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Studies on the structure of ferritin and apoferritin from horse spleen. I. Tryptic digestion of ferritin and apoferritin
34
BIOCHIMICAET BIOPHYSICAACTA
BBA 35442 S T U D I E S ON T H E S T R U C T U R E OF F E R R I T I N AND A P O F E R R I T I N FROM H O R S E S P L E E N I. T R Y P T I C D I G E S T I O N OF F E R R I T I N AND A P O F E R R I T I N
ROBERT R. CRICHTON Department of Biochemistry, University of Glasgow, Glasgow, W.2 (Great Britain) (Received May I9th, 1969)
SUMMARY
I. Proteolytic digestion with trypsin has been used to study the change in conformation of horse spleen apoferritin on binding of micellar iron to form ferritin. In the course of a 3o-min digestion, apoferritin was cleaved to about 2.5 times the extent of ferritin. 2. The products of digestion were analysed b y fingerprinting and b y ionexchange chromatography, and a number of peptides, which were absent in ferritin digests, were shown to be present in apoferritin digests. 3. The total number of tryptic peptides in overnight digests of apoferritin agreed well with the number of lysine plus arginine residues found by amino acid analysis. 4. The further investigation of these 'difference peptides' which were found in apoferritin, but not in ferritin, digests m a y yield useful information regarding the nature of the binding of the iron micelle within the apoferritin protein shell.
INTRODUCTION Ferritin, the principal iron storage protein of mammalian spleen and liver was first isolated b y LAUFBERGER1 in 1937. The iron is contained within a protein shell as a ferric hydroxyphosphate micelle of possible composition (FeO.OH)8.(FeOPO3H~)2, 3. The iron can be removed by reduction to the Fe e+ form and dialysis 4, leaving e m p t y shells of apoferritin. The ferritin molecule, completely filled with about 5000 iron atoms, has been found to have a molecular weight of 900 ooo (ref. 5), while that of apoferritin was found by a number of techniques to lie between 430 ooo and 480 ooo (ref. 6-8). X-ray diffraction studies together with chemical evidence have indicated that apoferritin consists of twenty identical subunits of mol. wt. 22 ooo24 ooo (refs. 9, IO) arranged at the vertices of a pentagonal dodecahedron with a small central space in each pentagonal facelX,L Biochim. Biophys. Acta, I94 (I969) 34-42
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
35
Globular proteins are known to exist in a number of different conformational states which are in dynamic equilibrium with each other TM. I f we shift the equilibrium between these states by addition of a ligand which stabilises one or a small number of conformations, this m a y not change molecular parameters sufficiently to be detectable by the usual hydrodynamic methods. However, the use of a technique such as proteolytic digestion, can enable us to detect changes in conformation, even though we cannot b y such a method define any particular conformation. By using this approach conformational changes in aspartate transcarbamylase on binding of ATP and CTP have been demonstrated is. MARKOS14 has demonstrated conformational changes which are manifested in increased resistance to proteolysis of human serum albumin on binding of methyl orange and of tryptophan, and several cases of increased resistance to proteolysis of enzymes on binding of their substrates have been observedlS, TM. It is clearly not unexpected that the binding of a 7o-,~ diameter iron micelle within the protein shell of apoferritin should lead to changes in the susceptibility of the protein to proteolytic digestion, as well as to changes in the hydrodynamic properties of the molecule. The object of the present investigation was to study the digestion of ferritin and apoferritin b y trypsin and to characterise the pattern of peptides produced in each case. The results indicate that not only are there differences in the extent of hydrolysis by trypsin but that there is also a difference in the peptide pattern produced, which m a y on further investigation yield useful information on the binding of the micellar iron within the apoferritin molecule. MATERIALS AND METHODS
Proteins and enzymes Ferritin was prepared from horse spleen b y the method of GRANICKx7 or was purchased from Mann Research (New York). Horse-heart cytochrome c was a product of Seravac Laboratories, Maidenhead, England and tosyl-L-phenylalanine chloromethylketone-treated trypsin was obtained from Worthington Biochemicals, New Jersey. Apoferritin was prepared from ferritin by two methods, the first of which was essentially the second method described b y GRANICK AND MICHAELIS4. The other method involved overnight dialysis of ferritin solutions against a 5% solution of sodium dithionite which had been adjusted to pH 4-5 with a 60% solution of acetic acid containing 0.5% 2,2'-bipyridyl. It was found best to carry out the dialysis under N 2 using deaerated water to prevent oxidation of the dithionite. The protein solutions were dialysed against frequent changes of dionised water in the cold for several days before estimation of the protein concentration. This was determined b y the microKjeldahl method TM which gave total nitrogen values. These were converted to protein concentrations b y use of the arbitrary factor 6.25. Attempts to use the Lowry method did not give consistent results with ferritin solutions.
Enzyme digestion Enzyme digestions were carried out at 37 ° using the Radiometer pH-stat (model TTTlb). The protein solution (lO-2O mg in 5 ml) was adjusted to p H 8.5, and once a steady base line was obtained trypsin dissolved in distilled water was added Biochim. Biophys. Acta, 194 (1969) 34-42
36
R.
R.
CRICHTON
to give an enzyme to protein ratio of 1:2o. The reaction was allowed to proceed, and the rate of uptake of o.oi M NaOH was recorded on a Titrigraph type SBR2c (Radiometer, Copenhagen). Overnight digestions were carried out using 0.06 M NaHCO 3 buffer (pH 8.0) with an enzyme to protein ratio of 1:20.
