This reconstituted holo-protein was then adsorbed on either bare gold substrate or pre-modified gold substrate. Native cytochrome b562 was also adsorbed to the same substrates for the control experiment. The reconstituted cytochrome b562 was successfully adsorbed on the pre-treated gold substrate without denaturation in structure, probably with homogeneous anisotropic orientation. Guanidine hydrochloride in the buffer of pH 5.0 was applied to remove the apo-protein from the surface, with the thiolated heme derivative remaining on the surfaces. Apo-protein was again introduced to the same surface for the reconstitution of holo-cyt.b562. The success of the association and dissociation reactions between apo-protein and tethered heme molecules on the surfaces was confirmed by the optical thickness changes.