Heterogeneity of horse spleen ferritin and apoferritin: Comparison of electrophoretic and chromatographic fractions

ARCHIVES

OF

BIOCHEMISTRY

Heterogeneity

AND

BIOPHYSICS

of Horse

Spleen

of Electrophoretic ANITA Department of Biochemistry,

399406

(1965)

Ferritin

and

111,

and

Chromatographic

A. SURAN University Received

Apoferritin:

AND oj

March

Comparison

Fractions

H. TARVER

California,

San Francisco, California

31, 1965

Horse spleen ferritin and apoferritin each separate into several analogous cornponents in gel electrophoresis. Sedimentation studies indicate that the major cornponents are related as monomer, dimer, and trimer, and that the electrophoretie separation is due to differences in molecular size or shape rather than to differences in charge. Disulfide bonds do not appear to be involved in the formation of the aggregates. Moreover, the pattern of aggregation could not be altered by various agents which might be expected to disrupt macromolecular aggregates. Chromatography of ferritin on DEAE-cellulose reveals a different and unstable order of heterogeneity, not related to aggregate size. No significant difference in amino acid composition or in “fingerprints” of tryptic peptides of the DEAE fractions could differences among ferritin be shown. Fractionation may be due to conformational molecules. Free boundary electrophoresis and sedimentation studies on ferritin and apoferritin have shown that their net surface charges are identical and are attributable to the

above 4’ was avoided. After freshly frozen horse spleen was homogenized in ice water, it was extracted at pH 4.75 (acetic acid) for 16 hours. Cellular debris was removed and ferritin was precipitated by the addition of 30 gm of ammonium suIfate per 100 ml, redissolved in water, and reprecipitated by addition of 0.25 volume of 0.3 M in cadmium sulfate in 0.4 M in sodium chloride. Apoferritin was prepared by a modification of the method of Granick and Michaelis (13). About 350 mg of sodium dithionite was added to 60-90 mg of ferritin in 10 ml of 0.05 M sodium acetate (pH 4.8). The solution was tightly covered, and when the red color of the ferritin disappeared, it was applied to a 20.gm Sephadex G-25 column (Pharmacia Fine Chemicals) equilibrated with the

protein moiety rat.her than to iron content (l-3).

Reports

have

appeared

which

indicate

that ferritin yields stable fractions in gel electrophoresis (4-9), and that similar fractions are present in apoferritin (8, 10, 11). We had made similar observat’ions, and t,his report describes studies which show that the fractions are molecules in various states of aggregation rather t’han with different charges. Fractionation of horse ferritin on DEAE-cellulose has been reported (4, lo), but the fract,ions do not correspond to those obtained by electrophoresis, and we have found t)hat upon recycling the fractions a redistribution of protein occurs, with a tendency to concentrate t’he protein into a single peak. MATERIALS

AND

METHODS

We used commercial ferritin from horse spleen (twice crystallized, Pentex, Inc.) and a preparation made in this laboratory based on the method of Granick (la), but any heating of t.he preparation

acetate buffer. Apoferritin appeared at the end of the void volume, well separated from ferrous ion and salts which followed. Starch-gel electrophoresis was conducted on horizontal plates according to the method of Smithies (14); Poulik’s buffer system of borate and tris-citrate (15) at pH 8.6 was used, or a similar buffer containing 7 M urea described by Wake and Baldwin (16) or a 0.11!4 glycine buffer adjusted to pH 3.0 with HCl. Starch concentration was that recommended (Connaught Laboratories, Toronto, Canada). In order to carry out analytical scale acryl-