Peptide analysis Fingerprinting was carried out on W h a t m a n 3MM papers of dimensions 46 cm × 57 cm (H. Reeve Angel, London, England). The electrophoresis was performed in 12.5 ml of pyridine, 12.5 ml of glacial acetic acid made up to i 1 for 16 h at 5 V/cm and descending chromatography for the same time in butanol-acetic acid-water (4:1:5, by vol.). The peptides were visualised using ninhydrin/cadmium acetate reagent (IOO mg of cadmium acetate dissolved in IO ml of water to which 5 ml of glacial acetic acid, ioo ml of acetone and I g of ninhydrin were added in the above order). Peptide chromatography was carried out on columns (15 cm x 0. 9 cm) of Technicon chromabeads, type B, using citrate buffers, on an amino acid analyser constructed in this Department b y Dr. G. Leaf. A nine-chamber varigrad was used, the composition of which is given in Table I. IO mg samples of tryptic digests were applied to the column dissolved in I ml of 0.2 M sodium citrate buffer (pH 2.1). TABLE
I
COMPOSITION
OF
VARIGRAD
Chamber
0.2 M sodium citrate, p H 3.25 (ml)
FOR PEPTIDE
0.2 M sodium citrate, p H 4.25 (ml)
0.35 M sodium citrate, p H 5.28 (ml) --
I
6o
--
2
60
--
--
3
4°
20
--
4
20
4°
--
5
--
60
--
6
--
60
--
7
--
4°
20
8
--
20
4°
9
--
--
60
ANALYSER
A mino acid analysis Amino acid analyses were carried out on protein samples which had been hydrolysed with 6 M HC1 at IiO ° for 16 h using a Locarte amino acid analyser (Locarte Co., Emperors Gate, London) with a single column system and stepwise elution. Cysteine and tryptophan were not determined.
RESULTS
Digestion of ferritin and apoferritin with trypsin gave the alkali uptakes illustrated in Fig. i. The results are for 20 mg of each protein with I m g of trypsin. The Biochim. Biophys. Acta,
194
(1969)
34-42
37
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
Apoferrit in
s
Ferritin ~2
I 0
0
2
&
4
~
;~
TIME
(rain
~
,'6
2'0 2'2
;8
'
)
Fig. I. R a t e of t r y p t i c digestion of ferritin and apoferritin. The u p t a k e of o.oi M N a O H as a function of time is s h o w n for the digestion of 20 m g samples of ferritin and apoferritin at p H 8. 5 with i mg of trypsin, as described in MATERIALS AND METHODS.
alkali uptake due to the trypsin itself, determined by addition of I mg of trypsin to 5 ml of water at pH 8.5, has been deducted from the values given in the figure. Fig. 2 presents the results obtained when IO mg of cytochrome c was digested with o. 5 mg of trypsin in the presence and absence of IO mg of ferritin. Fingerprints of the products of tryptic digestion of apoferritin and ferritin, both from overnight digests carried out in the presence of NaHCO 8 and from 3D-miD digests carried out on the Titrigraph, are illustrated in Fig. 3. The peptides have been numbered from I to 25 in the overnight digest of apoferritin, and additional peptides which appear in the other digests which are absent from this digest are given appropriate numbers. Thus there are three peptides present in 3D-miD digests of apoferritin
B
...""f~ /./
b
"
'
~
o
2
o
5
15
1o TIME
2
(min)
Fig. 2. Effect of ferritin on the t r y p t i c digestion of c y t o c h r o m e c. The u p t a k e of o.oi M N a O H as a function of time for the digestion of IO m g of horse-heart c y t o c h r o m e c in the presence ( ) a n d absence ( - - . - - ) of IO m g of ferritin is presented. The p H was 8. 5 and o. 5 mg of t r y p s i n was used in each case.
Biochim. Biophys. Acta, 194 (I969) 34-42
38
R. R. CRICHTON
0
'::;::"::::
0
26~ 27 ~'~'1322 0
;:2"
o?@
oe9 (b)
(a)
~)
0
"0, ~ 20°~2 o ~ "~
~
0
::'::"
0 o~
O,s
C-":::: (30 (c)
O0
0
~ O
.:::o"
®
Fig. 3- Fingerprints of t r y p t i c digests of ferritin and apoferritin. The fingerprints obtained as described in MATERIALS AI~D METHODS for overnight digests of apoferritin (a) and ferritin (c) and for 3o-min digests of apoferritin (b) and ferritin (d) are shown. Electrophoresis was from left to right and c h r o m a t o g r a p h y from t o p to b o t t o m of t h e figures. The origin is indicated b y a spot at t h e left h a n d corner. P e p t i d e s which were p r e s e n t in small a m o u n t s are indicated b y d o t t e d lines.
which do not correspond to any of the Peptides 1-25, and these are numbered 26, 27 and 28. Peptides which are present in small amounts are indicated by dotted lines. Peptide patterns obtained when ferritin and apoferritin digests were analysed on the peptide analyser system described in MATERIALSAND METHODSare presented in Fig. 4. The digests contained io mg of protein. Results of amino acid analysis of the apoferritin used in this study are given in Table II. The values in/,moles are the mean of five determinations. Tryptophan was not determined. The results are also expressed as residues of each amino acid per 22 500 dalton subunit, and the values of the final column of the table are the results of WILLIAMS AND HARRISON lg, which have also been expressed as residues per 22 500 dalton subunit. DISCUSSION
The use of proteolytic digestion as a probe of changes in protein conformation can be remarkably sensitive when compared with other methods for studying this problem. Thus, MARKUS14 has shown that the binding of one molecule of L-tryptophan per molecule of human serum albumin, whilst causing no change in the hydrodynamic l?iochim. Biophys. Zcta, 194 (1969) 34-42
39
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
(b) 2 14
o. 3
22 23
03 8
O 0"2
13
18
~
21
IB 3
O
11
15
23 21
II
12 01
0.1
=
5
t
t
'
10
15
20
Time (h)
Time (h)
13
(c)
t5 Z2 23
14 It
O3
e
1~
6
9
r 5
20
12
i 10
i
i
15
20
_
Time (h)
Fig. 4. Ion-exchange chromatography of tryptic peptides from ferritin and apoferritin. The elution profiles obtained when 3o-min tryptic digests of apoferritin and ferritin and an overnight tryptic digest of apoferritin were chromatographed on the peptide analyser described in MATERIALS AND METHODS are shown in (a), (b) and (c) respectively.
