An electrophoretical study of ferritin from rainbow trout (Salmo gairdneri, R) liver

Comp. Biochem. PhysioLVol. 106B,No. 4, pp. 937-942, 1993

0305-0491/93$6.00+ 0.00 Pergamon Press Ltd

Printed in Great Britain

AN ELECTROPHORETICAL STUDY OF FERRITIN FROM RAINBOW TROUT (SALMO GAIRDNERI, R) LIVER J. L. MIGUEL and M. I. PABLOS* Departamento de Bioqulmica, Biologla Molecular y Fisiologia, Facultad de Ciencias, Universidad de VaUadolid, 47005, Valladolid, Spain (Received 16 April 1993; accepted 2 June 1993)

Abstract--1. Ferritin from rainbow trout fiver has been characterized by electrophoresis and isoelectrofocusing. 2. Both ferritin and apoferritin subunits migrate upon SDS-PAGE as a single band with a molecular weight around 19 kDa while the polymer shows under native conditions an Mr of 435 kDa. 3. Ferritin and apoferritin and subunits of both of them contain carbohydrates, as revealed by carbohydrate-PAGE-specific stain. 4. Upon isoelectrofocusing ferritin resolves into four microheterogeneous bands with pI between 4.5 and 4.9. 5. This microheterogeneity represents polymorphic and not polymer forms.

INTRODUCTION

The capacity of cells to synthesize ferritin appears to have developed early in evolution and to be universally distributed, so that cells can utilize iron without being exposed to the toxic effects of free ionic iron. This protein has been identified in some vegetable, as well as in animal, species (Linder and Munro, 1972; Diez et al., 1985). Ferritins isolated from different sources may differ by single or multiple properties (pI, electrophoretical mobility, molecular weight, etc.). The generally assumed molecular weight is between 420 and 500 kDa for most tissue ferritin monomers in different animal models. PAGE, as described by Davis (1964), for example, is a useful technique for calculating the molecular weight and qualitatively demonstrating some protein-bound molecules, such as iron or carbohydrates. These atoms or molecules can be revealed by the use of appropiate staining procedures, e.g. Prussian Blue for iron and basic fucsin for carbohydrates. Ferritin can contain two subunit types: light subunit (L), which has a molecular weight around 19 kDa, and heavy subunit (H), which has a molecular weight around 21 kDa. Generally, iron-rich tissues contain higher proportions of L subunits (Munro and Linder, 1978). The molecular weight of these subunits is usually evaluated by SDS-PAGE using the Weber and Osborn (1969) SDS-phosphate continuous buffer system, or the Laemmli discontinu-

ous buffer system (Laemmli, 1970; Laemmli and F/tvre, 1973). Application of the technique of isoelectric focusing has demonstrated a further type of heterogeneity in ferritin, multiple band patterns (microheterogeneity) being observed in ferritins from several organs of human, horse and rat. The individual bands in such patterns are also frequently referred to as "isoferritins" (Russell and Harrison, 1978). The cause of such microheterogeneity is controversial. It has been demonstrated that ferritin molecules are constituted by two genetically distinct subunits and the proportions of these subunits vary from band to band. Others have considered the bands to be artifacts arising from incomplete equilibrium focusing (Crichton and Bryce, 1973), the interaction of the protein with the ampholytes used in IEF, or the existence of a surface charge in the protein (Miguel et al., 1991). Ferritin from rainbow trout liver contains a large proportion of charged amino acids such as glutamic acid, aspartic acid and lysine. Moreover, it contains charged carbohydrate-like sialic acids. Both charged amino acids and carbohydrates can result in surface charge shifts that can explain microheterogeneity (Miguel et al., 1991). This paper characterizes previously isolated ferritin from rainbow trout liver for molecular weight, carbohydrate content and ferritin pI, by IEF. We also demonstrate that isoferritins are not system artifacts and neither are they due to incomplete equilibrium focusing. MATERIALS AND METHODS

*To whom correspondence should be addressed. Abbreviations--PAGE: polyacrylamide gel electrophoresis;

SDS: sodium dodecyl sulphate; pI: isoelectric point; V/hr: volts per hour; F: ferritin; AF: apoferritin; FS: ferritin subunit; AS: apoferritin subunit; IF: isoferritins.