399

400

SURAN

AND

amide-gel electrophoresis, we used EC. Apparatus Co. reagents. Five gm of Cyanogum 41 (a mixture of acrylamide and N,N’-methylene-bisacrylamide) was dissolved in 100 ml of the appropriate buffer, either the tris-citrate or the glycine buffer. The solution was filtered and 0.3 ml dimethylaminopropionitrile (DMAPN) solution, and 25 mg ammonium persulfate were added successively with gentle swirling. The solution was immediately poured to fill a lucite mold 3 mm deep which was then tightly covered with a plate of glass greased at the edges. After gelation, 2.5 X 7.5.cm sections of the gel were cut out and stored at 4” in the appropriate buffer. Pieces of Whatman No. 3 MM paper (l-4 mm”) with up to 5 ~1 of protein solution on them were inserted in the gel by sliding them down a spatula tip into slits cut into the gel. The gel on a glass slide was connected to the electrode chambers via short lengths of cellulose sponge, and after an average run of 60 minutes at about 6-8 mA and 80-100 V the protein was stained for 5 minutes in a 1% solution of Naphthol Blue Black (Allied Chemical Corp.) in methanol-acetic acid-water (5:5:1). The gel was cleared in 10% acetic acid. Preparative electrophoresis was done in 5% acrylamide gel at pH 8.6 in a mold 13 X 13 X 2.5 cm. Three ml of a solution containing about 100 mg of ferritin was absorbed onto a piece of white felt which was inserted into a 2-3 mm wide slit in the gel. The gel was covered with a slightly shorter lucite plate (allowing for insertion of cellulose sponges at each end) which was bolted to the mold. Runs were made at 4” with the gel in a vertical position, at an average potential drop of 4-5 V per centimeter at 40-50 mA for 16-24 hours. The red zones were cut out of the gel, each was ground in a loose-fitting hand homogenizer with 0.2 M ammonium sulfate, and extracted one or more times with 46 volumes of this solution for 34 hours. The gel was removed by centrifugation and ferritin was precipitated with cadmium as before. Sedimentation velocity studies were made with a Spinco model E ultracentrifuge equipped with schlieren optics and wedge cells. Four to five mg per milliliter samples of apoferritin were dialyzed against 3-4 changes of 100 volumes of a 0.01 M sodium acetate buffer (pH 4.8) 0.1 M in potassium chloride. Sedimentation constants were calculated according to the method of Schachman (17). DEAE-cellulose (Schleicher and Schuell) was packed into a 2.5 X 59cm column under hand pressure exerted upon a perforated plunger which was then locked into place. Up to 100 mg of ferritin was applied to this column. Buffers were pumped at a constant volume of 6 ml per minute, and lo-ml samples were collected. Pooled fractions were precipitated wit,h cadmium.

TARVER For tryptic hydrolysis, a 3-5 mg sample of apoferritin in 10 ml of water was denatured by heat,ing. The precipitated protein was collected, resuspended in 4 ml of water, brought to pH 9.0, and digested under nitrogen wit,h 0.3-0.5 mg of trypsin (twice crystallized, salt-free; Worthington Biochemicals). The reaction was followed on a pHstat (Radiometer, Denmark) until uptake of 0.1 N NaOH ceased and the solution was clear. Aliquots of the hydrolyzate representing about 1 mg of protein were applied as 2 X 15-mm streaks on Whatman No. 3 MM paper. High voltage electrophoresis was conducted in an apparatus similar to that described by Katz el al. (18); one of the following solutions was used: (a) pH 2.1 solution of 2.57, formic acid, (b) pH 3.5 solution of acetic acid-pyridine-water (10: 1: 180), (c) pH 6.4 solution of acetic acid-pyridine-water (0.2:10:180), and (d) pH 8.5 solution of 0.05 M ammonium carbonate. Two thousand V was applied for 60 minutes. The dried papers were stained with 0.2% ninhydrin in acetone and then redried in a stream of air at 90”. Amino acid analyses were made with an automatic analysis system (Technicon Chromatography Corp.). Protein samples in redistilled HCI were frozen and sealed in evacuated tubes (19) and hydrolyzed for 16 hours at 105”. Hydrolyzates were taken to dryness at 45”~50”, redissolved in a small volume of water, re-evaporated, and finally dissolved in 0.1 N HCl for application to the column. In order to separate methionine sulfone from aspartic acid, we used the following modifications suggested by Technicon: the column was run at 55” instead of 60”, and the pH of the first buffer was 2.75 instead of 2.87. Protein was estimated by the method of Lowry et al. (20). RESULTS

