Jul 31, 1992 - binant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers ...
T H E JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 268, No. 17, Issue of June 15, pp. 12744-12748,1993 Printed in U.S.A.
Production and Characterizationof Recombinant Heteropolymersof Human Ferritin H and L Chains* (Received for publication, July 31, 1992, and in revised form, February 9, 1993)
Paolo SantambrogioS, Sonia Levi$, Anna CozziS, Ermanna Rovidaj, Albert0 AlbertiniT, and Paolo ArosioS11 From the $Department of Biological and Technological Research, Scientific Institute San Raffaele, Department of Biomedical Science and Technology, University of Milum, Via Olgettinu 60,20132 Milano, the glstituto Technologie Biomediche Avanzati, Consiglio Nazionale delle Richerche, Via Ampere 56, 20131 Milano, and the BInstitute of Chemistty, University of Brescin, Via Valsabbinu, 25124 Brescia, Italy
Vertebrate ferritins are iron storage proteins com- H- and L-type chains andof some variants indicated that: (i) posed by24 subunits of oneor more types. The recom- in vitro the H ferritin oxidizes iron at rates several-fold faster binant homopolymers of human ferritin H- and L-type than L ferritin (12-16), due to the presence of a particular chains differ in iron uptake and in physical stability, ferroxidase centre in H ferritin that is absent in L ferritin but the properties of heteropolymers with various pro- (11, 15); (ii) L ferritin appears to induce iron mineralization portions of H- and L-type chains cannot be predicted. with higher efficiency than the H ferritin (17); (iii)L is Present study shows that unfoldedhuman ferritin H- notably more stable to physical denaturation than H ferritin, and L- type chains renature under similar conditions a difference in part due to the saltbridge L ~ S ~ ~ - in G the ~U’~~ to form homopolymers indistinguishable from the naL subunitreplacing the ferroxidase centre of the H chain (18). tive ones and that, when mixed, the unfolded H and L Natural isoferritins are heteropolymers made by the cochainsrenature to formheteropolymers with reassembly of the two subunits. Because of the complexity of stricted heterogeneity and with the expected H:L rathe molecule, the knowledge of the functional/structuralproptios. Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunologi- erties of the two chains (or homopolymers) is not sufficient cal reactivity, and stability to guanidine denaturation to predict the structural and functional properties of any given heteropolymer. Studies performed on restricted popuvaried as predicted, based on the homopolymers. In lations of natural isoferritin heteropolymers indicated that contrast, the rate of iron uptake, monitored by the their immunological reactivity (19-21) and rateof iron uptake variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; (22) varied with the H:L proportion. However, whether the further increments in H chains had no additional ef- two chains have recognition specificities that may affect the fect. Thisfindingindicatesthat,underthepresent structure of the assembled ferritins was not clear. Nor was conditions,onlyalimited numberof H chains are the effect of a limited number of H chains (ie. of the ferroxneeded for the maximum rate of ferritin iron uptake. idase centres) on the overall activity of a determined ferritin Variations of L- and H-type chains in vivo may thus molecule. have biological relevance. Denatured ferritin subunits readily reassemble in protein cages with structural and functional properties analogous to the parent protein (23-27) and with a mechanism which is partially characterized (24, 26, 27). Most of the denaturation Ferritin is an almost spherical iron storage protein made and renaturation studies have been made on horse spleen by 24 subunits (1-4). Each subunit is a bundle of four long a ferritin, which is similar to a homopolymer (about 90% L helices, with a fifth short helix, a short nonhelical extension chain), buta previous report by Otsuka et al. (28) showed that at theC and N termini,and a long loop connecting helices B heteropolymers can be obtained in vitro by the co-renaturaand C (1, 3). In most vertebrates the ferritins are composed tion of denatured H and L chains. In the present study we show that human ferritin H and L of various proportions of two subunit types, H and L chains, genetically distinct and with about 50% sequence identity (5, chains renature in similar conditions to give products struc6). The expression of the two chains is under strict genetic turally and functionally analogous to the native homopolycontrol, leading to tissue-dependent variation of isoferritins mers. When mixed, the two denatured subunits reassemble to populations (5, 7, 8). Recent x-ray analyses of recombinant produce heteropolymers with low heterogeneity and with the ferritins and their variants overexpressed in Escherichia coli predicted subunit composition. The properties of the heteroshowed H and L chains to have homologous subunit confor- polymers were found to vary with the subunit content; stabilmations and subunit arrangements,at least at low resolution ity increased linearly with L chain proportion, and the rate (9-11). Biochemical studies of the homopolymers of human of iron uptake ( A A 3 1 0 n m ) increased from 0 to about 35% H chain proportion,when a plateauwas reached. Present results * The work was supported by the Commission of European Com- indicate a preferential recognition specificity of the subunits munities (BRIDGE Contract BIOT-CT91-0262) and Consiglio Na- for hetero-assembly over homo-assembly. The implications of zionale delle Richerche Target Projects on Biotechnology and Bioin- present findings on the mechanism of formation and funcstrumentation. The costs of publication of this article were defrayed tionality of natural ferritins are discussed.
