FERRITIN*

I. PHYSICAL AND CHEMICAL PROPERTIES OF HORSE SPLEEN FERRITIN

BY S. GRANICK

(From the Laboratories of The Rockefeller Institute for fife&al Research, New York)

(Received for publication, September 21, 1942)

In 1937 Laufberger (1) isolated a crystallizable protein from horse spleen which contained over 20 per cent by dry weight of iron. The protein crys- tallized out readily as a cadmium salt and was stable between pH 4 and 10. Kuhn, Siirensen, and Birkofcr (2) confirmed these findings, and from their experiments concluded that ferritin consisted of 54.5 per cent protein, 12.1 per cent nucleic acid, and 35 per cent Fe+++OOH. The prot,ein nature of the compound was corroborated by the analyses of amino acids after hydrolysis. Since there is an iron atom for almost every peptide group, they postulated that each iron atom was attached to each CONH group. (We may remark here that an alternative hypothesis will br suggested pres- ently.) A further study of ferritin, with regard to its chemical, physical, crystalline, and magnetic properties will be the subject of a series of papers, of which this first one is concerned mainly with the preparation of ferritin and some of its physicochemical properties.

The spleen of a normal horse is of a dark brown color, this being due in part to its large content of brown-black hemosiderin granules and in part to the brown color of ferritin. Temporarily one may classify the non- hematin iron of the spleen into three fractions: (a) the iron contained in the hemosiderin granules, (b) the iron in the soluble ferritin whichiscrystal- lizable with CdS04, designated simply as “ferritin,” (c) the iron in a soluble but non-crystallizable substance (or substances) which is present in the mother liquor resulting from the crystallization of ferritin with CdS04. We shall refer preliminarily to this fraction as “non-crystallizable ferritin.” The iron is in the ferric state, and it will be shown in a later paper dealing with magnetic measurements that the iron shows the same type of atomic structure in all of these fractions.

* This is the first communication on closely related topics, all concerned with ferritin and some other ferric compounds, worked out by the collaboration of S. Granick, A. Rothen, and I,. Michaelis, of The Rockefeller Institute for Medical Research, Sew l-ark, and Charles D. Coryell of the Chemical I,aboratory, University of California, Los Angclcs.

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452 FERRITIN. I

Method

Isolation of Few&-A few modifications were made in the method of isolation of ferritin as used by Laufberger and by Kuhn et al. Only one aqueous extract was made instead of two (2), since the yield on the second extraction was found to be negligible. After the aqueous spleen extract was heated to 80” (I), in order to expedite filtration, it was found con- venient to remove the heavy precipitate by first running the hot suspension through cheese-cloth and then onto large fluted filters. (To increase the yield of filtrate, the precipitate in the cheese-cloth was pressed.) A clear red-brown solution resulted. (Since no precipitate was observed to form on the addition of 10 gm. of ammonium sulfate to 100 cc. of this red-brown solution, this step in the procedure of Kuhn et al. was omitted.) 30 gm. of ammonium sulfate were now added directly to each 100 cc. of the solution

FIG. 1. Horse spleen ferritin; X 250

and the resulting suspension was kept in the ice box at 0” overnight. The precipitate was then centrifuged down and dissolved in distilled water. To crystallize out the ferritin, 4 to 5 gm. of cadmium sulfate (CdSOI.8/3H20) were added per 100 cc. of this solution. Crystallization began within several minutes. After the solution had stood overnight, the crystals were separated off by centrifugation from a dark brown mother liquor that will no longer yield crystals. This solution will be referred to as “non-crystal- lizable ferritin.” The crystals of ferritin (Fig. 1) which are sparingly soluble in distilled water we have found to be soluble in 2 per cent am- monium sulfate, yielding a clear deep red-brown solution. Ferritin in this solution can readily be crystallized by adding 4 to 5 gm. of cadmium sulfate per 100 cc. of solution.

The crystals of ferritin are not an artifact of the process of extracting and heating, since ferritin crystals can be seen to form on the under surface

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S. GRANICK 453

of a cover-slip by treating a piece of teased horse spleen with a few drops of 10 per cent CdSO,, on a microscope slide.