Electrophoretic fractions. Ferritin and apoferrit,in each contained 3-5 analogous components upon elect,rophoresis in starch or acrylamide gels at pH 8.6. The fastest moving component contained about 85% of the protein and was followed by bands containing about 10 and 5 %. These components were stable to the electrophoretjic procedure: A lengthwise section sliced from a preparative run, when embedded as the origin for a second run, showed only the same discrete spots. The ferrit’in prepared in the cold behaved identically. Horse ferrit’in is isoelectric at’ pH 4.4 (1) so it was of interest to observe its behavior at pH 3.0. The same number of components was seen, and migration was in the same

HETEROGENEITY

OF HORSE

SPLEEN

order as at pH 8.6, the largest band moving most rapidly (Fig. l), so the fractionation was probably due to discrete proteins which differed in size or shape being separated by seiving in the gel. Apoferritin behaved in the same manner. Preparative gel electrophoresis yielded the fast, major component (GI) 90-100% pure as seen in subsequent analytical scale gel electrophoresis. The second component (GII) was obtained 5%80% pure, accompanied mainly by GI and traces of the slower

FIG. 1. Top: Electrophoresis of apoferrit,in and ferritin on acrylamide gel at pH 8.6. Negative electrode to the right. Bottom: Electrophoresis of apoferritin and ferritin at pH 3.0. Positive electrode to the right.

FERRITIN

AND

APOFERRITIN

401

component. These fractions were reduced to apoferritin and st’udied in the ultracentrifuge. Figure 2 shows t’he sedimentation patterns for unfractioned apoferritin which contained 18 S and 26 S protein peaks in a ratio of about 9: 1 (2). Fraction GI showed a similar distribution of material; apoferritin GII contained a significant amount of fast sedimenting 34 S material (GIII), and 26 S (GII), and 18 S (GI) in the approximate ratio of 1:4: 5. The values of 18, 26, and 34 S are consistent with the apoferritin fractions existing as monomer, dimer, and trimer based on the approximation that the sedimentation constant is proportional to the N power of molecular weight. For a given apoferritin preparation the electrophoretic and ultracentrifugal patterns of protein distribution were always in close correspondence. In view of these findings, in the ensuing discussions the principal fractions, GI and GII, the 18 S and 26 S peaks, will be referred to as monomer and dimer, respectively. Chromatography on. DEAE-cellulose. Ferritin samples were applied to a column equilibrat,ed with 0.15 M sodium acetate (pH S.O), and elution was begun with this solution. Three fract,ions were obtained: a rapidly moving “A” followed by a small amount of “B” eluted with the initial buffer; a red band “C” remained at) t’he top of t,he column

FIG. 2. Left: Sedimentation diagram for unfractionated apoferritin showing about 90% of 18 S and 10% of 26 S components. Right: Apoferritin GII containing 18 S, 26 S, and 34 S peaks in the ratio of 5:4:1. These photographs have been masked to show only one of the patterns seen with the use of a wedge cell. For designation of fractions see Table I.

402

SURAN

F FIG.

ferritin details

TUBE

3. Comparison and apoferritin see text).

AND

NO

of the elution patterns from DEAE-cellulose

of

(For

TABLE I

Ferritin Al A2 Cl

FRACTIONS”

70 Recovery of protein as fraction B

A

48 (Al) 47 (A2) 47 (A3)

7 6 10 -

C

45 (Cl) 22 31 86 (C2)

a The progress of fractionation of ferritin and apoferritin on columns is shown as follows: Initial fractions designated A, B, C; recycled fractions as Al, C2, etc. Fractions prepared on acrylamide gels are designated Gl, G2, G3, with Gl being the fastest moving component. Combinations of fractionation procedures are indicated by the appropriate symbols shown in the order of the methods employed, e.g., Al-G1 indicates a component run twice through DEAE then separated on a gel.

c!

protein; so C was collected by changing to 0.15 M sodium acetate at pH 4.5. Hence C was expected to be heterogeneous. Under identical conditions, apoferritin was eluted solely as C (Fig. 3). Similar elution patterns were observed in preliminary experiments by using pH or ionic strength gradients with tris-citrate in columns equilibrated at pH 7.6. The stability of the chromatographic fractions