in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. I(To whom correspondence should be addressed DIBIT, Inst. San Raffaele, Via Olgettina 60, 20132 Milano, Italy. Tel.: 39-2-2643-4748 Fax: 39-2-2643-4844.
MATERIALS AND METHODS
Ferritins-Homopolymers of human H and Lchains were expressed by E. coli strains transformed with the plasmids pEMBLex2HFT and pEMBLexZLFT, described in Refs. 29 and 17,
12744
Recombinant Ferritin Heteropolymers respectively, which encode for the full and correct amino acid sequences of the two chains. The ferritins were overexpressed and purified with the procedures already described (12, 17)obtaining purified proteinsin the ranges of10-15 mg and 2-5 mg/liter of culture, for rHF' and rLF, respectively. Briefly, expression was induced by heat shock a t 42 "C, cells were disrupted by sonication, and the soluble homogenates were heated at 75 "C for 10 min, precipitated with ammonium sulfate (520 g/liter), and treated with DNase. The final purification steps consisted of gel filtration on a Sepharose 6B column for rHF and ofgel filtration on Sephacryl S-200 for rLF, followed by a DEAE column. All ferritins were electrophoretically pure, rHF contained 100-200 iron atoms/molecule, and rLFless than 10 atoms. Both ferritins were treated for iron removal by incubation with 1%thioglycollic acid, pH 5.5, and 2,2'-bipyridine followed by dialysis against 0.1 M Hepes, pH 7.0 (12). The apoferritins had280:260 nm absorbance ratio higher than 1.3 and they had an iron content below 10 iron atoms/molecule. Protein concentration was determined with BCA reagent (Pierce) using bovine serum albumin as standard. The iron concentration was determined spectrophotometrically as in Ref. 12. Electrophoresis-Nondenaturing electrophoreses were run on 7.5 or 6% polyacrylamide gels and stained with Coomassie Blue, silver stain (Bio-Rad), or Prussianblue (12). The products of renaturation experiments were directly loaded on gels, since we observed that GdnHCl up to 1.2 M in the samples did not affect electrophoretic mobility of assembled ferritin. Isoelectric focusing experiments were performed on 5% polyacrylamide slab gels, 1-mmthick, with 2% ampholines, pH range 3.5-10 (Pharmacia) asin Ref. 7. Cross-linking was performed essentially as described in Ref. 26; ferritin samples in 0.1 M phosphate, pH 7.4, were incubated with 0.1-0.25% glutaraldehyde for 2 min a t room temperature; the samples were then boiled in 1%SDS, 5% 2-mercaptoethanol, 0.25 M Tris-HC1, pH 6.8, run on 15% polyacrylamide gels, and stained with Coomassie Blue. Blotting experiments were performed as in Ref. 30; briefly the ferritins were run on polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, saturated with 10% solution of defatted drymilk, and incubated with the primary monoclonal antibodies for human H or L ferritins (10 pg/ml), washed, and further incubated with peroxidaselabeled anti-mouse Ig rabbit antibodies (Miles). Bound activity was developed as before (30) using 4-chloro-1-naphthol as a chromophore. Spectrophotometric Analyses-Far UV CD spectra were collected with a Jasco 500-A spectropolarimeter equipped with a Jasco DP500/PC data processor. The samples were at concentration of 0.030.06 mg/ml in 0.1-cm path length cells. Fluorescence spectra were collected with aPerkin-Elmer spectrofluorimeter with excitation window of 3 nm and emission window of 20 nm. Protein concentrations were in the range 0.02-0.07 mg/ml. Signal of the buffer background at different concentrations of GdnHCl was subtracted in the CD and fluorescence experiments. Denaturation plots were obtained by