A protein impurity, deep brown in color, may be removed from a dialyzed solution of crystallized ferritin by bringing it to pH 4.6 =t 0.1 with dilute acetate buffer (resulting ionic strength p = 0.1) and letting it stand over- night. In some of the preparations, this fraction may be quite voluminous, so much so, that one might be misled in believing that the precipitate represents ferritin at its isoelectric point. However, in other samples this precipitate at p1-I 4.6 may be very small. In any case, no crystalline ferritin can be obtained from this precipitate, although the supernatant fluid readily yields crystals with CdS04. Perhaps this material represents denatured protein. Preliminarily, one may classify it, together with “non-crystallizable ferritin,” into a group of “non-crystallizable ferritins.”

The recrystallized ferritin solutions, dissolved with the aid of ammonium sulfate, are dialyzed in cellophane tubes against slowly running distilled water until free from NHd+ and SO,=.

Analytical Methods-The analytical methods used were checked on known mixtures of Fe, N, P, and Cd in the amounts to be expected in the unknowns, in the presence of added sucrose and edestin with appropriate blanks. For Fe and Cd determinations on the same sample, 25 to 50 mg. of ferritin were ashcd wet with 1 cc. of concentrated HzS04 + 0.2 to 0.5 cc. of -concentrated HNOB in 100 cc. Kjeldahl flasks. The heating was continued gently at the stage of SOS fumes for at least 30 minutes after clearing in order to oxidize the last traces of organic compounds. After cooling, 10 cc. of water were added and the solution was boiled for 10 minutes. To separate Fe from Cd, the solution was made 0.5 N with respect, to H&SO4 by neutralizing with strong NaOH solution (iron-free), adding the requisite amount of acid, and passing in H$ (3). The precipi- tate of CdS was filtered through a modified Wintersteiner micro filter ap- paratus onto an asbestos mat contained in a 2 cc. capacity Gooch crucible. The filtrat,e was boiled to remove the H2S and titrated with 0.04 N KMnOa. The error in the determination of 4 to 10 mg. of Fe was ~0.5 per cent. The CdS on the mat was brought into solution with a few cc. of hot 4 N

HCI and filtered through another asbestos mat. For amounts of Cd between 0.5 and 2.0 mg. in this filtrate, isolation as

the 8-hydroxyquinoline complex was used. This reagent had been found satisfactory by l3erg (4) for the macrodetermination of Cd. The procedure for the semimicrodetermination of Cd is described here in detail, since the conditions necessary for this determination are not evident from the litera- ture (4). The hydrochloric acid solution containing CdClz was brought to pH 4 to 5 (methyl red indicator) and diluted to a volume of 20 cc. 1 cc. of 8-hydroxyquinoline sulfate containing 10 mg. of the reagent was added;

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454 FERRITIN. I

then 4 M potassium acetate was added dropwise until a faint turbidity ap- peared, when 1 cc. more was added. The pH of this solution was 6 to 7. The solution was heated 10 minutes in a boiling water bath and the Cd complex was permitted to crystallize overnight and then filtered on a tared micro filter. It was dried at between 120-130” and weighed on the micro balance. The compound is Cd(C9H60H)z (4). The error for 0.5 mg. of Cd sample was f2 per cent; for 1 to 2 mg. of Cd it was fl per cent.