properties of the molecule, reduces its susceptibility to proteolysis by 3O°/o. In addition, characterisation of the products of enzymic digestion can give useful information about these parts of the molecule which are involved in the conformation change. In the present investigation it was found that the presence of the iron micelle of ferritin considerably reduces the extent to which the protein is digested by trypsin. In 30 min apoferritin is digested (Fig. I) to an extent that is approx. 2.5 times that of ferritin. At this pH the uptake of alkali will be directly proportional to the number of peptide bonds split ~°. On the basis of a molecular weight of 22 500 per subunit, this corresponds to 6.30 bonds hydrolysed per subunit for apoferritin and 2.49 bonds per subunit for ferritin. From the results of Table II, this indicates that there has been cleavage of 27.5~/o and lO.8% of the possible lysyl plus arginyl bonds in the molecule respectively. In an effort to demonstrate that the presence of the micellar iron in the ferritin was not having an inhibitory effect on the trypsin, the experiment illustrated in Fig. 2 was undertaken. The result indicates that in the presence of ferritin, cytochrome Biochim. Biophys. Acta, 194 (I969) 34-42
4°
R.R. CRICHTON
TABLE II AMINO ACID COMPOSITION
Amino acid
OF APOFERRITIN
Amount (pmoles)
Residues per 22 50o daltons This R@ x6 investigation
Cysteine* A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
-0.777 o. 246 0.403 1.o71 o.127 o.443 0.627 o. 311 o. 124 o. 157 1.12o o. 224 0.329 o.259 0.392 0.424
-21.o5 6.67 lO.91 29.Ol 3.43 12.oi 16.99 8.43 3- 36 4-25 30.35 6.07 8.92 7.o3 lO.62 I 1.49
2.39 21.o3 6.7 ° i i .55 29.19 2.51 i 1.92 17.26 8.59 3.59 4.56 30.52 6.33 8.87 7.14 lO.9O I 1.98
* Not determined.
c was still split at approximately the same rate and to the same extent as in its absence. This is indicative but not conclusive evidence that the observed differences between ferritin and apoferritin digestion are related to conformational changes rather than to inhibition of the trypsin. It has been shown in this laboratory ~1 that the effect of pepsin on ferritin and apoferritin is virtually identical. However, these are not satisfactory criteria for the claim that the presence of the micellar iron does not inhibit the enzyme. The demonstration of this is fraught with technical difficulties in view of the problems associated with obtaining the micelle in its native state outside of the protein, and of studying Fe 8+ at neutral pH. The synthesis of the polymeric c o m p o u n d I F e a O 3 ( O H ) 4 ( N O s ) 2 ! n by SPIRO et al. 22,2a and BRADY et al. 2~ which appears to be a remarkable analogue of the iron containing core of ferritin, m a y serve as a useful model for confirmation of the hypothesis enunciated above. From the peptide fingerprints in Figs. 3b and 3d it can be seen that the major difference between short term digests of ferritin and apoferritin is the presence in apoferritin digests of five peptides (I, 4, 7, 16 and 27) which are absent from ferritin digests and of two peptides (3° and 31) which appear only in the ferritin digest. Comparison of fingerprints from the overnight digests (Figs. 3a and 3c) indicates that there are seven peptides unique to apoferritin viz. I, 4, 7, 9, IO, I I and 14, while one peptide, 29, is in the ferritin digest only. Of the seven unique apoferritin peptides, IO and I I are present in small amounts in the ferritin digest, and these two, together with 9 and 14 are also absent from short term digests of apoferritin. Peptides I, 4 and 7 are absent from both short and long term digests of ferritin. A comparison of the short term and overnight digests of apoferritin reveals that there are six peptides viz. 2, 9, io, I I , 14 and 17 which are present only in the overnight digest, while this digest lacks Peptides 26, 27 and 28. Biochim. Biophys. Acta, 194 (1969) 34-42
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
41
When the number of peptides found in overnight digests is compared with the lysine p l u s arginine content of the protein, based on the results of amino acid analysis (Table II) it is apparent that there are approximately the correct number of tryptic peptides. Twenty five peptides are found (Fig. 3a) and from the amino acid analysis twenty four would be expected. The use of ion-exchange chromatography (Figs. 4 a and 4 b) indicates that there are at least five peptides absent from ferritin digests viz. 5, 12, 14, 17 and 22 which are found in digests of apoferritin. Although 7 is indicated in Fig. 4 a as being present it is rarely more than a base line inflection. The Peptide 19 which appears to be absent from the apoferritin digest does appear from preliminary characterisation studies (R. R. Crichton, unpublished observations) to be present in the latter part of Peak 18. Thus the peptide patterns from both fingerprinting and ion-exchange chromatography confirm the presence of at least five peptides which appear in short term digests of apoferritin and are either absent or greatly reduced in ferritin. At present the results from the two methods have not been correlated so that it is not known which peptide on the analyser corresponds to which on the fingerprints. The importance of the overnight digests of apoferritin for determination of the sequence of the apoferritin molecule prompts the inclusion of Fig. 4c. It differs by intensification of a number of peptides, notable 5, 6, 9, i i and 14, and a diminution in the amount of I6 from short term apoferritin digests. Peptides 7 and 19 are present in this digest but not readily apparent in the short term apoferritin digest. Once again it has not yet been possible to correlate these changes with the results of fingerprinting, although this work is at present in progress in this laboratory. The results presented here are consistent with the optical rotatory dispersion studies of LISTOWSKY et al. 25 who were able to demonstrate differences in the conformation of ferritin and apoferritin. Their results indicate that approx. 