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Microheterogeneity of horse spleen ferritin and apoferritin
BIOCHIMICAET BIOPHYSICAACTA
I87
BBA 35909 M I C R O H E T E R O G E N E I T Y OF H O R S E S P L E E N F E R R I T I N AND APOFERRITIN
ICHIRO URUSHIZAKI, YOSHIRO NIITSU, KUNIHIKO ISHITANI, MIKIHITO MATSUDA AND MORIMICHI FUKUDA Department of Medicine, Cancer Research Institute, Sapporo bIedical College, Sapporo, Hokkaido (Japan)
(Received March I s t h
, I97I )
SUMMARY In the present experiments we attemped to fractionate horse spleen ferritin and apoferritin on an electrofocusing column and obtained eight or nine heterogenous peaks in a pH range of 4.22 to 4.6, which were grouped into three main components. There was no significant difference between the fraction patterns of horse spleen ferritin, monomeric ferritin and apoferritin. The heterogeneity demonstrated in the present paper represents polymorphic forms of ferritin rather than polymeric forms as hitherto described.
INTRODUCTION Gel electrophoresis of ferritin and apoferritin have revealed the existence of at least three fractions of different mobilitiesl, 2. It has been suggested that this might be due to the presence of polymeric forms of ferritin. SURAN AND TARVER3 also demonstrated that horse spleen ferritin could be fractionated by DEAE-cellulose column chromatography. This heterogeneity was estimated to be due to differences in conformation and shape rather than to differences in the iron content or in the charge of ferritin molecules. In I966 VESTERBERG AND SVENSSON4 proposed a new method of protein fractionation based on the differences in the isoelectric points of proteins. The method is characterized by the production of a natural pH gradient by passing an electric current through a solution of the carrier ampholytes with a suitable pH range on a sucrose density gradient; when a protein sample is applied to the column it moves with the carrier ampholytes to the site corresponding to the individual isoelectric points during passage of the electric current. We have preliminarily reported microheterogeneity in ferritin molecules of rat liver 1°. The present paper describes the microheterogeneity of horse spleen ferritin and apoferritin demonstrated by the above isoelectric fractionation method. Biochim. Biophys. Acta, 243 (1971) 187-192
I88
t. URUSHIZAKI 8l ell.
MATERIAL AND METHODS
Materials We used commercial ferritin from horse spleen (6 times crystallized, Pentex, Inc.). Apoferritin was prepared by a method based on that of GRANICK AND MICHAELIS 5.
Monomeric ferritin was prepared by Sepharose 6-B column chromatography of I % horse spleen ferritin, using o.i M Tris buffer, pH 7.o, containing I M Nat1.
Methods Electrofocusing column fractionation by carrier ampholytes with a narrow pH range was employed. Isoelectrie fractionation of ferritin samples was carried out, using an LKB model 81Ol column of I2o-ml capacity. The temperature was kept constant at 4 ° throughout the experiment by means of an electrothermostat connected to the column. A carrier ampholyte of 4oO/o, pH 4-6, was chosen for this experiment because horse spleen ferritin has its isoelectric point near pH 4.4. Under these conditions, ferritin was distributed over a very narrow range in the column and the elution pattern varied because of minor effects of the experimental conditions. To overcome this disadvantage, narrow pH range carrier empholytes were obtained by isoelectric fractionation of the pH 4-6 carrier ampholytes. For this purpose, pH 4-6 carrier ampholytes with a final concentration of 7% were subjected to 1Ioo V for 48 h on a sucrose density gradient, and the carrier ampholytes with a pH range of 4.2 to 4.65 were obtained by measuring the pH of the eluted fractions. The electrofocusing column fractionations of the ferritin samples were performed using this narrow pH range carrier ampholyte. One quarter of the volume of a solution of this ampholyte was added to a 50% sucrose solution to give a final volume of 60 ml. A