Subunit heterogeneity in ferritin

Vol. 55, No. 4, 1973

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SUBUNIT HETEROGENEITY IN FERRITIN Yoshiro Niltsu, Kunihlko Ishitani and Irving Listowsky Department of Biochemistry, Albert Einstein College of Medicine, Bronx, N. Y. Received October 29,1973

SUMMARY: Subunits of horse, human, rat and rabbit ferrltlns were examined by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Each ferritin preparation had multiple different subunit components, and the molecular weights of the three major polypeptide chains were approximately 19,000, 10-11,000 and 7-8,000. The subunits were isolated and purified, and it was demonstrated that the two lower molecular weight polypeptides originated from the higher molecular weight component. The 7-8,000 and 10-11,000 molecular weight components are probably linked together by very stable non covalent interactions and thus migrate together in the gels. It is suggested that ferrltln should be considered to contain at least two different polypeptide subunits, and this proposal contradicts the prevailing v i m that it consists solely of 24 identical subunits. I NTROD UCTI O N According to a number of recent reports, the protein moiety of ferritin consists of 24 identical polypeptlde chains (tool. wt. 18,500) (1-4) and these subunlts are uniformly arranged as a hollow sphere surrounding an inorganic iron oxide micelle in its central cavity (5). The icosahedral symmetry as determined by X-ray crystallography (6) and some earlier analytical data (7,8) are not consistent with a 24 subunit model.

Furthermore,

isoelectric focusing methods that show characteristic microheterogenelty in native ferrltin (9,10), and other indications that small amounts of minor subunlt components are present in ferritln (3,11), are also not entirely compatible with the assumption that the subunlts are identical. The heterogeneity in ferritln had become a controversial issue but it recently appeared that the controversy was finally resolved on the basis of claims that the minor subunit components were not found (4), and that the isoelectric focusing results were artifactual (12,13). In the present study, we have analyzed the subunit structure of ferritins obtained from several different sources.

Evidence is presented that all of the

ferritlns studied indeed contain more than one type of subunlt, and these subunits were 1134 Copyright ©1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

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characterized by electrophoresis in sodium dodecyl sulfate (SDS). It follows that any model of ferritin that is based on the premise that the protein consists exclusively of 24 identical subunlts, is oversimplified, and probably incorrect. MATERIALS AND METHODS Protein preparations: Human and rat liver ferritins were prepared according to the procedure of Drysdale and Munro (14), with some minor modifications(15). Rabbit liver ferritin was a gift from Drs. D. Shafritz and J.W. Drysdale. Horse spleen ferritin was obtained from Miles-Pentex (6X crystallized) or from Sigma (2X crystallized). The commercial ferritins were routinely purified by gel filtration using Sepharose 6B. Aggregates of whole ferritin molecules (dimer, trimers, etc.) which are commonly found in ferritins, were removed by the gel filtration to preclude ambiguity in the interpretation of results. The resulting ferritins were thus homogeneous chromatographic entities, and each migrated as a single component in polyacrylamide gel electrophoresis (5% gels) (16,17). PolyacryJamlde gel electrophoresis in SDS: The SDS gels (11% acrylamide) were usually prepared in trls--acetate buffer pH 7.4 by the procedure described by Fa~rbanks et. a1.(18). The protein samples were heated to 100° for 3 min. in 1% SDS and then applied directly to the SDS gels. The gels were stained with Coomassle Brilliant Blue, fixed and destalned by the stepwise procedure described in reference 18. Unstained gels were scanned at 280nm, and stained gels near 600nm, using a Gilford spectrophotometer with a model 2410 linear transport.

SDS from Schwartz-Mann was used, and dialysis procedures

were carried out using Spectropor #3 membrane tubing (tool. wt. cut off 3,500)(Spectrum Medical Industries, Los Angeles, Calif.), to minimize losses of the low molecular weight po lypeptides. RESULTS AND DISCUSSION Each ferritin preparation heated in SDS solution to dissociate the protein into

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subunits, was examined by electrophoresis in SDS polyacrylamide gels. Horse spleen ferritin exhibited four major protein bands with electrophoretic mobilities corresponding to molecular weights of about 19,000 (IV), 15,000 (111), 10-11,000 (11), and 7-8,000 (I) (Fig. 1). Lesseramounts of some additional bands of higher molecular weight were also

III II

N E o

w

Z

:

