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BIOCHEMICAL SOCIETY TRANSACTIONS 400 Intracellular low molecular weight iron N. DEIGHTON and R. C. HIDER Depurtmetit of Pharmacy, Kitig :s C’ollege, Mutiresu Rotid, London SW 3 hLX, U.K. L Summary A low molecular weight complex of an iron oligomer ( M , - 1000) has been isolated from rat livcr and characterized physically and chemically. The h.p.1.c.-purified material contains the free amino acids glutamate and aspartate. Chemical techniques suggest the iron present in the factor is as iron(ll1) and is readily donated t o high-affinity chelators such as the hydroxypyridinones and desferrioxamine. The isolation utid characterization of u low molecirlar weight iron complex f r o m rat liver In biological systems the majority o f oxygen-derived free radicals formed via metal complexes are produced by either the Cu(l):Cu(II)or Fe(II):Fe(lll)redox couples. The ability of metal complexes to generate hydroxyl radicals are dependent on both the stereochemical environment and the binding affinity of the ligand for the metal (Singh & Hider, 1Y88u, h).Furthermore. iron(l1)complexes tend to be more efficient than those of iron(ll1). as the metal is in the correct redox state to take part in thc reaction: Fe(ll)+H,O, - F e ( l l I i + H O ~+HO. For these reasons we argue that, to avoid toxicity, low molecular mass ‘transit‘ species (Jacobs, 1 Y77) are probably stable complexes of iron(lI1). These complexes must not, however, be s o stable as to prevent metal transfer to ironrequiring metalloproteins. A low molecular mass iron-containing fraction has been isolated from rat liver using a sequence of chromatographic procedures, including h.p.1.c. (Full details of purification and chromatograms are available on request.) Thc molecular mass of the species is estimated to be 1000- 1500. which is in close agreement to previous reports (Mulligan et ul., 1986; Jones Bi Grady. 1988). Glu and Asp (as the free acids) were found to be associated with the metal at a Glu:Asp ratio of 7:2. Atomic absorption spectroscopy reveals the liver concentration of iron in this form is approximately 2 ,UM. Also associated with the fraction are chromium (0.2 ,UM) and aluminium (0.4 ,uM). The iron contained in the fraction is in the iron(ll1)state and is rapidly chelated by 4-hydroxypyridinones, but less readily by the hcxadentate chelator, dcsferrioxamine. Bathophenanthroline, an ironjll) chelator, only removes iron from the fraction in the presence of dithionitc. The U.V. spectrum of the fraction is similar to those of a number of oligomeric iron cornpounds containing bridging carboxylate functions (Fig. 1) prepared by Lippard ( 1Y 88). The carboxylate-containing ligands may be simple anions such as acetate, or more complex. such as alanine (Holt et al.. 1 Y 74). Chromium(111) forms analogous compounds (Cotton Bi Wilkinson, lY80). A series o f compounds [Iron(111): acidic amino acids] have been prepared for comparison with the purified liver fraction. These possess spectra similar to both the isolated fraction and the tri-iron(1II) mateiials of Lippard. In particular, a compound, tentatively identified as Fe,O[Glu],, has been the most extensively studied. Iron in this form is rapidly chelated by 4-hydroxypyridinones, but more slowly by desferrioxamine. As is the case with the liver fraction, bathophenanthroline (in the absence of a reductant) will not remove iron from the material. This synthetic material. like the fraction isolated from liver, readily donates iron to ferritin in the presence o f phosphate. Preliminary e.s.r. studies have been unable to distinguish the iron in the liver fraction from that in the synthetic material. It is also known that iron(II1)-carboxylate bonds are facile, permitting rapid ligand exchange. The carboxylatesurrounded, 3-fold channels of ferritin are strikingly similar in size and shape t o the tri-iron unit (Rice CI (11.. lY83). The relative positions of the channel carboxylate functions suggest that ligand exchange with the Glu residues would be possible. Indeed, such a mechanism of iron uptake is supported by thc finding that Fe-0-Fe moieties form o n the surface o f ferritin (Chasteen et al., 1985; Yang el al., 1987). Further physical and chemical characterization of both the biological and synthetic materials is in progress. We thank the M.R.C.f o r financial support Chasteen. N . I).. Antanaitis, B. C . & Aisen, P. ( 1 0 x 5 ) .J. Iliol. C ’ h c r n . 260.2926-2929 Cotton. F. A. & Wilkinson. G. ( 19x0) Acli~riric~c~cl Inorgtrriic~ C’hwni.s/ry. Fourth Edition, John Wiley and Sons. New York Holt, E. M., Holt, S. L., Tucker, W. F., Asplund. R. 0.& Watson, K. J . ( 1974) J . Arner. C‘hern. SOC. 9 6 , 2 6 2 1-2623 Jacobs. A . ( 1077) Aloorl 50. 333-439 Jones. R. L. & Grady. K.W. ( 19x8) pi~r.\(~/r(i/c.otnr~rrr,ric.tr/rorr Lippard, S.J . ( I Y X X ) ilrrgcw. C’heni. 27. 334-30 I Mulligan. M.. Althaus, B. & Linder. M. C , ( I9X6) Inr. J . Hiochem. 18.791-79X Rice. .)L E., Ford. G. C., White. J . L.. Smith. J . M. A . & Harrison. P. M. ( 19x3) in Srrircrirrc, t i n t / Fiinc/iorr (J’Iron S/ortrgc trrrd Trcrnsport /’ro/eins(Urushizaki ef a/. eds.),p. I 1. Elsevier, New York Singh, S.& Hider. R. C. ( 1 9880) Anml. Hiocheni. 171. 47-54 Singh, S. SC Hider. K.C . ( 19x86) in Frw Kiit/ic.cih: Mc/hot/o/ogyu r i d C‘orrc,cp/.s (Rice-Evans, C. & Halliwell, R., eds.), p. 0 1-90, Richelieu Press Yang, C. Y.. Meagher. A , , Huynh, B. H., Sayen. I). E. SC Theil. E. C. ( 19x7) Hiochrmistry 26. 497-503 Received 24 November 1 V X X 108Y