further onequarter volume was made up to 60 ml with distilled water. The above two solutions and I ml of a I ~/O ferritin sample were applied to the column using a proportioning pump to make a continuous density gradient by the method of AYOD et el. a. Isoelectric fractionation was performed for 72 h at IlOO V with the cathode at the top of the column and the anode at the bottom. The samples were then eluted from the bottom of the column using a proportioning pump at a constant rate of I ml/min ; 0.5 ml of each fraction was obtained. The pH of each fraction was measured by a pH-recording electrometer at 20 ° and the A 280 nm of the fractions was determined by a UV 200 spectrophotometer. Disc electrophoresis of the fractions separated was also carried out. The fractions were concentrated using Diaflo membranes, type XM-5o, and purified by gel filtration in o.oi M phosphate buffer, pH 8.o, on a Sephadex G-2oo column. Electrophoresis on 5% polyacrylamide gels was performed for 3 h at 15o V using Tris-glycine buffer, pH 8. 3. RESULTS
Electric focusing column fractionation of horse spleen ferritin As shown in Fig. I, the horse spleen ferritin essentially gave a heterogenous pattern with at least eight or nine isoelectric peaks between pH 4.22 and 4.6. The Biochim. Biophys. ,qcta, 243 (1971 ) I 8 7 - I 9 2
I8()
MICROHETEROGENEITY OF FERRITIN
® 04
42
40
J 20
30
40 50 60 FRACTION NUMBER
70
B0
Fig. I. Electric f o c u s s i n g c o l u m n f r a c t i o n a t i o n of horsespleen ferritin. Fig. 2. P o l y a c r y l a m i d e disc electrophoresis of t h e s e p a r a t e d fractions of horse spleen ferritin on isoelectric focusing. F r o m t h e t o p down, F r a c t i o n s 39, 48, 51, 56, 59, 69 a n d 71, s t a i n e d for iron.
pH values of these peaks were 4.27, 4.29, 4-35, 4.38, 4.40, 4.43, 4.47, 4.50 and 4.57, respectively. Moreover, the heterogenous peaks were generally grouped into three main components.
Polyacrylamide disc electrophoresis of fractions of horse spleen ferritin separated by isoelectric focusing Polyacrylamide disc electrophoresis was performed on the separated fractions of horse spleen ferritin by isoeleetric focusing. As shown in Fig. 2, each fraction gave two different bands.
Electric focusing column fractionation of monomeric ferritin of horse spleen The separation of monomeric ferritin from horse spleen ferritin was performed by Sepharose 6-B chromatography. Fig. 3 shows the monomeric dimeric and oligomeric forms of ferritin. As shown in Fig. 4, monomeric ferritin from horse spleen gave essentially the same pattern as horse spleen ferritin, with at least eight or nine isoelectric peaks between pH 4.22 and 4.6. The pH values of the peaks were 4.27, 4.29, 4.34, 4-37, 4.39, 4.42, 4.46, 4.51 and 4.58 (4- o.o2), respectively.
Polyacrylamidc disc electrophoresis of fractions of monomeric ferritin separated by isoelectric focusing The fractions of monomeric ferritin obtained by isoelectrie focusing were subBiochim. Biophys. Acta, 243 (1971) I 8 7 - I 9 2
I. URUSHIZAKI el al.
I90
~C
A O8
O2
,
........ 10
=J",-.-: , ~~ 20
30
~ 40
FRACTION
50
60
...... 70
96
80
NUMBER
Fig. 3. The preparation of m o n o m e r i c ferritin from horse spleen ferritin by Sepharose 6-B column chromatography, using o.i M Tris buffer containing I M NaC], p H 7.0. The last peak is the monomeric form of ferritin.
jected to polyacrylamide disc electrophoresis. As shown in Fig. 5, each fraction gave one band.
Electric focusing column fractionation of horse spleen apoferritin As shown in Fig. 6, apoferritin from horse spleen gave a pattern similar to those of horse spleen ferritin and monomeric ferritin, having eight isoelectric peaks between pH 4.22 and 4.6. The pH values of the peaks were 4.29, 4.34, 4-39, 4.42, 4.44, 4-46, 4.5I and 4.57 (:k o.o2), respectively. pH 46
0,4
,4 03
44
02
42
40
20
30
40
50
FRACTION
60
70
80
NUMBER
Fig. 4- Electric focusing column fractionation of monomeric ferritin from horse spleen.
Biochim. Biophys. Acta, 243 (1971) 187-192
191
MICROHETEROGENEITY OF FERRITIN
....."