incubating ferritin samples (50 pg/ml) for 18h a t 4 'C with various GdnHCl concentrations in 0.1 M phosphate buffer, pH 7.4, and 3 mM DTT. The conformational status of ferritin was derived from the ellipticity values at 222 nm and from fluorescence spectra with excitation at 295 nm, using as controls the ferritin in0.1 M phosphate, pH 7.4 (native status), and in 6 M GdnHCl, 0.1 M phosphate buffer, pH 3.5 (denatured status) (18). Fluorescence spectra were analyzed for the excitation maxima, which are at 330 and 325 nm in native rHF andrLF, respectively, and a t 355 nm in both denatured ferritins, and for the ratios of the emission at 355 and 330 nm. Both CD and fluorescence experiments provided analogous indication of protein conformation. Renaturation plots were obtained by diluting at least 10-fold the denatured ferritins in 0.1 M phosphate, pH 7.4, 3 mM DTT with various GdnHCl concentrations and by recording, after a 2-h rest, the ellipticity values a t 222 nm and thefluorescence spectra for analysis of conformational status. Iron Uptake-The kinetics of iron uptake were studied as in Refs. 12 and 13using a molar ferritin:iron ratio of 1:lOOO. A freshly prepared 100 mM solution of ferrous ammonium sulfate in water was added to 0.1 p M apoferritins (50 pg/ml) in 0.1 M Hepes buffer, pH 7.0 or 6.5, a t 30 "cto a final 0.1 mM concentration. The formation of the amber iron product was monitored by the increase of optical reading a t 310 nm. When iron incorporation was to be monitored by gel electrophoresis, apoferritins (1wM,in 0.1 M Hepes, pH 7.0 or 6.5) wereincubated at room temperature with 1 mM of freshly prepared ferrous ammonium sulfate for 2 h and then loaded on gel electrophoresis (17).
' The abbreviations used are: rHF, recombinant human H chain ferritin; rLF, recombinant human L chain ferritin; GdnHC1: guanidine hydrochloride; DTT, dithiothreitol.
12745 RESULTS
Renaturation of H and L Chains-Homopolymers of the human H and L chains were equilibrated with 6 M GdnHCl, pH 3.5, to ensure complete unfolding (18) and diluted in phosphate buffer, pH 7.4, to a final GdnHCl concentration below 0.6M. Electrophoretic analysis showed that both chains reassemble in the ferritin cages with high yields in less than 2 h, with the formation of dimers and oligomers typical of native ferritin (not shown). Further analyses were carried out to test the quality of the renatured proteins. Tryptophan fluorescence spectra show that the differences between aporLF and rHFin emission intensity and maxima (325 and 330 nm, respectively) remained essentially unchanged in renatured samples. A minor (2 nm) red shift was observed for renatured rHF, but not in rLF (Fig. lA). Protein stabilitywas probed by the variation in fluorescence of samples equilibrated with various concentration of GdnHCl at pH 7.4. Fig. 1B shows that renatured ferritins conserved the difference between rHF and rLF(18), with no substantial variationfrom the native samples. Apoferritins were incubated with ferrous iron at 1:lOOO molar ratio, andelectrophoretic analysis followed byPrussian blue staining showed that natural and renatured samples incorporated analogous amounts of iron (not shown). The rates of iron uptake at pH 6.5 were similar for the native and renatured apoferritins with major differences between rHF and rLF (Fig. IC). These data indicate that, under these renaturation conditions, H and Lhomopolymers are produced with a high recovery (about 70%) and with chemico-physical and functional propertiesvery close to thenative proteins. Next we constructedrenaturationplots to compare the patterns