50% of the native protein in ferritin exists in the helical form, and that the rotatory properties are independent of the iron concentration. Apoferritin was found by them to exhibit rotational changes consistent with additional folding of the molecule, and the percentage helix content of apoferritin was calculated to be 8-9% higher than for ferritin. It is interesting that in the more ordered apoferritin structure there is a greater potential for tryptic digestion, but the two studies clearly underline that some parts of the apoferritin molecule change their conformation considerably when the iron micelle is bound. The results of amino acid analysis (Table II) show such close agreement with those of WILLIAMS AND HARRISON 19, with the exception of the proline value, that it seems quite unlikely that there can be more than one kind of subunit in apoferritin. This is underlined by the good agreement between the number of tryptic peptides found both by fingerprinting and by ion-exchange chromatography and lends strong support to the previous arguments 9,26 in favour of the presence of twenty identical subunits in the apoferritin molecule. At present there are two conflicting views regarding the way in which iron is introduced into the apoferritin molecule. One, based on the fact that the shape of the micelle is largely imposed by the shape of the interior of the protein, while the atomic structures of core and protein are not specifically related, suggests that the micelle grows within the protein molecules at a number of different sites and in a number of different directions ~7-29. The other is that the micelle forms and then Biochim. Biophys. Acta, 194 (1969) 34-42
42
R. R. CRICHTON
interacts with the subunits of the apoferritin by noncovalent forces to form ferritin 3° This latter hypothesis would perhaps more readily explain the stabilisation of the protein subunits against proteolytic digestion in ferritin, and in turn the stimulation of ferritin synthesis observed when iron is administered in vivo 31-33. In any event it seems that an investigation of the difference peptides, found only in tryptic digests of apoferritin, may yield information about the nature of attachment of the micellar iron within the apoferritin molecule. This investigation, which we are at present pursuing may help to clarify the controversy outlined above. ACKNOWLEDGEMENTS
I should like to thank Mr. James Blackstock and Miss Veronica Griffin for their technical assistance, Professor J. N. Davidson and Professor R. M. S. Smellie for the provision of facilities and Dr. George Leaf for his advice and criticisms. REFERENCES i :2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
V. LAUFBERGER, Bull. Soc. Chim. Biol., 19 (1937) 1575. L. MICHAELIS, C. D. CORYELL AND S. GRANICK, J. Biol. Chem., 148 (1943) 463 • S. GRANICK AND P. F. HAHN, J. Biol. Chem., 155 (1944) 661. S. GRANICK AND L. MICHAELIS, J. Biol. Chem., 147 (1943) 91. F. A. FISCHBACH AND J. W. ANDEREGG, J. Mol. Biol., 14 (1965) 458. A. ROTHEN, J. Biol. Chem., 152 (1944) 679. P. M. HARRISON, J. Mol. Biol., 6 (1963) 404 . G. W. RICHTER AND G. F. WALKER, Biochemistry, 6 (1967) 2871. P. M. HARRISON AND T. HOFMANN, J. Mol. Biol., 4 (1962) 239. P. M. HARRISON, T. HOFMANN AND W. I. P. MAINWARING, J. Mol. Biol., 4 (1962) 251" P. M. HARRISON, Acta Cryst., 13 (196o) lO6O. C. TANFORD, Abstr. 6th Intern. Congr. Biochemistry, New York, 1964, p. 119. D. K. McLINTOCK AND G. MARKUS, J. Biol. Chem., 243 (1968) 2855. G. MARKUS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 253. G. SZABOLCSI, Acta Physiol. Acad. Sci. Hung., 13 (1958) 213. K. A. TRAYSER AND S. P. COLOWICK, Arch. Biochem. Biophys., 94 (1961) 169. S. GRANICK, J. Biol. Chem., 146 (1942) 451. A. FLECK AND H. N. MUNRO, Clin. Chim. Acta, i i (1965) 2. M. A. WILLIAMS AND P. M. HARRISON, Biochem. J., IiO (1968) 265. S. G. WALE¥ AND J. WATSON, Biochem. J., 55 (1953) 328. R. R. CRICHTON, Abstr. 6th Federation European Biochem. Soc. Meeting, Madrid, 2969, p. 171. T. G. SPIRO, S. E. ALLERTON, J. RENNER, A. TERZlS, R. BILS AND P. SALTMAN, J. Am. Chem. Soc., 88 (1966) 2721. T. G. SPIRO, S. E. ALLERTON, J. RENNER, A. TERZlS, R. BILS AND P. SALTMAN, J. Am. Chem. Soc., 88 (1966) 3147. G. W. BRADY, C. R. KURKJIAN, E. V. X. LYDEN, M. n . ROBIN, P. SALTMAN, T. SPIRO AND A. TERZlS, Biochemistry, 7 (1968) 2185. I. LISTOWSKY, J. J. BETHEIL AND S. ENGLARD, Biochemistry, 6 (1967) 1341. T. HOFMANN AND P. M. HARRISON, J. Mol. Biol., 6 (1963) 256. F. A. FISCHBACH, P. M. HARRISON AND T. G. HOV, J. Mol. Biol., 39 (1969) 235. G. H. HAGGIS, J. Mol. Biol., 14 (1965) 598. G. H. HAGGIS, 6th Intern. Congr. Electron Microscopy, Kyoto, 1966, Vol. 2, Maruzen, T o k y o , 1966, p. 127. L. PAPE, J. S. MULTANI, C. STITT AND P. SALTMAN, Biochemistry, 7 (1968) 6o6. J. W. DRYSDALE AND H. N. MUNRO, J. Biol. Chem., 241 (1966) 363 o. R. A. FINEBERG AND D. M. GREENBERG, J. Biol. Chem., 214 (1955) 97R. A. FINEBERG AND D. M. GREENBERG, J. Biol. Chem., 214 (1955) lO7.