42
40
20
30
40 50 60 FRACTION NUMBER
70
BO
90
Fig. 5. Polyacrylamide disc electrophoresis of the fractions of monomeric ferritin separated by isoelectric focusing. From the top down, horse spleen ferritin; eluates from Fractions 3o-39; eluates from Fractions 42-48; eluates from Fractions 53 46; eluates from Fractions 68-73; and eluates from Fractions 75-8o. Fig. 6. Electric focusing column fractionation of apoferritin from horse spleen.
DISCUSSION
Until recently, it was generally accepted that all apoferritin molecules in any one animal were of the same composition. However, ALFREY et a l d found some definite electrophoretic differences between ferritins from different human organs of the same subject, and GABUZDAAND GARDNER8 also observed a similar phenomenon in rabbit. The separation of both horse spleen ferritin and apoferritin into several analogous components by gel electrophoresis was reported by KoPP et al. 9 and SURAN AND TARVERa. However, sedimentation studies indicated that the major components are related as monomer, dimer and trimer, and that their electrophoretic separation is due to differences in molecular size or shape rather than to differences in charge . St;RAN AND TARVERa reported a different and unstable order of heterogeneity of horse spleen ferritin using DEAE-cellulose chromatography, but no significant difference in amino acid composition or in tryptic peptide fingerprints could be shown. This fractionation may be due to conformational differences in ferritin molecules. We briefly reported microheterogeneity in rat liver ferritin molecules as detected by isoelectric focusing 1°. DRYSDALE11 developed a small-scale analytical method for isoelectric focusing in a polyacrylamide gel column and found a similar microheterogeneity in a crystalline sample of horse spleen ferritin. Biochim. Biophys. Acta, 243 (1971) 187-192
192
1. I.'F.USHIZAI{1 C[ et[.
111 tile p r e s e n t e x l ) e r i m e n t s , we h a v e d e m o n s t r a t e d t h a t h o r s e s p l e e n f e r r i t i n monomeric ferritin and apoferritin have the same microheterogeneity of protein m o i e t i e s o n e l e c t r o f o c u s i n g c o l u m n f r a c t i o n a t i o n . T h e y w e r e c o m p o s e d o f e i g h t or n i n e p e a k s w i t h a c o n s t a n t p H r a n g e . T h e s e f e r r i t i n p e a k s w e r e also g r o u p e d i n t o t h r e e m a j o r c o m p o n e n t s . T h e m i c r o h e t e r o g e n e i t y is n o t d u e t o d i f f e r e n c e s in i r o n c o n t e n t or a g g r e g a t e size a n d m a y r e p r e s e n t , in p a r t , s t a b l e c o n f o r m e r s or s o m e f o r m o f p o l y m o r p h i s m d u e t o t h e i n t r i n s i c a l l y d i f f e r e n t p r o t e i n shells o f a p o f e r r i t i n . F u r t h e r i n v e s t i g a t i o n is n e c e s s a r y a n d w e are n o w p e r f o r m i n g e x p e r i m e n t s c o n cerning the amino acid compositions of the heterogenous peaks of apoferritin. REFERENCES J. J. THERON, A. (). HAWTREY AND V. SCHIRREN, Clin. Chim. Acta, 8 (1963) 165. F. CARNEVALI AND G. TECCE, Arch. Biochem. Biophys., lO5 (1964) 207. A. A. SURAN AND H. TARVER, Arch. Biochem. Biophys., I I I (1965) 399. O. VESTERBERG AND H. SVENSSON, Acta Chem. Scan&, 20 (1966) 820. S. GRANICK AZ~D L. MICHAELIS, J. Biol. Chem., 147 (1943) 91. S. R. AYOD, R. W. BONSALL AND S. HUNT, Science Tools, 14 (1967) 4° . C. P. ALFREY, g. C. LYNCH AND C. F.. WHITLEY, J. Lab. Clin. Med., 7° (1967) 419. T. G. GABUZDA AND F. H. GARDNER, Blood, 29 (1967) 77 o. R. KoPP, A. VOGT AND G. MAASS, Nature, 202 (1964) 1211. I. URUSHIZAKI, M. FUKUDA, M. MATSUDA, W. NIITSU, M. YOKOTA AND M. KITAGO, .]a/~..]. Meal., 9 (~97 o) 47II J. 'v~,r. DRYSDALE, Biochim. Biophys. Acta, 207 (197o) 256.