of renaturation of the two chains. Unfolded H and L chains were diluted a t various concentrations of GdnHC1, pH 7.4, and analyzed for tryptophan fluorescence (Fig. 2) and ellipticity at 222 nm (not shown). The results show that rHF and rLF have very similar refolding transitions and that at GdnHCl below 1.2 M both ferritins are fully assembled (Fig. 2). Assembly of H and L Chains Heteropolymers-Thetwo denatured chains were mixed at 1:1 molar ratio and allowed to renature under the conditions described for the homopolymers. The reassembled proteins moved as a single monomeric band intermediate between H and Lhomopolymers, in nondenaturing gel electrophoresis (not shown), whereas in isoelectric focusing they were resolved in three to four bands with a PI in between the H and Lhomopolymers (Fig. 3, lanes c, d, and e). The same samples eluted from a Superose 6 column as a major peak at 13.17 ml, intermediate between H and L homopolymers (12.99 and 13.30 ml, respectively). SDS electrophoresis showed that the subunit composition of the fractions did not change from the head through the tail of the peak, whereas in a controlmixture of equimolecular amounts of rHF and rLF, which eluted in the same volume (13.19 ml), the H chain proportion decreased from the head to the tail fractions of the peak (not shown). These heteropolymer preparations were subjected to crosslinkingexperiments with glutaraldehyde, under conditions which were shown to stabilize subunit dimers and oligomers (26). Dimers and trimers of H and L chains can be clearly separated by SDS electrophoresis, and the results show that most of the dimers are mixed H and L chains, whereas the H-homodimers are almost undetectable and the L-homodimers arepresentin minor proportion (Fig. 4). Thus, the distribution of the subunit dimers in these experiments is significantly different from the one expected by a random interaction between the H and L chains, which should produce equivalent amounts of homo- and heterodimers.
Recombinant Ferritin Heteropolymers
12746
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FIG.1. Analyses of renatured H and L homopolymers. A, fluorescence emission spectra of native (n)and renatured ( r ) rLF and rHF with excitation a t 295 nm; 50 pg/ml apoferritins in 0.1 M phosphate, pH 7.4, 3 mM DTT. B, denaturation plots of native and renatured ferritins monitored by the ratio of fluorescence emission a t 355 and 330 nm with excitation a t 295 nm; apoferritins a t 50 pg/ml in 0.1 M phosphate, pH 7.4; D.N., denatured in 6 M GdnHC1, 0.1 M phosphate, pH 3.5. C,progression plots of iron uptake monitored by absorbance at 310 nm. Conditions: 0.1 p M apoferritins, 0.1 M Hepes, pH 6.5, 0.1 mM ferrous ammonium sulfate, 30 "C.
In otherexperiments unfolded H and L chains were mixed in various proportions and renatured. Eachpreparations moved in electrophoresis as a single monomeric band with mobility intermediate between the slower L and the fasterH homopolymers, in agreementwith the subunitcomposition of the mixture (Fig. 5 A ) . In blotting experiments,the antibodies for H (rH02) and L chains (LF03)stained the renatured heteropolymers with intensity related to the H:L proportion (Fig. 5B). Even this sensitive method did not reveal the
FIG.3. Isoelectric focusing of renatured ferritins. Controls of native rHF (lanea ) and rLF (lane b ) and the mixture of the two (lane f ) renatured homopolymers of H chain (lane c) and L chain (lane e ) , renatured heteropolymers from 1:1 ratio of H and L chains (lane d). Ampholine pH range 3.5-10, 5-10-pg protein samples, stained with Coomassie Blue.