For amounts of Cd below 0.5 mg., a titrimetric dithizone procedure was used. This titrimetric method was suggested by Fischer (5) but the conditions for the quantitative determination of Cd by this means have not hitherto been described. The conditions to be described here were primarily worked out for the determination of Cd and iron in the same sample of ferritin. To separate small amounts of Cd from Fe, the CdS was left standing overnight before being filtered in order to form a coarser pre- cipitate. The reagents were prepared for titration of solutions containing about 0.02 mg. of Cd per cc., although the procedure may be modified to determine as little as a few micrograms of Cd per cc. The dithizone solu- tion was made up to contain 3 mg. of dithizone in 100 cc. of CCL. The reaction of Cd with dithizone was found to be quantitative between pH 6 and 7.5. In order to attain this region of pll readily, a solution of 4 M potassium acetate freed from metals was needed. This was prepared by shaking the 4 M potassium acetate in a separatory funnel for 5 minutes with 20 cc. of Ccl, containing 10 mg. of dithizone. The aqueous layer was then filtered through a moistened filter paper. A stop-cock grease insoluble in CC& was also prepared (6). The standardization of the dithizone solution is given as a typical example of the titrimetric dithizone procedure. To a 25 cc. separatory funnel were added 1 cc. of CC14, 1 cc. of a standard Cd solution containing 0.02 mg. of Cd in 0.01 N HCl, 2 cc. of HzO, and 0.5 cc. of 4 M potassium acetate. The dithizone solution was now added from the burette in 0.5 cc. portions, and shaken for 15 seconds after each addition. The Cd reacted with the green CCL solution of dithizone to form anorange- pink Cd complex which was soluble in CCL but insoluble in water. When so much dithizone had been added that the CC14 layer no longer turned orange-pink but remained green, the end-point had been exceeded. A slight orange scum formed in the funnel during the titration and this was removed as completely as possible by dissolving it in 3 to 5 cc. of CC14, shaking, and drawing off the CC14 layer. With a little practice the titration end-point on two succeeding titrations could bc determined within 0.05 cc. Since the amount of dithizone added was in the neighborhood of 3 cc., the error in the titration end-point was around 1.7 per cent.

Phosphorus was determined by ashing with sulfuric and nitric acids and by precipitation as ammonium phosphomolybdate, the precipitate being weighed on the micro balance (3). Phosphorus was also determined by the

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S. GRANICK 455

calorimetric method of Youngburg and Youngburg (7). Nitrogen was determined by the Kjeldahl method with CuSO4 as catalyst and perhydrol to complete the digestion.

Physical Properties of Crystallized Fe&tin

Changes on Heating Ferritin-When a dialyzed ferritin solution con- taining 5 mg. of ferritin per cc. is heated slowly, at the rate of approximately 3-4” per minute, it remains clear at 80”. At 82” it becomes cloudy. When allowed to cool, the suspension becomes completely clear again in 20 min- utes. If it is heated to 90”, an insoluble residue remains even on the next day, this residue constituting less than 25 per cent of the ferritin; the clear filtrate is readily crystallizable with CdSO,. Still higher temperatures bring about a coagulation no longer reversible on cooling.

When dialyzed ferritin in a higher concentration (20 mg. per cc.) is heated, it becomes cloudy at 66” and when cool it clears completely within 5 minutes. Heating to higher temperatures delays the clearing. For example, if kept at 80-82” for 5 minutes, the ferritin becomes granular and requires 30 minutes to become clear, leaving only a trace of insoluble residue. If the hot solution containing the granular material is treated with an equal volume of 10 per cent CdS04, small irregular crystals are formed immediately. As the temperature decreases, well formed crystals begin to grow and the poorly formed ones disappear.

According to Chick and Martin (8), two phenomena are involved in heat coagulation of a protein. In modern terminology we may describe the one as a “denaturation,” i.e. a chemical or structural change of the protein molecule itself, the second being an aggregation or “agglutination” of the individual protein molecules into particles larger than the wave-length of visible light. With many proteins, under certain conditions, especially of pH, denaturation by heat may occur before or without aggregation by heat. In the case of ferritin at higher concentrations (20 mg. per cc.), heat in the range between 60-80” causes the molecules to aggregate, this ag- gregation being reversible on cooling. Above 80”, changes in the indi- vidual molecules occur which are not reversible; such changed molecules agglutinate to insoluble granules. The special peculiarity of ferritin is that at sufficiently high concentrations the normal unchanged molecules can agglutinate before they denature. Others may interpret the factsby assuming two kinds of “denaturation,” a reversible one occurring at 80” and an irreversible one occurring at higher temperatures. This depends on the definition of denaturation. It should be emphasized that the exact figure, 80”, depends on the conditions of these experiments; the temperature of denaturation, of course, varies with the concentration of protein and the rate of heating.