I 2 3 4 5 6 7 S 9 IO
Biochim. Biophys. Acta, 243 (i97 I) 187-192
I87
BBA 35909 M I C R O H E T E R O G E N E I T Y OF H O R S E S P L E E N F E R R I T I N AND APOFERRITIN
ICHIRO URUSHIZAKI, YOSHIRO NIITSU, KUNIHIKO ISHITANI, MIKIHITO MATSUDA AND MORIMICHI FUKUDA Department of Medicine, Cancer Research Institute, Sapporo bIedical College, Sapporo, Hokkaido (Japan)
(Received March I s t h
, I97I )
SUMMARY In the present experiments we attemped to fractionate horse spleen ferritin and apoferritin on an electrofocusing column and obtained eight or nine heterogenous peaks in a pH range of 4.22 to 4.6, which were grouped into three main components. There was no significant difference between the fraction patterns of horse spleen ferritin, monomeric ferritin and apoferritin. The heterogeneity demonstrated in the present paper represents polymorphic forms of ferritin rather than polymeric forms as hitherto described.
INTRODUCTION Gel electrophoresis of ferritin and apoferritin have revealed the existence of at least three fractions of different mobilitiesl, 2. It has been suggested that this might be due to the presence of polymeric forms of ferritin. SURAN AND TARVER3 also demonstrated that horse spleen ferritin could be fractionated by DEAE-cellulose column chromatography. This heterogeneity was estimated to be due to differences in conformation and shape rather than to differences in the iron content or in the charge of ferritin molecules. In I966 VESTERBERG AND SVENSSON4 proposed a new method of protein fractionation based on the differences in the isoelectric points of proteins. The method is characterized by the production of a natural pH gradient by passing an electric current through a solution of the carrier ampholytes with a suitable pH range on a sucrose density gradient; when a protein sample is applied to the column it moves with the carrier ampholytes to the site corresponding to the individual isoelectric points during passage of the electric current. We have preliminarily reported microheterogeneity in ferritin molecules of rat liver 1°. The present paper describes the microheterogeneity of horse spleen ferritin and apoferritin demonstrated by the above isoelectric fractionation method. Biochim. Biophys. Acta, 243 (1971) 187-192
I88
t. URUSHIZAKI 8l ell.
MATERIAL AND METHODS
Materials We used commercial ferritin from horse spleen (6 times crystallized, Pentex, Inc.). Apoferritin was prepared by a method based on that of GRANICK AND MICHAELIS 5.
Monomeric ferritin was prepared by Sepharose 6-B column chromatography of I % horse spleen ferritin, using o.i M Tris buffer, pH 7.o, containing I M Nat1.
Methods Electrofocusing column fractionation by carrier ampholytes with a narrow pH range was employed. Isoelectrie fractionation of ferritin samples was carried out, using an LKB model 81Ol column of I2o-ml capacity. The temperature was kept constant at 4 ° throughout the experiment by means of an electrothermostat connected to the column. A carrier ampholyte of 4oO/o, pH 4-6, was chosen for this experiment because horse spleen ferritin has its isoelectric point near pH 4.4. Under these conditions, ferritin was distributed over a very narrow range in the column and the elution pattern varied because of minor effects of the experimental conditions. To overcome this disadvantage, narrow pH range carrier empholytes were obtained by isoelectric fractionation of the pH 4-6 carrier ampholytes. For this purpose, pH 4-6 carrier ampholytes with a final concentration of 7% were subjected to 1Ioo V for 48 h on a sucrose density gradient, and the carrier ampholytes with a pH range of 4.2 to 4.65 were obtained by measuring the pH of the eluted fractions. The electrofocusing column fractionations of the ferritin samples were performed using this narrow pH range carrier ampholyte. One quarter of the volume of a solution of this ampholyte was added to a 50% sucrose solution to give a final volume of 60 ml. A further onequarter volume was made up to 