RECHROMATOGRAPHYOF FERRITIN Material applied to column = 100%

TARVER

to

the

fractionation

procedure

was

examined by recycling experiments. Rechromatography of ferritin fraction A yielded about half of the material eluted from the column as A (here called Al), and in turn, rechromatography of Al gave A2 with a similar yield. In contrast, recycling fraction C yielded all the recoverable protein as Cl (Table I). Comparison of electrophoretic and chromatoqraphic fractions. Since gel electrophoresis of ferritin produced two major fractions, GI and GII, and column chromatography gave three fractions, A, B, and C, we used various combined procedures to investigate the relat’ionship between the two classes. Selected samples were checked by sediment,ation. Ferritin A3 was pure monomer in

, pH 4.5

TUBE

NO.

4. Chromatography on DEAE-cellulose of major gel electrophoresis fractions. GI was mainly 18 S monomer and eluted in two peaks, GI-A and GI-C. GII, enriched in aggregates and containing about 50% of monomer, eluted as GII-C. For designation of fractions see Table I. FIG.

and the length of the column was similarly stained. Applications of gradients, either of pH, ionic strength, or combined pH and ionic

strength,

latter material

merely

“rolled-up”

all

this

int,o a broad peak of dilute

FIG. 5. Top: Ferritin GI (main fraction in gel electrophoresis, chiefly monomer) yielded DEAE fractions, GI-A and GI-C, which were again examined by analytical scale electrophoresis. Monomeric ferritin predominates in both chromatographic fractions. Bottom: Ferritin GII, enriched in aggregated forms, was eluted from DEAE as GII-C and still contained the mixture of size classes. For designation of fractions see Table I.

HETEROGENEITY

OF HORSE

SPLEEN

FIG. 6. Sedimentation patterns of apoferritins prepared from ferritins GI-A (lower) and GI-C (upper). The distribution of protein into size classes corresponds to that seen in the upper gel of Fig. 4. For designation of fractions see Table I.

gel electrophoresis as was B2, but C2 was significantly enriched in aggregated forms. Apoferritin, which was eluted as C, also contained monomer plus aggregates. Since the DEAE fractions all contained monomeric ferritin, t)he effect of t,he chromatographic procedure on purified fractions isolated from preparative gel electrophoresis was investigated. Electrophoretic component GI, 90 % pure as monomer, yielded peak A (GI-A), a small amount of B (not further investigated), and peak C (GI-C). Component GII (enriched in dimer and larger aggregates) was eluted predominantly as C (GII-C) (Fig. 4). Analyt’ical gel electrophoresis of GI-A, GI-C, GII-C, and unfractionated ferritin showed that both of the GI fractions were mainly monomer, but GII still contained a mixture of monomeric and polymeric forms (Fig. 5). Sedimentat’icn patterns shown for fractions GI-A and GI-C

FERRITIN

AND

APOFERRITIN

403

in Fig. 6 correspond with the protein distribution seen in gel electrophoresis in Fig. 4. Conversion of ferritin fractions to apoferritin sometimes produced changes in the degree of aggregation. In the case of ferritin A3 (pure monomer in gel electrophoresis) this was particularly notable; apoferritin A3 contained about 15 % dimer and 5 % trimer as seen both in gel electrophoresis and in the ultracentrifuge. Table II summarizes sedimentation data for apoferritin fractions. Sediment’ation constants are not corrected for concentration dependence since early studies showed this to have little effect (2). Composition of DEAE fractions. Monomeric ferritin fractions after reduction to apoferritin and hydrolysis were analyzed for amino acid composition. The composition of each of the fractions was identical; differences in amounts of a given amino acid between the samples were no greater than those found within a group of replicates for a given sample (Table III). The values are in fair agreement with t’hose of Harrison et al. (al), assuming 24 subunits per molecule of apoferritin. Tryptic hydrolyzates of apoferritin monomers A3, B2, and C2-GI showed virtually ident’ical electrophoretic fingerprints in high voltage paper electrophoresis at four different pH values. Figure 7 shows those obtained at pH 3.5. Stability of aggregates. We test,ed the stabilit>y of aggregates as follows: TABLE SEDIMENTATION

Nature of samplea

Apoferritin, tionated A B2 C2-GI GI-C GII GII-C

II

CONSTANTS PREPARATIONS

unfrac-

OF

APOFERRITIN

s; x 10’8

18.2, 27.3 17.8,” 25.3,* 34* 17.9,b 27.5” 17.5, 27.8 17.8, 25.4 17.5, 25.8, 34 18.6, 26.6

a For key to the designation of fractions see Table I. *Average of data from duplicate determinations; a wedge cell was used.