L

© 03 o3

I ' - - - Z ............3

4

5

RELATIVE

6

7

8

g

MOBILITY

Fig. 1 - Densitometer tracing of an unstained gel of native horse spleen ferritln (70pg). The stained gels shown above, were tandem gels of the same preparation. Gel 1 is identical to the gel shown in the 280nm scan. Gel 2 is ferrltin pretreated with mercaptoethanol. Gel 3 contains some molecular weight markers: i, insulin; c, cyctochrome-c; m, myoglobln; cp carboxypeptidase; and o, ovalbumin.

observed. The latter minor components probably represented aggregates of ferritln subunits linked by disulfide bridges, since they disappeared if the protein was preincubated in the presence of mercaptoethanol or dithiothreitol in SDSo The mercaptoethanol treat-

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ment also eliminated band (111)(mol. wt. 15,000) which was a prominent component in the original electrophoretic profile, leaving components I, II and IV(Figo 1). Ferritins obtained from human, rat and rabbit livers, and prepared by diverse experimental methods all showed similar patterns of heterogeneity in the SDS gel systems employed. Band III was however, conspicuously absent in the human ferritin. On the basis of this observation, it appeared to us that the component with a molecular weight corresponding to 15,000 (111)was probably not a native subunit, but may randomly form via disulfide cross links during the course of the SDS denaturation of the protein. In this regard it is noteworthy that human liver ferritins have a lower cysteine content than the ferritins from the other species studied (19). In the presence of mercaptoethanol however, the three band types common to horse spleen ferritin (I, II and IV), were also consistently found in the other ferritin preparations. The properties of our purified ferritins ( i . e . , morphological appearance (15), CD spectra (20), electrophoretic mobilities (16), amino acid composition (19)) were consistent with those reported in the literature for this well characterized protein, yet the observed subunit heterogeneity appears to contradict the prevalent view regarding its subunit structure° We considered the possibility that heating the sample to 100° or other fortuitous circumstances may have induced the cleavage of a specific, particularly labile, peptide bond, although no fragmentation of any of our purified molecular weight marker proteins was observed under these conditions. Ferritins were therefore treated with 7 M guanidinlum chloride pH 4.0, and subsequently dialyzed against 8 M urea and tken 1% SDS. These preparations were not heated at all, and still showed substantial amounts of components 1 and I1 on the SDS gels. The iron also appeared to play no role in generating the multiple bands since apoferritins, which were essentially iron free, also exhibited similar multiple bands on SDS gels. In an attempt to explain the heterogeneous patterns and further characterize the

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ma jor subunit components of horse spleen ferrltin, components I-IV were isolated by preparatlve gel electrophoresls in SDS. The isolated components were then reexamined separately on the analytical gels. Purified bands i and II each migrated as a single species, and their e lectrophoretic mobilities were unchanged in the presence of mercaptoethanol, or by fui'ther heating and/or longer periods of incubation (5 days) in SDS. In the absence of mercaptoethanol component III (tool. wt. 15,000)also migrated as a single band, and the electrophoretic mobility of this band was identical to that of component III in the original ferritin subunit pattern. In the presence of mercaptoethanol however, purified component I11 unexpectedly showed 3 bands that corresponded to original components I, II and IV (tool. Wto 7-8,000, 10-11,000 and 19,000 respectively). Component III is therefore identical to component IV except that it probably contains a disulfide bridge. The disulfide bridge in III, may confer a more compact structure to the molecule~ and the electrophoretic mobility of this component appears to correspond to that of a peptide of lower molecular weight because of the molecular sieving effect of the gels. This result also implies that components I and II may originate from component IV. Component IV (tool. wt. 19,000) isolated from preparative gels, showed 3 bands, I, II, and IV when reexamined on the analytical gels. If the 19,000 mol. wt. component was reisolated again from this sample and the analytical process repeated, a further breakdown of component IV occurred as more of the lower molecular weight components (I and II) appeared.

It was evident therefore, that under the proper conditions, the 19,000 tool.

wt. component (IV) may ultimately break down entirely into the two constituent polypeptide chains with molecular weights of 10-11,000 and 7-8,000. It is noteworthy that an earlier report (21) suggested that ferritin subunits may have molecular weights of less than 12,000. The present results (summarized in Fig° 2) also rule out the possibillty that the low molecular weight peptides were impurities that were carried along with the ferritin in the course of its isolation and purification.