Biochim. Biophys. Acta, 194 (1969) 34-42
BIOCHIMICAET BIOPHYSICAACTA
BBA 35442 S T U D I E S ON T H E S T R U C T U R E OF F E R R I T I N AND A P O F E R R I T I N FROM H O R S E S P L E E N I. T R Y P T I C D I G E S T I O N OF F E R R I T I N AND A P O F E R R I T I N
ROBERT R. CRICHTON Department of Biochemistry, University of Glasgow, Glasgow, W.2 (Great Britain) (Received May I9th, 1969)
SUMMARY
I. Proteolytic digestion with trypsin has been used to study the change in conformation of horse spleen apoferritin on binding of micellar iron to form ferritin. In the course of a 3o-min digestion, apoferritin was cleaved to about 2.5 times the extent of ferritin. 2. The products of digestion were analysed b y fingerprinting and b y ionexchange chromatography, and a number of peptides, which were absent in ferritin digests, were shown to be present in apoferritin digests. 3. The total number of tryptic peptides in overnight digests of apoferritin agreed well with the number of lysine plus arginine residues found by amino acid analysis. 4. The further investigation of these 'difference peptides' which were found in apoferritin, but not in ferritin, digests m a y yield useful information regarding the nature of the binding of the iron micelle within the apoferritin protein shell.
INTRODUCTION Ferritin, the principal iron storage protein of mammalian spleen and liver was first isolated b y LAUFBERGER1 in 1937. The iron is contained within a protein shell as a ferric hydroxyphosphate micelle of possible composition (FeO.OH)8.(FeOPO3H~)2, 3. The iron can be removed by reduction to the Fe e+ form and dialysis 4, leaving e m p t y shells of apoferritin. The ferritin molecule, completely filled with about 5000 iron atoms, has been found to have a molecular weight of 900 ooo (ref. 5), while that of apoferritin was found by a number of techniques to lie between 430 ooo and 480 ooo (ref. 6-8). X-ray diffraction studies together with chemical evidence have indicated that apoferritin consists of twenty identical subunits of mol. wt. 22 ooo24 ooo (refs. 9, IO) arranged at the vertices of a pentagonal dodecahedron with a small central space in each pentagonal facelX,L Biochim. Biophys. Acta, I94 (I969) 34-42
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
35
Globular proteins are known to exist in a number of different conformational states which are in dynamic equilibrium with each other TM. I f we shift the equilibrium between these states by addition of a ligand which stabilises one or a small number of conformations, this m a y not change molecular parameters sufficiently to be detectable by the usual hydrodynamic methods. However, the use of a technique such as proteolytic digestion, can enable us to detect changes in conformation, even though we cannot b y such a method define any particular conformation. By using this approach conformational changes in aspartate transcarbamylase on binding of ATP and CTP have been demonstrated is. MARKOS14 has demonstrated conformational changes which are manifested in increased resistance to proteolysis of human serum albumin on binding of methyl orange and of tryptophan, and several cases of increased resistance to proteolysis of enzymes on binding of their substrates have been observedlS, TM. It is clearly not unexpected that the binding of a 7o-,~ diameter iron micelle within the protein shell of apoferritin should lead to changes in the susceptibility of the protein to proteolytic digestion, as well as to changes in the hydrodynamic properties of the molecule. The object of the present investigation was to study the digestion of ferritin and apoferritin b y trypsin and to characterise the pattern of peptides produced in each case. The results indicate that not only are there differences in the extent of hydrolysis by trypsin but that there is also a difference in the peptide pattern produced, which m a y on further investigation yield useful information on the binding of the micellar iron within the apoferritin molecule. MATERIALS AND METHODS
Proteins and enzymes Ferritin was prepared from horse spleen b y the method of GRANICKx7 or was purchased from Mann Research (New York). Horse-heart cytochrome c was a product of Seravac Laboratories, Maidenhead, England and tosyl-L-phenylalanine chloromethylketone-treated trypsin was obtained from Worthington Biochemicals, New Jersey. Apoferritin was prepared from ferritin by two methods, the first of which was essentially the second method described b y GRANICK AND MICHAELIS4. The other method involved overnight dialysis of ferritin solutions against a 5% solution of sodium dithionite which had been adjusted to pH 4-5 with a 60% solution of acetic acid containing 0.5% 2,2'-bipyridyl. It was found best to carry out the dialysis under N 2 using deaerated water to prevent oxidation of the dithionite. The protein solutions were dialysed against frequent changes of dionised water in the cold for several days before estimation of the protein concentration. This was determined b y the microKjeldahl method TM which gave total nitrogen values. These were converted to protein concentrations b y use of the arbitrary factor 6.25. Attempts to use the Lowry method did not give consistent results with ferritin solutions.
Enzyme digestion Enzyme digestions were carried out at 37 ° using the Radiometer pH-stat (model TTTlb). The protein solution (lO-2O mg in 5 ml) was adjusted to p H 8.5, and once a steady base line was obtained trypsin dissolved in distilled water was added Biochim. Biophys. Acta, 194 (1969) 34-42
36
R.
R.
CRICHTON
to give an enzyme to protein ratio of 1:2o. The reaction was allowed to proceed, and the rate of uptake of o.oi M NaOH was recorded on a Titrigraph type SBR2c (Radiometer, Copenhagen). Overnight digestions were carried out using 0.06 M NaHCO 3 buffer (pH 8.0) with an enzyme to protein ratio of 1:20.