Crystallization of Ferritin-Crystals of ferritin form most readily when

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456 FERRITIN. I

the cadmium sulfate solution is added to a dialyzed ferritin solution. The rate of crystallization is high; for example, within 30 seconds after an equal volume of 10 per cent CdSO, solution is added to a dialyzed aqueous ferritin solution (containing 2 per cent ferritin) one can observe well formed, growing crystals under the microscope. These crystals arc optically iso- tropic, usually with slightly curved edges. The smaller ones are well formed octahedra; the large ones are twinned octahedra formed on the plan of a cube, on each face of which is set a tetrahedral pyramid. They are very soft, being easily crushed under a cover-slip; yet when crushed, they splinter into distinct fragments rather than smear out into a gel.

Crystals of the same shape may be obtained with ZnS04 instead of CdSO, but crystallization is more difficult. Laufberger reports obtaining crystals of ferritin even with cobalt and nickel sulfates. The crystalliza- tion of ferritin by means of Zn, Cd, Ni, or Co salts reminds one of the peculiarity of insulin which needs traces of any of these elements to form crystals. Ferritin, in contrast to insulin, requires over 3 per cent CdS04 or ZnSOl for crystallization.

When lower concentrations of CdS04 are used for crystallization, several interesting phenomena are observed. Addition of CdS04 to a ferritin solu- tion so that the final solution contains 0.5 per cent CdS04 (Experiment 1, Table I) results in the production of a precipitate which appears to be amorphous even under the oil immersion lens, and which remains so for weeks; results with a 0.7 per cent CdS04 solution are similar (Experiment 2, Table I). At 1 per cent CdS04, flat plates are gradually formed. At 3 per cent CdS04, crystals of the typical octahedra and twinned crystals are formed rapidly and no amorphous precipitate is visible, the supernatant solution being colorless.

The amorphous precipitate formed at the lower concentrations of CdSOd is not an impurity, for if 5 per cent CdS04 is added directly to this amor- phous precipitate (freed from its mother liquor) one may observe under the microscope that it dissolves and octahedral and twinned crystals arise rapidly.’

In Experiment 5 octahedral crystals of ferritin formed in 5 per cent CdSO, were rapidly washed with 0.6 per cent CdS04 and suspended in this solution. After several days, the original crystals had disappeared, giving way to small square plates which were optically anisotropic. This is the only case in which we have observed anisotropy in ferritin crystals. When these plates were brought into solution and 5 per cent CdS04 was added, iso- tropic ferritin crystals were formed.

In order to find out whether these various amorphous precipitates and crystal forms represented substances of different cadmium content, the

1 Dr. K. G. Stern had previously obscrvcd this phcnomcnon and mentioned it to us.

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S. GRANICK 457

iron-cadmium ratios in the preparations were determined. Assuming an iron content in ferritin of 22 per cent, one can calculate from the Fe: Cd ratio the approximate percentage of Cd in the crystals. To remove the adhering mother liquor containing CdS04 in the preparation of ferritin samples for analysis, the samples (Experiments 1 to 5) were centrifuged down, spread on porous porcelain to dry overnight, and then dried at 110”. The analyses (Table I) indicate that the amorphous precipitates (Experi- ment 1) contain not less than half the cadmium that the crystals contain (Experiment 4).

TABLE I

Cd&O, Concentrations and Character of Fe&tin Precipitates

E.K- peri- ment No.

1

2

3

4

5

6

Composition

5 cc. Preparation VII (3 time crystallized fcrritin), 0.5 cc 0.4 M CdSO,, diluted to 10 cc. (0.50/, CdS04)

5 cc. Preparation VII, 0.7 cc. 0.4 M CdSOa, diluted to 10 cc. (0.7% CdSO1)

.5 cc. Preparation VII, 1.0 cc. 0.4 M CdSOa, diluted to 10 cc. (lo/, CdSOd)

5 cc. Preparation VII, 3.0 cc. 0.4 M CdS04, diluted to 10 cc. (3% CdSO,)

Octahedra of Preparation VI (6 times crystallized), sus- pended in 0.6% CdS04

Octahedra of Preparation VII washed with saturated KC1

Character of ppts.