Ferritin isolation

Ferritin isolation was performed as described by Linder and Munro (1972): precipitation by heating of

937

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J.L. MIGUELand M. I. PAaLOS

the initial homogenate is followed by precipitation by pH change and by exclusion chromatography on Sephadex G-200 and Sepharose CL-6B. The eluates were concentrated by ultracentrifugation at 100,000g. All isolation steps were controlled by PAGE in a 7% polyacrylamide gel. Tris-glycine buffer (0.2 M, pH 8.1) was present in both chambers and a current of 8 mA per tube was applied, with Bromophenol Blue for internal migration control. Gels were stained with Coomassie Brilliant Blue or Amido Black for proteins and Prussian Blue reaction for iron. For the last reaction, the gels were immersed in a fresh solution of 2% potassium ferrocyanide in 2% HCI for 30min, washed in distilled water and stored in 2% HC1 (gels are stable for 3 or 4 hr). The amount of applied protein (for this stain) was 2.5/~g/gel.

Apoferritin preparation Apoferritin was prepared by dialysis against three changes of 0.1 M thioglycolic acid/0.1 M sodium acetate at pH 4.4, followed by dialysis against three changes of 0.1 M KC1 and two changes of 0.1 M imidazole, pH 7.0, as described Hoy et al. (Hoy et al., 1974).

Ferritin molecular weight determination Molecular weight determination was performed by PAGE with Tris-glycine buffer at pH 8.3 and with Bromophenol Blue as saline marker. Standards were thyroglobulin, horse ferritin, catalase, phosphorylase b and bovine serum albumin. Staining was performed with Coomassie Brilliant Blue.

Ferritin and apoferritin subunit molecular weight determination Ferritin and apoferritin were boiled at 100°C for 15 min in a solution containing 2% SDS and fl-mercaptoethanol. Molecular weight determination was performed by SDS-PAGE, as described by Weber and Osborn (1969) with sodium phosphate-SDS buffer at pH 7.0. Standards were phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and lactoalbumin. Staining was performed with Coomassie Brilliant Blue.

Carbohydrate qualitative determination Carbohydrate determination was performed by PAGE without saline marker and with Tris-glycine buffer at pH 8.3. Carbohydrate-specific stain was by Schiff's reagent and basic fucsin, as described Zacharius et al. (1969).

Carbohydrate quantitative determination Purified ferritin was hydrolysed by boiling in 2% HC1 solution for 30rain in a bulb. After, it was passed through a C18-Pak column, and detected in a Waters HPLC, with a Sugar-Pak column, which detected hexoses and hexosamines. Sialic acids were

determined by the modified resorcinol procedure as used by Cabezas et aL (1964).

Ferritin isoelectric point determination IEF in thin layers of polyacrylamide gels at 5% (w/v) with 5% ampholyte (w/v) was performed in the pH range 3.5-10 (Ampholines, LKB Produkter, Bromma, Sweden). An LKB Multiphor apparatus was used for the focusing, with an LKB 2197 Constant Power Supply unit. The gel was maintained at 15°C. The gel was prefocused for 130V/hr and was run at 3300 V/hr. The gradient pH was determined at 15°C using a contact electrode and an IEF Calibration Kit (Pharmacia, Uppsala, Sweden). Immediately after this, the gels were fixed for 1 hr in an aqueous solution of 10% trichloracetic acid, 5% sulphosalieylic acid (w/v); washed for 30min in aqueous solution of 25% methanol, 5% acetic acid (v/v); stained during 20-30 min in 0.1% Coomassie Blue G-250 in aqueous solution of 25% methanol, 5% acetic acid (w/v) or in potassium ferrocyanide; destained in an aqueous solution of 25% methanol, 5% acetic acid (v/v) until the background was clear; and equilibrated for 1 hr in 5% glycerol, 25% methanol, water (v/v). Finally, a cellophane sheet soaked in preserving solution, was applied to the surface of the gel. The plates were then dried at room temperature. RESULTS AND DISCUSSION