Peptide analysis Fingerprinting was carried out on W h a t m a n 3MM papers of dimensions 46 cm × 57 cm (H. Reeve Angel, London, England). The electrophoresis was performed in 12.5 ml of pyridine, 12.5 ml of glacial acetic acid made up to i 1 for 16 h at 5 V/cm and descending chromatography for the same time in butanol-acetic acid-water (4:1:5, by vol.). The peptides were visualised using ninhydrin/cadmium acetate reagent (IOO mg of cadmium acetate dissolved in IO ml of water to which 5 ml of glacial acetic acid, ioo ml of acetone and I g of ninhydrin were added in the above order). Peptide chromatography was carried out on columns (15 cm x 0. 9 cm) of Technicon chromabeads, type B, using citrate buffers, on an amino acid analyser constructed in this Department b y Dr. G. Leaf. A nine-chamber varigrad was used, the composition of which is given in Table I. IO mg samples of tryptic digests were applied to the column dissolved in I ml of 0.2 M sodium citrate buffer (pH 2.1). TABLE
I
COMPOSITION
OF
VARIGRAD
Chamber
0.2 M sodium citrate, p H 3.25 (ml)
FOR PEPTIDE
0.2 M sodium citrate, p H 4.25 (ml)
0.35 M sodium citrate, p H 5.28 (ml) --
I
6o
--
2
60
--
--
3
4°
20
--
4
20
4°
--
5
--
60
--
6
--
60
--
7
--
4°
20
8
--
20
4°
9
--
--
60
ANALYSER
A mino acid analysis Amino acid analyses were carried out on protein samples which had been hydrolysed with 6 M HC1 at IiO ° for 16 h using a Locarte amino acid analyser (Locarte Co., Emperors Gate, London) with a single column system and stepwise elution. Cysteine and tryptophan were not determined.
RESULTS
Digestion of ferritin and apoferritin with trypsin gave the alkali uptakes illustrated in Fig. i. The results are for 20 mg of each protein with I m g of trypsin. The Biochim. Biophys. Acta,
194
(1969)
34-42
37
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
Apoferrit in
s
Ferritin ~2
I 0
0
2
&
4
~
;~
TIME
(rain
~
,'6
2'0 2'2
;8
'
)
Fig. I. R a t e of t r y p t i c digestion of ferritin and apoferritin. The u p t a k e of o.oi M N a O H as a function of time is s h o w n for the digestion of 20 m g samples of ferritin and apoferritin at p H 8. 5 with i mg of trypsin, as described in MATERIALS AND METHODS.
alkali uptake due to the trypsin itself, determined by addition of I mg of trypsin to 5 ml of water at pH 8.5, has been deducted from the values given in the figure. Fig. 2 presents the results obtained when IO mg of cytochrome c was digested with o. 5 mg of trypsin in the presence and absence of IO mg of ferritin. Fingerprints of the products of tryptic digestion of apoferritin and ferritin, both from overnight digests carried out in the presence of NaHCO 8 and from 3D-miD digests carried out on the Titrigraph, are illustrated in Fig. 3. The peptides have been numbered from I to 25 in the overnight digest of apoferritin, and additional peptides which appear in the other digests which are absent from this digest are given appropriate numbers. Thus there are three peptides present in 3D-miD digests of apoferritin
B
...""f~ /./
b
"
'
~
o
2
o
5
15
1o TIME
2
(min)
Fig. 2. Effect of ferritin on the t r y p t i c digestion of c y t o c h r o m e c. The u p t a k e of o.oi M N a O H as a function of time for the digestion of IO m g of horse-heart c y t o c h r o m e c in the presence ( ) a n d absence ( - - . - - ) of IO m g of ferritin is presented. The p H was 8. 5 and o. 5 mg of t r y p s i n was used in each case.
Biochim. Biophys. Acta, 194 (I969) 34-42
38
R. R. CRICHTON
0
'::;::"::::
0
26~ 27 ~'~'1322 0
;:2"
o?@
oe9 (b)
(a)
~)
0
"0, ~ 20°~2 o ~ "~
~
0
::'::"
0 o~
O,s
C-":::: (30 (c)
O0
0
~ O
.:::o"
®
Fig. 3- Fingerprints of t r y p t i c digests of ferritin and apoferritin. The fingerprints obtained as described in MATERIALS AI~D METHODS for overnight digests of apoferritin (a) and ferritin (c) and for 3o-min digests of apoferritin (b) and ferritin (d) are shown. Electrophoresis was from left to right and c h r o m a t o g r a p h y from t o p to b o t t o m of t h e figures. The origin is indicated b y a spot at t h e left h a n d corner. P e p t i d e s which were p r e s e n t in small a m o u n t s are indicated b y d o t t e d lines.
which do not correspond to any of the Peptides 1-25, and these are numbered 26, 27 and 28. Peptides which are present in small amounts are indicated by dotted lines. Peptide patterns obtained when ferritin and apoferritin digests were analysed on the peptide analyser system described in MATERIALSAND METHODSare presented in Fig. 4. The digests contained io mg of protein. Results of amino acid analysis of the apoferritin used in this study are given in Table II. The values in/,moles are the mean of five determinations. Tryptophan was not determined. The results are also expressed as residues of each amino acid per 22 500 dalton subunit, and the values of the final column of the table are the results of WILLIAMS AND HARRISON lg, which have also been expressed as residues per 22 500 dalton subunit. DISCUSSION
The use of proteolytic digestion as a probe of changes in protein conformation can be remarkably sensitive when compared with other methods for studying this problem. Thus, MARKUS14 has shown that the binding of one molecule of L-tryptophan per molecule of human serum albumin, whilst causing no change in the hydrodynamic l?iochim. Biophys. Zcta, 194 (1969) 34-42
39
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
(b) 2 14
o. 3
22 23
03 8
O 0"2
13
18
~
21
IB 3
O
11
15
23 21
II
12 01
0.1
=
5
t
t
'
10
15
20
Time (h)
Time (h)
13
(c)
t5 Z2 23
14 It
O3
e
1~
6
9
r 5
20
12
i 10
i
i
15
20
_
Time (h)
Fig. 4. Ion-exchange chromatography of tryptic peptides from ferritin and apoferritin. The elution profiles obtained when 3o-min tryptic digests of apoferritin and ferritin and an overnight tryptic digest of apoferritin were chromatographed on the peptide analyser described in MATERIALS AND METHODS are shown in (a), (b) and (c) respectively.