404

SURAN

AND TABLE

TARVER III

AMINO ACID COMPOSITION OF APOFERRITIN

--

B-2

A-3

GI-A

Acid

FRBCTIONW d

I

-Asp Thr Ser Glu Pro Gly Ala Val %CYS” Metb Ileu Leu TY~ Phe NH, LYS His Arg

18.0 5.44 8.76 24.5 2.1 9.71 13.1 5.91 2.40 24.8 4.61 7.11 8.59 5.79 10.3

18.0 5.74 9.05 24.5 2.1 9.57 13.1 5.85 0.9 3.1 2.64 24.0 4.72 6.71 15.4 7.57 5.35 9.20

-

C2-GI

GI-c -'_

18.0 5.86 8.83 24.0 2.3 9.85 13.2 5.60 -

18.0 5.35 8.90 26.6 2.3 10.0 13.8 5.71 1.6 3.4 2.51 24.7 4.71 6.70 15.2 8.04 5.50 9.45

18.0 5.54 8.89 24.8 2.1 8.93 13.3 7.35 1.6 2.62 24.8 4.94 7.05 14.8 7.66 5.53 9.77

18.0 5.49 8.91 25.0 2.4 9.80 13.4 5.74 1.3

18.0 5.48 9.36 25.2 2.3 10.2 13.4 5.72 2.3 -

2 :61 25.7 5.10 6.91 15.6 8.14 5.29 9.27

2.63 24.7 5.16 7.02 14.7 7.65 5.29 9.30

-

18.0 5.67 9.18 24.1 3.0 9.49 13.2 5.55 0.9 3.3 2.47 23.4 4.91 6.75 18.7 7.71 5.30 9.13

18.0 5.64 9.10 25.2 2.5 9.71 13.4 6.20 1.6 3.3 2.81 23.2 5.05 6.86 15.3 7.71 5.45 9.15

18.0 5.63 9.04 25.2 2.3 9.45 13.2 5.76 1.7 3.5 2.68 23.5 5.01 6.70 14.6 7.48 5.29 8.75

-

18 5.8 8.1c 24 2.7” 10.5 14.5 7.2 2 3c 3.6” 25c 5.6 7.3 19 8.1 5.2 8.9

0 For designation of fractions see Table I. b Total values for cystine and cysteic acid and methionine plus methionine sulfone. c Average of range reported in Ref. (21). d Replicate determinations for a given sample are under the subheadings. Values are expressed as residues per subunit of molecular weight 20,000, and are normalized to the value of 18 residues of asp per subunits (21).

FIG. 7. High voltage electrophoresis of tryptic peptides at pH 3.5. Comparison of finger prints of peptides derived from unfractionated apoferritin and apoferritins from DEAE fractions A3, B2, and CS-GI. For designation of fractions see Table I.

(a) Electrophoresis of ferritin in starch and acrylamide gels in 7 M urea at pH 8.6 and pH 3.0 produced a pattern of distribution of prot,ein indistinguishable from that seen in the absence of urea. (b) The presence of 20-25 % ethylene glycol in the chromatographic system altered neither the characteristic elution pattern of ferritin nor t’he electropherogram of the elut)ed fractions.

