FORMATION AND MOVEMENT OF FE(III) IN HORSE SPLEEN, H-RECOMBINANT AND L-RECOMBINANT FERRITINS - A FLUORESCENCE STUDY

Iron oxidation and incorporation into apoferritins of different subuni t composition, namely the recombinant H and L homopolymers and the nat ural horse spleen heteropolymer (10-15% H), have been followed by stea dy-state and time-resolved fluorescence. After aerobic addition of 100 Fe(ni) atoms/polymer, markedly different kinetic profiles are observe d. In the rL-homopolymer a slow monotonic fluorescence quenching is ob served which reflects binding? slow oxidation at the threefold apoferr itin channels, and diffusion into the protein cavity. In the rH-homopo lymer a fast fluorescence quenching is followed by a partial, slow rec overy. The two processes have been attributed to Fe(II) binding and ox idation at the ferroxidase centers and to Fe(IU:) released into the ca vity, respectively. The fluorescence kinetics of horse spleen apoferri tin is dominated by the H chain contribution and resembles that of the Il homopolymer. It brings out clearly that the rate of the overall pr ocess is limited by the rate at which Fe(III) leaves the ferroxidase c enters of the H chains where binding of incoming Fe(II) and its oxidat ion take place. The data obtained upon stepwise addition of iron and t he results of optical absorption measurements confirm this picture. Th e correspondence between steady-state and time-resolved data is remark ably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measu rements.