60 ml with distilled water. The above two solutions and I ml of a I ~/O ferritin sample were applied to the column using a proportioning pump to make a continuous density gradient by the method of AYOD et el. a. Isoelectric fractionation was performed for 72 h at IlOO V with the cathode at the top of the column and the anode at the bottom. The samples were then eluted from the bottom of the column using a proportioning pump at a constant rate of I ml/min ; 0.5 ml of each fraction was obtained. The pH of each fraction was measured by a pH-recording electrometer at 20 ° and the A 280 nm of the fractions was determined by a UV 200 spectrophotometer. Disc electrophoresis of the fractions separated was also carried out. The fractions were concentrated using Diaflo membranes, type XM-5o, and purified by gel filtration in o.oi M phosphate buffer, pH 8.o, on a Sephadex G-2oo column. Electrophoresis on 5% polyacrylamide gels was performed for 3 h at 15o V using Tris-glycine buffer, pH 8. 3. RESULTS
Electric focusing column fractionation of horse spleen ferritin As shown in Fig. I, the horse spleen ferritin essentially gave a heterogenous pattern with at least eight or nine isoelectric peaks between pH 4.22 and 4.6. The Biochim. Biophys. ,qcta, 243 (1971 ) I 8 7 - I 9 2
I8()
MICROHETEROGENEITY OF FERRITIN
® 04
42
40
J 20
30
40 50 60 FRACTION NUMBER
70
B0
Fig. I. Electric f o c u s s i n g c o l u m n f r a c t i o n a t i o n of horsespleen ferritin. Fig. 2. P o l y a c r y l a m i d e disc electrophoresis of t h e s e p a r a t e d fractions of horse spleen ferritin on isoelectric focusing. F r o m t h e t o p down, F r a c t i o n s 39, 48, 51, 56, 59, 69 a n d 71, s t a i n e d for iron.
pH values of these peaks were 4.27, 4.29, 4-35, 4.38, 4.40, 4.43, 4.47, 4.50 and 4.57, respectively. Moreover, the heterogenous peaks were generally grouped into three main components.
Polyacrylamide disc electrophoresis of fractions of horse spleen ferritin separated by isoelectric focusing Polyacrylamide disc electrophoresis was performed on the separated fractions of horse spleen ferritin by isoeleetric focusing. As shown in Fig. 2, each fraction gave two different bands.
Electric focusing column fractionation of monomeric ferritin of horse spleen The separation of monomeric ferritin from horse spleen ferritin was performed by Sepharose 6-B chromatography. Fig. 3 shows the monomeric dimeric and oligomeric forms of ferritin. As shown in Fig. 4, monomeric ferritin from horse spleen gave essentially the same pattern as horse spleen ferritin, with at least eight or nine isoelectric peaks between pH 4.22 and 4.6. The pH values of the peaks were 4.27, 4.29, 4.34, 4-37, 4.39, 4.42, 4.46, 4.51 and 4.58 (4- o.o2), respectively.
Polyacrylamidc disc electrophoresis of fractions of monomeric ferritin separated by isoelectric focusing The fractions of monomeric ferritin obtained by isoelectrie focusing were subBiochim. Biophys. Acta, 243 (1971) I 8 7 - I 9 2
I. URUSHIZAKI el al.
I90
~C
A O8
O2
,
........ 10
=J",-.-: , ~~ 20
30
~ 40
FRACTION
50
60
...... 70
96
80
NUMBER
Fig. 3. The preparation of m o n o m e r i c ferritin from horse spleen ferritin by Sepharose 6-B column chromatography, using o.i M Tris buffer containing I M NaC], p H 7.0. The last peak is the monomeric form of ferritin.
jected to polyacrylamide disc electrophoresis. As shown in Fig. 5, each fraction gave one band.
Electric focusing column fractionation of horse spleen apoferritin As shown in Fig. 6, apoferritin from horse spleen gave a pattern similar to those of horse spleen ferritin and monomeric ferritin, having eight isoelectric peaks between pH 4.22 and 4.6. The pH values of the peaks were 4.29, 4.34, 4-39, 4.42, 4.44, 4-46, 4.5I and 4.57 (:k o.o2), respectively. pH 46
0,4
,4 03
44
02
42
40
20
30
40
50
FRACTION
60
70
80
NUMBER
Fig. 4- Electric focusing column fractionation of monomeric ferritin from horse spleen.
Biochim. Biophys. Acta, 243 (1971) 187-192
191
MICROHETEROGENEITY OF FERRITIN
....."