(c) Apoferritin dialyzed for 24 hours at 20” in 1 M sodium chloride was examined in Dhe ultracentrifuge and appeared identical with undialyzed control apoferritin. (d) Ferritin and apoferritin incubated 16 hours in Yj% mercapt’oethanol at pH 3.0 showed no changes upon electrophoresis in the same system. Apoferritins A3 and GII (having different proportIions of aggregates) were incubat,ed in 0.1 M thioglycolate (pH

HETEROGENEITY

OF HORSE

SPLEEN

8.6) for 7 days, and samples showed no significant changes in t,he proportions of aggregated forms on electrophoresis in gels containing t’hioglycolate. The t.reated preparat’ion of A3 was also examined in the ultracentrifuge and found to be identical with cont,rol apoferrit,in A3. (e) A DEAE column was equilibrat,ed with a solution 0.05 M in t’hioglycolate and 0.10 M in acetate at pH 6.0. One-hundred mg of ferritin was preincubated with the same solution at’ pH 8.4 for 2 hours and applied to the column. The elution pattern was similar to Ohat usually observed for ferritin. However, the purple protein fractions contained apoferritin, as iron was removed during the procedure. The proport’ions of aggregated forms in the gel electJrophoresis bands remained the same. DISCUSSION

The fractionation of apoferritin in gel electrophoresis might be explained in several ways : (a) The fractions are artifacts produced during the isolat,ion of ferritin. This seems unlikely since like patterns of fractiona,tion have been observed in several laboratories employing various control procedures. Our preparation that, was isolated in the cold gave an identical pattern with the commercial preparation that had undergone heating at 80”. Ten minutes of addit’ional heating at. 80” did not alt’er the proportions of t,he components. (b) Reduction of ferritin to apoferritin may produce the fractions. However, as seen in Fig. 1 and reported elsewhere (8, 10, II), ferritin and apoferritin each show the same number of components, which contain about the same proportions of protein and which migrate with equal velocit’ies in gels. (c) Electrophoresis may itself produce t’he fractions as artifacts. Our finding that ferritin is stable to two 16-hour preparative runs agrees with the observations of others (4, 5, 7). (d) The fractions may separate due to differences in net charge. However, since identical orders of migration are observed on both sides of the isoelectric point (Fig.

FERRITIN

AND

APOFERRITIN

405

l), it is highly unlikely t,hat this is true. It should also be noted that only a single component appears on free boundary elect,rophoresis (1). (e) Fractionation may be due to the existence of molecules of like net charge density, which, because they differ in size and shape, are separated by t,he seiving effect of the gel. Our finding t>hat sedimenta,tion data are in agreement with the distribution of protein in the gel fract’ions confirms this conclusion. Sediment’ation constants are consistent with the fractions corresponding to monomer, dimer, and trimer.’ High concentrations of urea or ethylene glycol or salt failed to disaggregate the aggregat’es, and the sedimentation pattern of an apoferrit,in preparation enriched in aggregates was unchanged by 24 hours in 1 M sodium chloride. In addition, treatment of both the ferritin and apoferritin preparations with either mercaptoethanol or t,hioglycolate produced no changes in sedimentation or gel electrophoretic patterns. Hence it is not clear what forces are involved in maint’aining t’he aggregated forms of apoferritin since these treatments should have caused dissociation of various types of weak bonds and disulfide bonds. The behavior of ferritin upon chromatography on DEAE is complex. Carnevale and Tecce (9) obtained a fraction similar to our A which migrated as a single spot in gel electrophoresis. Theron et al. (4) effected a partial resolution of ferritin into a triple peak, each section of which still contained the three bands seen in gel electrophoresis of ferritin. We obt’ained t’hree fractions; A and B are distinct and mainly monomer, while fraction C is heterogeneous containing monomer, dimer, and trimer. Moreover, samples of the C region taken during gradient elution showed t.hat all of the aggregates were present, over the entire peak. 1 In the Svedberg equation, the sedimentation constant (S) is proportional to the product of molecular weight (M) and diffusion constant (D), and D in turn is inversely proportional to Ml/s. Therefore S N M2’3. Assuming the molecular weight of 18 S monomer to be 480,000, and the aggregatesto have equivalent shapes, we can calculate sedimentation constants of 28 S and 37 S for dimer and trimer.