presence of homopolymers in the heteropolymer preparations. Table I shows that, in two renaturation experiments, the total protein recovery after renaturation and concentration was analogous for all preparations (64-74%) and that thesubunit proportion found in the renatured samples was close to the expected one, except for a minor loss of H chain in the Hrich preparations. The different heteropolymer preparations showed parallel denaturation curves, constructed as in Fig. 1B, and their relative stability, indicated by the GdnHCl concentration of the 50% transitionpoints(Table I), decreased linearly with increasing H chain proportion ( R = 0.98). The seven apoheteropolymers incorporated equivalent amounts of iron when incubated aerobically with a 1000 atoms of ferrous iron/molecule for 2 h at pH 7.0 (not shown),
12747
Recombinant Ferritin Heteropolymers
indicating thattheyallarefunctionalferritins.Inother experiments we measured the initial ratesof iron uptake (at pH 6.5 and with a 1OOO:l iron:protein ratio) by the variation of optical reading a t 310 nm. The results (Fig. 6) show that the rate of uptake increases nonlinearly with the increase of H chain content: a sharp raise from0 to about35% and then it remains constant in the range 35-100% H chain. DISCUSSION
Assembly of Ferritin Homo- and Heteropolymers-Present data confirm that ferritins readily refold and reassemble in vitro to yield proteins which are functionally and structurally FIG. 4. Subunit cross-linking. H and L homopolymers (rHF analogous to the native ones(23-27). The H and L chains of and rLF) and the heteropolymer with 1:l of H and L chains ( H + L) human ferritin were found to have almost overlapping renawere incubated with 0.1% glutaraldehyde and analyzed by SDS-gel turation plots (Fig. 2), in contrast with their denaturation electrophoresis. MW, molecular mass standards expressed in kilodaltons; the arrows indicate H and L subunit monomers; the arrowheads plots which are remarkably different (Fig. 1B). This finding indicate the subunit dimers: H-H, H-L, and L-L, respectively, from indicates that thephysical stabilities of ferritins are unrelated to their renaturation conditions and suggests that ferritin the top of the gel. unfolding and refolding/reassembly follow differentpathways. This is consistent with the largehysteresisbetween A denaturation and renaturation plots of ferritins (see Figs. 1B 1 2 3 4 5 6 7 H I L and 2 and Ref. 26), which indicates that the conformation changes from unfolded to renatured protein arecomplex and not reversible, as it may be expected for a large multimeric protein. The evidence that H and L chains renature in the same conditions prompted usto study whether theyco-assemble in vitro. In fact they do (Figs. 3 and 5, Ref. 28), possibly with the same mechanism in which isoferritins are assembled in B tissues. When H and L chains are renatured together they H1 2 3 4 5 6 7 L H I L show a clear preference for the formationof heteropolymers over the homopolymers, and the distribution of the heterorH02 polymers is remarkably narrow. For instance from the renaturation of H and L chains a t 1:1 ratio, we obtained samples which behaved as homogeneous ferritin preparations on gel filtration columns and gel electrophoresis and which, more importantly, covered only a limitedrange of the possible isoferritin spectrum: four bands in isoelectric focusing to be compared with the two to three bands of the homopolymers (Fig. 3). A randominteractions betweenH and L chains LF03 of various species (e.g. FIG. 5. Nondenaturing electrophoresis of heteropolymers should lead totheformation with various H:L proportions. A , Coomassie Blue stain of the H&.. .Hl2LI2... ..HsL16) covering a significant proportion finding that the species seven renatured heteropolymers described in Table I and of a mixture of theisoferritinspectrum.The of rHF and rLF ( H / L ) . B, immunoblotting of the samesamples observed are fewer than expected suggests the existence of stained with the anti-H chain monoclonal antibody rH02, with the preferential interactionsbetween H and L chains, and it is in anti-L chain LF03 and sequentiallywith both antibodies.H , rHF; L, agreement with the cross-linking experiments showing a prefrLF; the arrows indicate the mobility of the ferritin monomers. erential formation of H-L chain dimersover the homodimers (Fig. 4). The demonstration of recognition specificities between H and L chains goes some way to explain why H and TABLE I Reassembly of H and L heteropolymers Sample
H chain content Expected"
Detectedb %
32
1 2 3 4 5 6 7
100 67 83 67 6450
33 17 0
100 76 59 47 6.6 14 0
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68
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40 io 60 i o 80 90 1 I % H chain FIG. 6. Initial rates of iron uptake of the heteropolymers with differentH:L ratios. Apoferritins,0.1 p M in 0.1 M Hepes, pH 6.5, 30 "C, were added of 0.1 mM ferrous ammonium sulfate and the rates of iron uptake monitored in the first minute by an optical reading at 310 nm. The mean of three experiments with less than 10% deviation is shown.