Amorphous ppt., optically 12.3:1 3.5 isotropic

Amorphous ppt., somewhat lO.O:l 4.4 greater than abovc, opt,i- tally isotropic

Mostly flat, large plates or 5.5:1 5.2 twinned, opt,ically isotropic slight amorphous ppt. (an- alyscs made on plates)

Star-shaped twinned crystals 6.6:1 6.7 and octahedra optically iso- tropic; no amorphous ppt.

15 X 15 p, small square plates 9.2:1 4.8 optically anisotropic

Crystals unchanged; opti- 26:l 1.7 cally isotropic

Ratio, Fe: Cd atoms

In Experiment G, the adhering mother liquor was removed by rapidly washing ferritin crystals (originally precipitated with 5 per cent CdS04) with saturated KC1 in which these crystals were only slightly soluble. The Cd content decreased from 6.7 to 1.7 per cent. Since protein crystals ap- pear to contain relatively large pores or capillary spaces (9), it appears that the CdSO., present in aqueous solution in the pores of the crystal was swept out by the KC1 solution without visibly affecting the crystalline form.

The CdS04 may be considered to serve two functions: the first, to coordi- nate the molecules of ferritin into a definite lattice pattern; the second, to

er cent Cd in rystals

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458 FERRITIN. I

decrease the solubility of ferritin, thus favoring crystallization. This latter function can be taken over by saturated KCl. Crystallization of ferritin could not be obtained, however, by using as the crystallizing solution 1 per cent CdSOI in the presence of saturated KCI.

Homogeneity of Ferritin--In order to determine whether ferritin repre- sented a pure protein, it was studied by the ultracentrifuge, electrophoretic, and solubility methods. The results of ultracentrifugatior? indicated ferritin to be an inhomogeneous material consisting of large brown particles of molecular weight in the neighborhood of several million, grading down finally to a colorless fraction which appeared to make up 30 per cent of the protein. If these small colorless particles were considered to be spherical in shape, their molecular weight would be around half a million. Electro- phoretic studies in the Tiselius apparatus,2 however, indicated ferritin to be

TABLE II

Solubility Studies on Ferritin

Tube No.

Ferritin in 0.4 per cent CdS04.8/3%0

Ferritin -

Suspension 1 Filtrate

ntg. per cc. mg. +er cc.

0.260 0.256 1.44 0.990 2.10 1.34 3.92 1.85 4.39 1.88 5.77 1.97 8.19 2.21

Ferritin in 0.6 per cent CdS04,8/3HzO

Ferritin

Suspension Filtrate

11zg. fier cc. mg. per cc.

0.678 0.591 1.18 1.05 2.79 2.06 5.32 2.45 6.50 2.50

10.2 2.64 13.0 2.73 17.9 3.02

a completely homogeneous substance with an isoelectric point below pH 5.4. The solubility method of Kunitz and Northrop (10) indicated ferritin to be inhomogeneous. This latter method was tested at two different con- centrations of CdS04. Twice recrystallized ferritin was precipitated with 5 per cent CdSO,; the crystals were centrifuged down and washed twice by centrifugation with 0.4 per cent CdS04.8/3HzO. Increasing amounts of these washed crystals were then suspended in a series of tubes together with the same 0.4 per cent CdSOd solution. After 24 hours of shaking at room temperature, which preliminary tests indicated as sufficient for the es- tablishment of equilibrium, the tubes were centrifuged and aliquots of the

2 Ultracentrifuge and electrophoretic studies on ferritin have been made by Dr. A. Rothen, who will report his data in another paper of this series.

