Rapid isolation of ferritin by means of ultracentrifugation

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S H O R T COI~IMUNICATIONS

BBA 33118

Rapid isolation of ferritin by means of ultracentrifugation For studies of iron metabolism in rabbit 1, we need a rapid isolation technique of ferritin resulting in a pure product with a high yield. The methods available are based on heat treatment of a liver homogenate at 8o ° during IO min (ref. 2) followed by concentration with ammonium sulfate and final purification by means of cadmium sulfate crystallisation or column chromatography 3-'~. However, there appeared some difficulties, the isolation of ferritin by means of cadmium sulfate crystallisation resulted in a low yield, while the splitting up into slightly different ferritin fractions seemed to be the disadvantage of the column chromatographic procedure 3. We tried to attain our purpose by means of two alternative ways: heat treatment followed by gel filtration or heat treatment followed by ultracentrifugation. The ultracentrifugation technique is applied to the separation of the ferritin polymers 6 or in the separation of ferritin molecules with a different iron content v. The gel filtration was applied to a partly purified ferritin fraction, obtained from liver after heat treatment 2 and a concentration procedure of the filtrate by means of ammonium sulfate precipitation. The precipitate obtained was dissolved in water and dialyzed against o.oI M ammonium acetate, pH 7.4, and applied to a coluinn (9o cm × 3.5 cm) filled with a gel suspension equilibrated with the same buffer. Three different types of Sephadex (Pharmacia, Sweden) were used in these experiments,

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589

SHORT COMMUNICATIONS

- FERRITIN_MONOMER

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Fig. 2. P o l y a c r y l a m i d e gel electrophoresis of the heat-treated liver homogenate and the ferritin fractions resulted from the purification procedures. The polyacrylamide gels were 6 cm long, contained 5% p o l y a c r y l a m i d e and were equilibrated with buffer of p H 9.5. The gels were stained with Amidoblack for protein {hatched bands) and with o.2 % potassium ferrocyanide (solid bands) for iron 4. The arrow indicates the place of application• The second slower m o v i n g band {solid) is a ferritin dimer. Current, 5 m A / t u b e ; time of electrophoresis, 45 min. A. Heat-treated liver homogenate. B. Purified ferritin Fraction AI, after gel filtration over Sephadex G-IsO. ('. Purified ferritin Fraction B1, after gel filtration over Sephadex G-2oo. D. Purified ferritin Fraction CI, after gel filtration over Sepharose 4-B, the contamination represents less then I ° ' o of the total protein. E. The purified ferritin fraction obtained from the ultracentrifugation technique {precipitate). For B, C and D see Fig. I. Ihtbi~[t I : ~ c t IIHn,~gu]ultL'

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Scheme 1. The purification procedure for rabbit ferritin with the use of ultracentrifugation. N o ferritin was detected in the supernatant arising from centrifugation at 78 ooo × g. The polyacrylamide gel electropheretogram of the final product is given in Fig. 2E.

Sephadex G-I5O, Sephadex G-2oo and Sepharose 4-B. The fractions eluted were analysed for protein content, by determining the absorbance at 280 nm, and for iron by means of the method of RAMSAY8. The elution patterns of these separations are shown in Fig. I and the fractions collected are indicated. These analyses of the ferritin-rich Fractions AI, BI and CI by means of polyacrylamide gel electrophoresis are given in Fig. 2. Satisfactory results by means of gel Biochim. Biophys..4eta, 168 (1968) 588 59 °

5()0

SHORT COMMUNICATIONS

filtration were o b t a i n e d with Sepharose 4-B (Fraction CI). However, the technique is u n s u i t a b l e for rapid analyses of a n u m b e r of livers. B y using two characteristics of ferritin, stability to h e a t i n g at 8o ° a n d the relative high s e d i m e n t a t i o n rate (see Scheme I), we succeeded in preparing ferritin in a rapid way. After heat t r e a t m e n t a n d filtration, the filtrate was subjected to u l t r a c e n t r i f u g a t i o n in a Spinco centrifuge model L, rotor 3o. After a z-h r u n at 78 ooo x g, a b r o w n ferritin-rich precipitate was obtained. This precipitate was dissolved in water a n d the solution was centrifuged at 7ooo x g for I h. A faint precipitate was discarded. Thereafter, ferritin was again precipitated b y centrifugation in a rotor 4 ° at 95 ooo x g for I h. This centrifugation procedure at 95 ooo × g was repeated after the precipitate was dissolved in water. Since we were not able to detect a n y ferritin in the s u p e r n a t a n t s collected, the yield of this isolation seemed to be high. I n order to check the yield of this procedure a n d e v e n t u a l l y losses of ferritin, we added ferritin labelled with 7aSe in the protein part to the filtrate after heat t r e a t m e n t . In the precipitate from centrifugation at 7ooo )< g only traces of r a d i o a c t i v i t y were present. The yield of the centrifugation procedure was a b o u t o °'/'o. As judged from immunoelectrophoreses with a n t i - n o r m a l h m n a n serum a n d polyaerylamide gel electrophoreses the ferritin preparation did not c o n t a i n sermn proteins. The time c o n s m n i n g step is the first centrifugation at 7 8 ooo X g; in one run, the filtrates of two h e a t - t r e a t e d liver homogenates could be centrifuged. The next centrifugation steps can be carried out with six liver homogenates. We wish to t h a n k Dr. H. G. VAN EIJK for helpful discussions.

Rotterdam Medical Faculty, Department of Chemical Pathology, University Hospital Dijkzigt, Rotterdam (The Netherlands)

T. J. PENDERS H. H. DE R o o I j - D U K B. LEIJNSE

I [I. (J. \rAN JGYKAND B. LEIJNSE, Biochim. Biophys. Acta, 16o (~908) 120. 2 S. GRAN1CK,Chem. Rev., 38 (1946) 379. 3 J\. SURAN, Steadies on Ferritin and Apoferritin, Thesis, University of California, U.S.A., 19t,~u 4 J. \ v , DRYSDALE AND I-t. N. MUNRO, Biochem., J. 95 (I965) 85I. .5 N. ~'[URAOKA,S. NAKAJIMA,T. I~ATOAND T. SHIMADA,J. Biochem. Tokyo, 60 (1960) 489 . (, A. O. A. MILLER, Biochim. Biophys. Acta, 155 (I968) 202. 7 J. W. I)RYSDALE AND H. N. MUNRO, dr. Biol. Chem., 24~ (1966) 303 o, ,g W. N. M. RAMSAY,CIin. Chim. Acla, 2 (I957) 214. Received J u l y i 7 t h , i968

Biochim. Biophys. Ac&, r68 (~968) 588 590