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20
30
12748
Recombinant Ferritin Heteropolymers
L homopolymers are poorly populated in mammalian tissues in iron detoxifications and cellular protection (14) and are (6, 31-33). probably more fitted for the needs of non-iron storage tissues, Present data demonstrate that it is possible to produce in whereas the “L-like” ferritinswith slower rates of iron uptake uitro, and with yields above 60%, restricted populations of form larger iron cores in uitro (16) and are probably more recombinant ferritin heteropolymers with predefined H:L ra- suited to the needs of the iron-rich tissues. Present data also tios. This allows the investigation of the effect of different suggest that thesmall changes in H:L ratio that occur during proportions of the H and L chains on the biochemical and cellular proliferation or differentiation (34, 35), in developfunctional properties of the ferritin cages. ment (36), or which are induced by chemical agents (8, 37, Functionality of the Heteropolymers-We approached the 38) have a functional effect that is related to the isoferritin characterization of the recombinant heteropolymers by pro- content of the tissues: a strong one when they are rich in L ducing seven ferritin preparations with different H:L ratios. chain and a minor one when they are H-rich. Their electrophoretic mobilities and immunologicalproperties Acknowledgment-We are grateful to Dr. G. Vecchio (Hormone varied with the subunit content, as expected, and their stability to denaturationvaried linearly with their H (or L chain) Chemistry, Consiglio Nazionale delle Ricerche, Milano) for CD meascontent. In addition they incorporated similar amounts of urements. iron, under conditions (pH 6.5-7.0, 1OOO:l iron:ferritin molar REFERENCES ratio) which do not differentiate between H and L homopol1. Ford, G.C., Harrison, P. M., Rice, D.W., Smith, J. M. A,, Treffry, A., White, J. L. & Yariv, J. (1984) Philos. Trans. R. SOC.L o n d . B Biol. Sci. ymers. These findings suggested that some of the properties 304,551-565 of the heteropolymers can be predicted knowing their H:L 2. Theil, E. C. (1987) Annu. Reu. Biochem. 56,289-315 3. Harrison, P. M., Andrews, S. C., Artymiuk, P. J., Ford, G. C., Guest, J. R., ratios, but this does not apply to the kinetics of iron uptake Hirzmann, J., Lawson, D. M., Livingstone, J. C., Smith, J. M. A, Treffry, ( A A 3 1 0 n m ) . In fact, under the conditions in which the H A. & Yewdall, S. J. (1991) Adu. Inorg. Chem. 36,449-486 4. Aisen, P. & Listowsky, I. (1980) Annu. Reo. Biochem. 49,357-393 homopolymer takes up iron at rates severalfold faster than 5. Boyd, D., Vecoli, C., Belcher, D. M., Jain, S. K. & Drysdale, J. W. (1985) the L homopolymer (22), the rate of uptake of the heteropoJ. Biol. Chem. 2 6 0 , 11755-11761 6. Arosio, P., Cairo, G. & Levi, S. (1989) in Iron in Immunity, Cancer and lymers varies, but not linearly, with the subunit content; it Inflammation (deSousa, M., and Brock,J. H., eds) pp. 55-79, John Wiley shows a fast increase in the range from 0 to about 35% H & Sons, Inc., Chichester, United Kingdom 7. Arosio, P., Adelman, T. G. & Drysdale, J. W. (1978) J. Biol. Chem. 2 5 3 , chain and a plateaufrom 35 to 100% H chain. 