properties of the molecule, reduces its susceptibility to proteolysis by 3O°/o. In addition, characterisation of the products of enzymic digestion can give useful information about these parts of the molecule which are involved in the conformation change. In the present investigation it was found that the presence of the iron micelle of ferritin considerably reduces the extent to which the protein is digested by trypsin. In 30 min apoferritin is digested (Fig. I) to an extent that is approx. 2.5 times that of ferritin. At this pH the uptake of alkali will be directly proportional to the number of peptide bonds split ~°. On the basis of a molecular weight of 22 500 per subunit, this corresponds to 6.30 bonds hydrolysed per subunit for apoferritin and 2.49 bonds per subunit for ferritin. From the results of Table II, this indicates that there has been cleavage of 27.5~/o and lO.8% of the possible lysyl plus arginyl bonds in the molecule respectively. In an effort to demonstrate that the presence of the micellar iron in the ferritin was not having an inhibitory effect on the trypsin, the experiment illustrated in Fig. 2 was undertaken. The result indicates that in the presence of ferritin, cytochrome Biochim. Biophys. Acta, 194 (I969) 34-42
4°
R.R. CRICHTON
TABLE II AMINO ACID COMPOSITION
Amino acid
OF APOFERRITIN
Amount (pmoles)
Residues per 22 50o daltons This R@ x6 investigation
Cysteine* A s p a r t i c acid Threonine Serine G l u t a m i c acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
-0.777 o. 246 0.403 1.o71 o.127 o.443 0.627 o. 311 o. 124 o. 157 1.12o o. 224 0.329 o.259 0.392 0.424
-21.o5 6.67 lO.91 29.Ol 3.43 12.oi 16.99 8.43 3- 36 4-25 30.35 6.07 8.92 7.o3 lO.62 I 1.49
2.39 21.o3 6.7 ° i i .55 29.19 2.51 i 1.92 17.26 8.59 3.59 4.56 30.52 6.33 8.87 7.14 lO.9O I 1.98
* Not determined.
c was still split at approximately the same rate and to the same extent as in its absence. This is indicative but not conclusive evidence that the observed differences between ferritin and apoferritin digestion are related to conformational changes rather than to inhibition of the trypsin. It has been shown in this laboratory ~1 that the effect of pepsin on ferritin and apoferritin is virtually identical. However, these are not satisfactory criteria for the claim that the presence of the micellar iron does not inhibit the enzyme. The demonstration of this is fraught with technical difficulties in view of the problems associated with obtaining the micelle in its native state outside of the protein, and of studying Fe 8+ at neutral pH. The synthesis of the polymeric c o m p o u n d I F e a O 3 ( O H ) 4 ( N O s ) 2 ! n by SPIRO et al. 22,2a and BRADY et al. 2~ which appears to be a remarkable analogue of the iron containing core of ferritin, m a y serve as a useful model for confirmation of the hypothesis enunciated above. From the peptide fingerprints in Figs. 3b and 3d it can be seen that the major difference between short term digests of ferritin and apoferritin is the presence in apoferritin digests of five peptides (I, 4, 7, 16 and 27) which are absent from ferritin digests and of two peptides (3° and 31) which appear only in the ferritin digest. Comparison of fingerprints from the overnight digests (Figs. 3a and 3c) indicates that there are seven peptides unique to apoferritin viz. I, 4, 7, 9, IO, I I and 14, while one peptide, 29, is in the ferritin digest only. Of the seven unique apoferritin peptides, IO and I I are present in small amounts in the ferritin digest, and these two, together with 9 and 14 are also absent from short term digests of apoferritin. Peptides I, 4 and 7 are absent from both short and long term digests of ferritin. A comparison of the short term and overnight digests of apoferritin reveals that there are six peptides viz. 2, 9, io, I I , 14 and 17 which are present only in the overnight digest, while this digest lacks Peptides 26, 27 and 28. Biochim. Biophys. Acta, 194 (1969) 34-42
TRYPTIC DIGESTION OF FERRITIN AND APOFERRITIN
41
When the number of peptides found in overnight digests is compared with the lysine p l u s arginine content of the protein, based on the results of amino acid analysis (Table II) it is apparent that there are approximately the correct number of tryptic peptides. Twenty five peptides are found (Fig. 3a) and from the amino acid analysis twenty four would be expected. The use of ion-exchange chromatography (Figs. 4 a and 4 b) indicates that there are at least five peptides absent from ferritin digests viz. 5, 12, 14, 17 and 22 which are found in digests of apoferritin. Although 7 is indicated in Fig. 4 a as being present it is rarely more than a base line inflection. The Peptide 19 which appears to be absent from the apoferritin digest does appear from preliminary characterisation studies (R. R. Crichton, unpublished observations) to be present in the latter part of Peak 18. Thus the peptide patterns from both fingerprinting and ion-exchange chromatography confirm the presence of at least five peptides which appear in short term digests of apoferritin and are either absent or greatly reduced in ferritin. At present the results from the two methods have not been correlated so that it is not known which peptide on the analyser corresponds to which on the fingerprints. The importance of the overnight digests of apoferritin for determination of the sequence of the apoferritin molecule prompts the inclusion of Fig. 4c. It differs by intensification of a number of peptides, notable 5, 6, 9, i i and 14, and a diminution in the amount of I6 from short term apoferritin digests. Peptides 7 and 19 are present in this digest but not readily apparent in the short term apoferritin digest. Once again it has not yet been possible to correlate these changes with the results of fingerprinting, although this work is at present in progress in this laboratory. The results presented here are consistent with the optical rotatory dispersion studies of LISTOWSKY et al. 25 who were able to demonstrate differences in the conformation of ferritin and apoferritin. Their results indicate that approx. 