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! 11101. Wt. m

.....

m

I ,.°.,~

19000 15000 I0000 7000

BAND IV (19000)

BAND III (15000)

Fig. 2 - Schematic representation of the breakdown of purified components IV and III. Component IV ultimately breaks down to I and Ii, component II1 is converted to IV in the presence of mercaptoethanol, which then breaks down to ! and II.

Although the mechanisms for the formation of peptldes, I and II cannot be defined at thls time, it is clear that well characterized ferritin preparations that are chromatographically and electrophoretically homogeneous should no longer be considered to consist solely of 24 identical polypeptide chains with molecular weights of 18,500. The possibility that the multiple polypeptide chains may have originated from a proteolytic or other type of cleavage of a peptide bond that occurs after isolation of the ferrltln from the tissue should not however be ruled out. The cleavage, if it does occur, would have to be of a very specific nature and is apparently ubiquitous in different ferrltlns. On the other hand, it is concelvable that the multiple subunits may be indigenous in ferritins which may in reality be comprised of 2 distinct polypeptlde chains of molecular weight 10-11,000 and 7-8,000. These two chains could interact via strong non covalent linkages that are not easily disrupted in the SDS. Native ferritin may therefore consist of many more than 24 subunlts. The observed microheterogeneity of undissoclated native ferritins, which has been difficult to rationalize in terms of the 24 identical subunit models, (trivial effects such as non specific deamldations could explain these results) may na~be

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considered in terms of possible minor variations in combinations of these different polypeptide chains to yield hybrid like ferritin molecules. Acknowledgment: Y.N. and K.I. are on leave from the Cancer Research Institute, Sapl:~'o Medical College, Sapporo, Japan. This work was supported by grant HL 11511, and a Career Development Award to I.L. from the National Institutes of Health. We also thank Mr. Mitchell Weiss for his technical assistance. References: 1o 2. 3. 4. 5. 6. 7.

Crlchton, R.R. and Bryce, C.F.A. (1970) FEBS Letters, 6_, 121-124o Bryce, C. FoA. and Crichton, R.R. (1971) J. Biol. Chem., 246, 4198-4205. Bjork, I. and Fish, W.W. (1971) Biochemistry, 10, 2844-288z~'~'. Crlchton, R.R. (1973) Angew. Chem., !2, 57~-5"o Farrant,J.L. (1954) Biochim. Biophys.'-,~'cta., 13, 569-576. Harrison, P.M. (1963). J. Mol. Biol., 6, 404-472. Harrison, P.M., Hoffman, T. and Mainwaring, W.I.P. (1962) J. Mol. Biol., 4, 251-256. Malnwarlng, W.I.P. and Hoffman, T. (1968)Arch. Biochemo Biophys., 125, 975-980. Drysdale,J.Wo (1970) Biochim. Biophyso Acta., 207, 256-258. Urushizaki, I., Niitsu, Y., Ishltani, K., Matsuda,~. and Morlmichi, F. (1971) Biochim. Biophys. Acta., 243, 187-t97. Shafritz, D.A., Drysdale, J.--~. and Isselbacher, K.J. (1973) J. Biol. Chem., 248, 3220-3227. Tee--,J.C.K. and Richter, G.W. (1971) Comp. Biochem. Physiol., 29B, 325-333. Bryce, C.F.A. and Crichton, R.Ro (1973) Hoppe-Seylers Z. Physiol. Chem., 354, 344-346. Drysdale, J.W. and Munro, H.W. (1965) Biochem. J., 95, 851-858. Niitsu, Y. and Listowsky, I. (1973)Arch. Biochem. Biop-~'ys., 158, 276-281. Niltsu, Y. and Listowsky, I. (1973) Biochemistry (in press). Listowsky,I., Betheil, J.J. and Englard, S. (1967) Biochemistry, 6, 1341-1348o Falrbanks, G., Steck, T. L. and Wallach, D.F.H. (1971) Biochemistry, 10.._., 2606-2617. Crichton, RoR., Millar, J.A., Cumming, R. L.C. and Bryce, C.F.A. (1973) Biochem. J., 131, 51-59. Listowsky,I., Blauer, G., Englard, S. and Betheil, J.J. (1972) Biochemistry, 11_, 2176-2182° Smith-Johannsen, H. and Drysdale, J.Wo (1969) Biochim. Biophys. Actao, 194, 43-49. m

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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