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S. GRANICK 459

brown supernatant solution were used for calorimetry in a Pulfrich photom- eter to determine the concentration of ferritin in solution. The results are given in Table II. In another experiment 0.6 per cent CdS04*8/3HzO was used as solvent with a ferritin preparation that had been recrystallized six times; the results are also reported in Table II.

Some Chemical Properties of Ferritin

Iron Content-Further evidence for the inhomogeneity of ferritin is the variability of the iron, phosphorous, and nitrogen content of different crystallized ferritin samples. Iron analyses of these ferritin samples after

TABLE III

Analyses of Non-Hemin Iron Components of Horse Spleen in Per Cent of Dry Weight*

Preparation Corrected

for Cd for Cd Uncorrected for Cd

7 ficr cent #tv cent per ce?d +e* cent per cent

I. Ferritin crystallized twice. 22.2 22.7 2.88 1.60 10.55 II. “ IL 3 times;

removal of Cd by ammoni- um sulfate and dialysis.. 22.7 22.3 0.724 1.26

IX. Ferritin crystallized 4 times. 19.7 20.2 2.74 1.42 11.1 x. ‘I “ once;

from a horse severely bled several mos.. 22.6 23.1 1.93 1.99 12.6

IV. Fraction pptd. with NazSOa (not crystallized). 24.5 1.45 11.0

VIII. Hemosiderin granules 8.29 1.59 12.9 VII. “Noncrystallizableferritin”

from mother liquor. 19.8 20.2 2.02 1.52 9.12

* All preparations were dried in thin layers at 80” for 24 hours and then for 3 hours at 110”.

dialysis vary from 20.2 to 23.1 per cent if the Cd adhering to the ferritin is considered as an impurity and is corrected for.

It was interesting to see what the iron content of a preparation would be if it were not crystallizedwith CdSO+ but merely fractionated with Na2S04. Preparation IV (Table III) was therefore made in the following manner. The filtrate of the horse spleen extract after being heated to 80” was pre- cipitated with ammonium sulfate in the usual manner. This precipitate was then dissolved in water to give a deep brown, clear solution which was fractionated into three parts by Na2S04 of increasing concentration up to saturation. There is no particularly sharp separation with this procedure, since the precipitates form in a concentration of NazSOl near saturation.

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460 FERRITIN. I

The intermediate fraction was arbitrarily chosen and was dialyzed free from salts. This Preparation IV had an iron content of 24.5 per cent.

The brown mother liquor from a CdSO, precipitation (designated as “non-crystallizable ferritin”) was precipitated with ammonium sulfate, dialyzed, and centrifuged. The supernatant liquid soluble in the absence of ammonium sulfate yields a preparation, No. VII, having 19.8 per cent iron and somewhat less nitrogen than the crystallized ferritin preparation.

Hemosiderin granules isolated by differential centrifugation in a partial state of purity had an iron content of 8.29 per cent, a nitrogen content of 12.9 per cent, and a phosphorus content of 1.6 per cent. It is not possible to say whether this phosphorus is a component of the granules or not.

Phosphorus Content-The phosphorus content of crystallized ferritin preparations was also variable, ranging from 1.26 to 2.00 per cent. Kuhn and his coworkers have postulated this phosphorus to be a constituent of a desoxyribonucleic acid. We have been unable to confirm the presence of any nucleic acid in any of our crystallized samples of ferritin. When ferritin was made 1 N with NaOH, within a few minutes at room tempera- ture a dark brown, flocculent precipitate formed. This precipitate, Frac- tion A, contained all of the iron, 5 per cent of the nitrogen, and 23 per cent of the phosphorus. The supernatant liquid, Fraction B, was colorless and contained 77 per cent of the phosphorus in the form of inorganic ortho- phosphate. Fraction B also contained 85 per cent of the total N in the form of a substance precipitated at pH 4.6 and identified as a protein by the biuret, ninhydrin, and Millon tests. Both Fractions A and B were tested for pentoses with Bial’s reagent, for desoxypentose by the Kiliani method, and with the Dische diphenylamine reagent;all tests were negative. To detect purines, absorption spectra in the ultraviolet region were taken by Dr. G. Lavin on both fractions, and again with negative results. It is not yet possible to say whether the phosphate is in some very labile organic combination or whether the phosphate, perhaps as a basic ferric phosphate, is a part of a colloidal micelle of ferric hydroxide. Consequently, if some preparations of ferritin, such as Kuhn’s, should contain nucleic acid, this should be considered as a non-essential admixture.