50% of the native protein in ferritin exists in the helical form, and that the rotatory properties are independent of the iron concentration. Apoferritin was found by them to exhibit rotational changes consistent with additional folding of the molecule, and the percentage helix content of apoferritin was calculated to be 8-9% higher than for ferritin. It is interesting that in the more ordered apoferritin structure there is a greater potential for tryptic digestion, but the two studies clearly underline that some parts of the apoferritin molecule change their conformation considerably when the iron micelle is bound. The results of amino acid analysis (Table II) show such close agreement with those of WILLIAMS AND HARRISON 19, with the exception of the proline value, that it seems quite unlikely that there can be more than one kind of subunit in apoferritin. This is underlined by the good agreement between the number of tryptic peptides found both by fingerprinting and by ion-exchange chromatography and lends strong support to the previous arguments 9,26 in favour of the presence of twenty identical subunits in the apoferritin molecule. At present there are two conflicting views regarding the way in which iron is introduced into the apoferritin molecule. One, based on the fact that the shape of the micelle is largely imposed by the shape of the interior of the protein, while the atomic structures of core and protein are not specifically related, suggests that the micelle grows within the protein molecules at a number of different sites and in a number of different directions ~7-29. The other is that the micelle forms and then Biochim. Biophys. Acta, 194 (1969) 34-42
42
R. R. CRICHTON
interacts with the subunits of the apoferritin by noncovalent forces to form ferritin 3° This latter hypothesis would perhaps more readily explain the stabilisation of the protein subunits against proteolytic digestion in ferritin, and in turn the stimulation of ferritin synthesis observed when iron is administered in vivo 31-33. In any event it seems that an investigation of the difference peptides, found only in tryptic digests of apoferritin, may yield information about the nature of attachment of the micellar iron within the apoferritin molecule. This investigation, which we are at present pursuing may help to clarify the controversy outlined above. ACKNOWLEDGEMENTS
I should like to thank Mr. James Blackstock and Miss Veronica Griffin for their technical assistance, Professor J. N. Davidson and Professor R. M. S. Smellie for the provision of facilities and Dr. George Leaf for his advice and criticisms. REFERENCES i :2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
V. LAUFBERGER, Bull. Soc. Chim. Biol., 19 (1937) 1575. L. MICHAELIS, C. D. CORYELL AND S. GRANICK, J. Biol. Chem., 148 (1943) 463 • S. GRANICK AND P. F. HAHN, J. Biol. Chem., 155 (1944) 661. S. GRANICK AND L. MICHAELIS, J. Biol. Chem., 147 (1943) 91. F. A. FISCHBACH AND J. W. ANDEREGG, J. Mol. Biol., 14 (1965) 458. A. ROTHEN, J. Biol. Chem., 152 (1944) 679. P. M. HARRISON, J. Mol. Biol., 6 (1963) 404 . G. W. RICHTER AND G. F. WALKER, Biochemistry, 6 (1967) 2871. P. M. HARRISON AND T. HOFMANN, J. Mol. Biol., 4 (1962) 239. P. M. HARRISON, T. HOFMANN AND W. I. P. MAINWARING, J. Mol. Biol., 4 (1962) 251" P. M. HARRISON, Acta Cryst., 13 (196o) lO6O. C. TANFORD, Abstr. 6th Intern. Congr. Biochemistry, New York, 1964, p. 119. D. K. McLINTOCK AND G. MARKUS, J. Biol. Chem., 243 (1968) 2855. G. MARKUS, Proc. Natl. Acad. Sci. U.S., 54 (1965) 253. G. SZABOLCSI, Acta Physiol. Acad. Sci. Hung., 13 (1958) 213. K. A. TRAYSER AND S. P. COLOWICK, Arch. Biochem. Biophys., 94 (1961) 169. S. GRANICK, J. Biol. Chem., 146 (1942) 451. A. FLECK AND H. N. MUNRO, Clin. Chim. Acta, i i (1965) 2. M. A. WILLIAMS AND P. M. HARRISON, Biochem. J., IiO (1968) 265. S. G. WALE¥ AND J. WATSON, Biochem. J., 55 (1953) 328. R. R. CRICHTON, Abstr. 6th Federation European Biochem. Soc. Meeting, Madrid, 2969, p. 171. T. G. SPIRO, S. E. ALLERTON, J. RENNER, A. TERZlS, R. BILS AND P. SALTMAN, J. Am. Chem. Soc., 88 (1966) 2721. T. G. SPIRO, S. E. ALLERTON, J. RENNER, A. TERZlS, R. BILS AND P. SALTMAN, J. Am. Chem. Soc., 88 (1966) 3147. G. W. BRADY, C. R. KURKJIAN, E. V. X. LYDEN, M. n . ROBIN, P. SALTMAN, T. SPIRO AND A. TERZlS, Biochemistry, 7 (1968) 2185. I. LISTOWSKY, J. J. BETHEIL AND S. ENGLARD, Biochemistry, 6 (1967) 1341. T. HOFMANN AND P. M. HARRISON, J. Mol. Biol., 6 (1963) 256. F. A. FISCHBACH, P. M. HARRISON AND T. G. HOV, J. Mol. Biol., 39 (1969) 235. G. H. HAGGIS, J. Mol. Biol., 14 (1965) 598. G. H. HAGGIS, 6th Intern. Congr. Electron Microscopy, Kyoto, 1966, Vol. 2, Maruzen, T o k y o , 1966, p. 127. L. PAPE, J. S. MULTANI, C. STITT AND P. SALTMAN, Biochemistry, 7 (1968) 6o6. J. W. DRYSDALE AND H. N. MUNRO, J. Biol. Chem., 241 (1966) 363 o. R. A. FINEBERG AND D. M. GREENBERG, J. Biol. Chem., 214 (1955) 97R. A. FINEBERG AND D. M. GREENBERG, J. Biol. Chem., 214 (1955) lO7.
Biochim. Biophys. Acta, 194 (1969) 34-42