42
40
20
30
40 50 60 FRACTION NUMBER
70
BO
90
Fig. 5. Polyacrylamide disc electrophoresis of the fractions of monomeric ferritin separated by isoelectric focusing. From the top down, horse spleen ferritin; eluates from Fractions 3o-39; eluates from Fractions 42-48; eluates from Fractions 53 46; eluates from Fractions 68-73; and eluates from Fractions 75-8o. Fig. 6. Electric focusing column fractionation of apoferritin from horse spleen.
DISCUSSION
Until recently, it was generally accepted that all apoferritin molecules in any one animal were of the same composition. However, ALFREY et a l d found some definite electrophoretic differences between ferritins from different human organs of the same subject, and GABUZDAAND GARDNER8 also observed a similar phenomenon in rabbit. The separation of both horse spleen ferritin and apoferritin into several analogous components by gel electrophoresis was reported by KoPP et al. 9 and SURAN AND TARVERa. However, sedimentation studies indicated that the major components are related as monomer, dimer and trimer, and that their electrophoretic separation is due to differences in molecular size or shape rather than to differences in charge . St;RAN AND TARVERa reported a different and unstable order of heterogeneity of horse spleen ferritin using DEAE-cellulose chromatography, but no significant difference in amino acid composition or in tryptic peptide fingerprints could be shown. This fractionation may be due to conformational differences in ferritin molecules. We briefly reported microheterogeneity in rat liver ferritin molecules as detected by isoelectric focusing 1°. DRYSDALE11 developed a small-scale analytical method for isoelectric focusing in a polyacrylamide gel column and found a similar microheterogeneity in a crystalline sample of horse spleen ferritin. Biochim. Biophys. Acta, 243 (1971) 187-192
192
1. I.'F.USHIZAI{1 C[ et[.
111 tile p r e s e n t e x l ) e r i m e n t s , we h a v e d e m o n s t r a t e d t h a t h o r s e s p l e e n f e r r i t i n monomeric ferritin and apoferritin have the same microheterogeneity of protein m o i e t i e s o n e l e c t r o f o c u s i n g c o l u m n f r a c t i o n a t i o n . T h e y w e r e c o m p o s e d o f e i g h t or n i n e p e a k s w i t h a c o n s t a n t p H r a n g e . T h e s e f e r r i t i n p e a k s w e r e also g r o u p e d i n t o t h r e e m a j o r c o m p o n e n t s . T h e m i c r o h e t e r o g e n e i t y is n o t d u e t o d i f f e r e n c e s in i r o n c o n t e n t or a g g r e g a t e size a n d m a y r e p r e s e n t , in p a r t , s t a b l e c o n f o r m e r s or s o m e f o r m o f p o l y m o r p h i s m d u e t o t h e i n t r i n s i c a l l y d i f f e r e n t p r o t e i n shells o f a p o f e r r i t i n . F u r t h e r i n v e s t i g a t i o n is n e c e s s a r y a n d w e are n o w p e r f o r m i n g e x p e r i m e n t s c o n cerning the amino acid compositions of the heterogenous peaks of apoferritin. REFERENCES J. J. THERON, A. (). HAWTREY AND V. SCHIRREN, Clin. Chim. Acta, 8 (1963) 165. F. CARNEVALI AND G. TECCE, Arch. Biochem. Biophys., lO5 (1964) 207. A. A. SURAN AND H. TARVER, Arch. Biochem. Biophys., I I I (1965) 399. O. VESTERBERG AND H. SVENSSON, Acta Chem. Scan&, 20 (1966) 820. S. GRANICK AZ~D L. MICHAELIS, J. Biol. Chem., 147 (1943) 91. S. R. AYOD, R. W. BONSALL AND S. HUNT, Science Tools, 14 (1967) 4° . C. P. ALFREY, g. C. LYNCH AND C. F.. WHITLEY, J. Lab. Clin. Med., 7° (1967) 419. T. G. GABUZDA AND F. H. GARDNER, Blood, 29 (1967) 77 o. R. KoPP, A. VOGT AND G. MAASS, Nature, 202 (1964) 1211. I. URUSHIZAKI, M. FUKUDA, M. MATSUDA, W. NIITSU, M. YOKOTA AND M. KITAGO, .]a/~..]. Meal., 9 (~97 o) 47II J. 'v~,r. DRYSDALE, Biochim. Biophys. Acta, 207 (197o) 256.
I 2 3 4 5 6 7 S 9 IO
Biochim. Biophys. Acta, 243 (i97 I) 187-192