Cadmium Content-It has not been found possible, by dialysis against distilled water, to remove all the cadmium from a solution prepared from ferritin-cadmium crystals. Prolonged dialysis until the dialysate is free from sulfate ions gives a preparation containing from 2 to 3 per cent cad- mium (Preparations I, IX, X, Table III). We have been able to decrease the cadmium content to 0.72 per cent by washing the ferritin-cadmium with ammonium sulfate (Preparation II) in the following manner. A thrice crystallized ferritin solution was precipitated with 30 per cent ammonium sulfate, the flocculent precipitate was washed with a fresh solution of am-

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S. GRANICK 461

monium sulfate, the ferritin was redissolved, and the precipitation and washing repeated. Finally the precipitate was dissolved and dialyzed until free from ammonium ions and its cadmium content determined. Ferritin seems to hold cadmium as tenaciously as, according to Schorn (II), egg albumin treated with zinc ions holds the zinc, of which 0.63 per cent still remains even on electrodialysis.

SUMMARY

Horse spleen ferritin, a protein containing over 20 per cent iron, and crystallizable as a cadmium salt is not an artifact of the methods of isola- tion and heat’ing, since crystals can be obtained directly by treating a piece of teased horse spleen with CdSOI. Ferritin is homogeneous according to the Tiselius electrophoretic method. It is, however, inhomogeneous in the ultracentrifuge, being made up of brown particles having a molecular weight in the neighborhood of several million and grading down to colorless parti- cles of the size of large globulin molecules. Solubility studies also give evidence for the inhomogeneity of crystallized fcrritin. Inhomogeneity is further indicated by the variation in the iron and phosphorus content of different ferritin samples. No evidence was obtained for the presence of nucleic acid in fcrritin. The effect of heat on ferritin is peculiar, since a 3 per cent solution of ferritin when heated to 60” gives a coagulum which redissolves on cooling; when heated to higher temperatures, a coagulum is produced having the appearance of an irreversibly denatured protein. Peculiarities of crystallization and the cadmium content of the crystals are discussed. Methods for cadmium analyses by means of 8-hydroxyquino- line and by a titrimetric dithizone procedure are described.

BIBLIOGRAPHY

1. Lnufbergcr, &I., Bull. Sot. c&m. biol., 19, 1575 (1937). 2. Kuhn, R., Sorensen, N. A., and Birkofer, L., Ber. them. GM., ‘73 B, 823 (1940). 3. Treadwell, I?. P., and Hall, W. T., Analytical chemistry, Quantitative analysis,

New York and London, 7th edition (1930). 4. Berg, R., 8. anal. Chem., 71, 321 (1927). 5. Fischer, H., Bngew. Chem., 46,442 (1933); 47, 685 (1934); 50, 919 (1937). 6. Meloche, C. C., and Frederick, W. G., J. ilm. Chem. Sot., 64, 3264 (1932). 7. Youngburg, G., and Youngburg, N., J. Lab. and Clin. Med., 16, 158 (1930). 8. Chick, H., and Martin, C., J. Physiol., 40, 404 (1910); 43, 1 (1911). 9. Granick, S., J. Gen. Physiol., 26, 571 (1942).

10. Kunitz, RI., and Northrop, J. I-I., Colnpt.-rend. tmu. Lab. Curlsberg, 22,288 (1938). 11. Schorn, H., Biochem. Z., 199, 459 (1928).

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S. GranickSPLEEN FERRITIN

CHEMICAL PROPERTIES OF HORSE FERRITIN: I. PHYSICAL AND

1942, 146:451-461.J. Biol. Chem. 

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