4451-4458 Each H chain has a ferroxidase center and the ferroxidase 8. Miller, L. L., Miller, S. C., Torti, S. V., Tsuji, Y. & Torti, F. M. (1991) Proc. Natl. Acud. Sci. U.S. A. 88, 4946-4950 activity of ferritins was expected to be related to theconcenof Recombinant 9. Lawson, D. M. (1990) X-rayStructureDetermination Ferritins. Ph.D. thesis, University of Sheffield, Sheffield, United Kingtration of catalytic sites, under the conditions of iron and dom oxygen excess used. However, the mechanism of ferritin iron 10. Harrison, P. M., Ford, G. C., Rice, D. W., Smith, J. M. A., Treffry, A. & White, J. L. (1987) Biochem. Soc. Trans. 16,744-748 uptake is more complex than a simple oxidation, since the D. M., Artymiuk, P. J., Yewdall, S. J., Livingstone, J. C., Treffry, iron atoms oxidized on the ferroxidase centers (which can be 11. Lawson, A,, Luzzago, A., Levi, S., Arosio, P., Cesareni, G., Thomas, C. D., Shaw, W. & Harrison, P. M. (1991) Nature 349,541-544 as many as 24/molecule) must be transferred to the core in S., Luzzago, A,, Cesareni, G., Cozzi, A., Franceschinelli, F., Albertini, the ferritin cavity. These two steps appearto occur in different 12. Levi, A. & Arosio, P. (1988) J. Biol. Chem. 2 6 3 , 18086-18092 sites of the molecule (12, 16, 17), and it is likely that the rate 13. Levi, S., Salfeld, J., Franceschinelli, F., Cozzi, A., Dorner, M. H. & Arosio, P. (1989) Biochemistry 28,5179-5184 of iron oxidation and that of transfer/core formation may be 14. Cozzi, A,, Santambrogio, P., Levi, S. & Arosio, P. (1990) FEBS Lett. 277, 119-122 limiting in different experimental conditions. This appearsto Lawson, D. M., Treffry, A,, Artymiuk, P. J., Harrison, P. M., Yewdall, S. 15. be the case with the heteropolymers; the ferroxidase capacity J.. Luzzaco. A,. Cesareni., G... Levi.. S. & Arosio., P. (1989) . . FEBS Lett. 264,2071210 ’ is rate-limiting from zero to about eight H chains/molecules V. J., Levi, S., Arosio, P., Treffry, A,, Harrison, P. M. & Mann, S. and then thecore formation capacity becomes saturated and 16. Wade, (1991) J. Mol. BioL 2 2 1 , 1443-1452 it becomes rate-limiting, under the present conditions. The 17. Levi, S., Santambrogio, P., Cozzi, A., Rovida, E., Albertini, A., Yewdall, S. J., Harrison, P. M. & Arosio, P. (1992) Biochem. J. 288,591-596 increase in rate of iron uptake from 0 to 35% H chain may be 18. Santambrogio, P. , Levi, S., Arosio, P., Palagi, L., Vecchio, G., Lawson, D. M., Yewdall, S. J., Artymiuk, P. J.,Harrison, P. M., Jappelli, R. & also due to a synergistic activity of H and L chains; the L Cesareni, G. (1992) J.Biol. Chem. 267,14077-14083 homopolymers have a higher efficiency to form the core than 19. Ruggeri, G., Iacobello, C., Albertini, A,, Brocchl, E., Levi, S.,,Gabri, E. & Arosio, P. (1984) in Ferritins and Isoferritins as Biochemacal Markers the H ones (17), a property possibly localized on carboxyl (Albertini, A., Arosio, P., Chiancone, E., and Drysdale, J. W., eds) pp. groups of the cavity. If this characteristic remains in the L67-78, Elsevier Science Publishers B. V., Amsterdam rich molecules (e.g. 100-65% L chain), itwould facilitate the 20. 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