406

SURAN

AND

The results of the recycling experimentas suggest that init’ially the chromatographic fractions of ferritin are separated by differences in surface properties, since with each successive cycle a significant amount of A is converted to C. Perhaps ferritin aggregabes exist in conformations-A, B, and ?!! which are converted to a stable conformat)ion, C, during chromatography.2 Since A and B fractions are not found in apoferritin, they may be stabilized by the presence of iron in ferritin. Alternatively, during the conversion of apoferritin to ferritin, the most st,able conformation may be achieved. Although the amino acid composition of a protein like ferritin with subunit)s of molecular weight 20,000-24,000 (21) can hardly reveal minor differences in primary structure, the fact that the chromatographic fractions all have what appears to be an identical composition, suggests that, all the subunits are identical. This conclusion receives support from the fact that, aft,er high voltage electrophoresis on paper, at four different pH values, t)he tryptic peptide fingerprints of the monomeric fra&ions of A, B, and C were virtually identical. The number of trypt,ic peptides is consistent’ with the arginine and lysine content of the subunit and suggests t,hat apoferritin consists either entirely or predominantly of one type of subunit), in agreement with published data (22, 23).

TARVER ferritin in the cold aud collaborated in the Sephadex procedure. Dr. R. A. Fineberg contributed mauy valuable discussions and criticisms t.o this study. This work was aided by a grant. from the National Science Foundation (G-21261). REFERENCES

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

ACKNOWLEDGMENTS We wish to thank Dr. Dale Gross for his assistance in the use of the ultracentrifuge. Captain Brendan Joyce (U.S.A.) made the preparation of 2 Yu and Fineberg (this laboratory) have found that rat ferritin also yields three fractions in chromatography on DEAE-cellulose. Each contains an 18 S protein. In contrast to our findings with horse ferritin, in subsequent gel electrophoresis small but definite differences in mobilities have been noted. It may be that chromatography causes conformational changes of sufficient magnitude to be reflected in the electrophoretic mobility of DEAE fractions.

18. 19. 20.

21. 22. 23.

Mazun, A., .IND SHORH, E., J. Biol. Chem. 182. 607 (1950). KOTHEN, A., J. Biol. Chem. 162, 679 (1944). MAZUR, A., LITT, I., AND SHORR, E., J. Biol. Chem. 187, 473 (1950). THERON, J. J., HAIVTREY, A. O., .~NU SCHIRREN, V., Clin. Chim. Acta 8, 165 (1963). KOPP, Ii., VOGT, A., ANDM.lass, G., Nature 198, 892 (1963). FINE, J. M., AND HARRIS, H., clilz. chim. Acta 8, 794 (1963). RICHTER, G. W., Lab. Invest. 12, 1026 (1963). RICHTER, G. W., Brit. J. Exptl. Pathol. 46, 88 0964). S~DDI, It., Rev. Franc. Etudes Clin. Biol. 7. 408 (1962). CARNEVALI, F., A4~~ TECCE, G., Arch. Biochem. Biophys. 106, 207 (1964). KOPP, R., VOGT, A., SND MA~SS, G., Nature 202, 1211 (1964). GRANICK, S., J. Biol. Chem. 146, 451 (1942). GRANICK, S., .~ND MICHAELIS, L., J. Biol. Chem. 147, 91 (1943). SMITHIES, O., Biochem. J. 61, 629 (1955); Biochem. J. 71, 585 (1959). POULIK, M. O., Nature 180, 1477 (1957). WAKE, R. G., AND B~LDJVIN, R. L., Biochim. Biophys. Acta 47, 225 (1961). SCHXHM.~N, H. K., Meth. Enzymol. 4, 32 (1957). Katz, A. M., DREYER, W. J., AND ANFINSEN, C. B., J. Biol. Chem. 234, 2897 (1959). MOORE, S., AND STEIN, W. H., Meth. Enzymol. 6, 819 (1963). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R., J. Biol. Chem. 193, 265 (1951). HARRISON, P. M., HOFMANN, T., AND MAINWARING, W. I. P., J. Mol. Biol. 4, 251 (1962). HARRISON, P. M., AND HOFM.4NN, T., J. Mol. Biol. 4, 239 (1962). SADDI, R., SHAPIR.Z, G., AND DREYFUS, J. C., Bull. Sot. Chim. Biol. 43, 409 (1961).