Holoprotein-staining (Fig. la) and iron-staining (Fig. I b) P A G E was carried out during several steps of the purification procedure to determine the location and purity of the ferritin. Pure band apoferritin (lane V, Fig. la) and ferritin (lane IV, Fig. la and lane IV, Fig. lb) showed a major, fast-moving band, together with minor, slow-moving bands. The major band is generally accepted as consisting of monomeric ferritin molecules, and the minor ones, as consisting of dimers. Sometimes, a third band, consisting of trimer molecules, was observed. Pure apoferritin and ferritin showed the same electrophoretic behaviour (Fig. lb, lanes IV and V). PAGE was used to determine ferritin molecular weight. Ferritin electrophoresis were performed with standards which were represented forming a calibration line (logarithm of relative mass vs mobility relative, Rf) as seen in Fig. 2(b). A ferritin molecular mass was obtained of between 425 and 435 kDa. The generally accepted molecular weight is between 420 and 500 kDa for most tissue ferritin monomers in different animal models. The evidence of a molecular weight of around 430 kDa for rainbow trout liver ferritin is agreement with previous reports. The similar electrophoretical behaviour of ferritin and apoferritin may indicate that the presence or absence of iron does not influence this behaviour. The lack of difference in migration between ferritin and apoferritin may indicate a similar net charge, not a similar

Ferritin in rainbow trout liver

939

I II III IV V I U III IV Fig. 1. PAGE at different purification steps of trout liver ferritin. Figure l(a) shows PAGE with protein stain of initial homogenate (lane I), cooled extract after heating at 70°C (lane II), change of supematant pH (lane III), ferritin purified by exclusion chromatography and ultracentrifugation at 100,000 g (lane IV) and apoferritin (lane V). Figure 1(b) shows iron stain for the same samples as in Fig. l(a) except apoferritin (this protein's lack of iron).

molecular weight. Really, when full, holoferritin can accommodate about 4500 Fe atoms as ferric oxyhydroxide, thereby doubling the molecular weight to 900,000 Da. Most ferritins are composed of two subunit types which have a molecular weight of between 19 and 21 kDa. Both ferritin and apoferritin subunit molecular weight determination by S D S - P A G E yield only one electrophoretical band with a molecular weight of around 19 kDa. Figure 3(a) shows the standards and ferritin subunit and in Fig. 3(b) a plot of the logarithm of standard molecular weight vs Rf. This

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evidence is in agreement with previous reports which show that the L subunit is the most abundant compound in ferritins from iron-rich tissues (Munro and Linder, 1978). Similar data have been obtained in ferritins from molluscs and bean seeds which contained only one subunit. Molluscs had two ferritin forms and one of them contained only one subunit type, with a molecular weight of around 19 kDa (Bottke, 1985), while bean seed ferritin contained only a 26 kDa subunit (Van der Mark and Van der Briel, 1985). Ferritin, apoferritin and subunits of both contain

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. . . . 0"5 ' ' ' ' 1'.0 FS AS at Fig. 3. Relative mass (Da) evaluation of ferritin and apoferritin subunits. Both ferritin and apoferritin subunits were dissociated in 2% SDS and 1% p-mereaptoethanol at 100°C over 15 min. Figure 2(a) shows SDS-PAGE of standards (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and lactalbumin) and both ferritin, and apoferritin subunits. Figure 3(b) shows standard calibration line (logarithm of standards relative mass vs Rf).

carbohydrates which have been qualitatively characterized by PAGE and Schiff's reagent stain as shown Fig. 4. It can be seen that ferritin and both apoferritin and ferritin subunits contain neutral and charged carbohydrates which can influence charge microheterogeneity, as well as participate in iron-linking to ferritin, contribute to the acidic isoelectric point (sialic acids) and extend protein time-life. Both ferritin and apoferritin contain 4.4 mol sialic acids, 16.5 mol hexosamines and 130 mol hexoses per

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mol protein. An HPLC technique was used to differentiate hexose types. We have used two column types (Sugar-Pak and Carbohydrate Analysis Columns) as the Sugar-Pak column was not able to differentiate between glucose and mannose. Both ferritin and apoferritin contain 86 mol glucose, 24mol fucose, 12 mol galactose, and 8 mol mannose per mol protein. These data represent a proportion of 5.4% (w/w) for ferritin of the trout liver. This percentage is the greatest found in ferritins from different sources except for ferritin from human heart which has a 5.1% (w/w), similar to our results (Munro and Linder, 1978). IEF in thin layers of polyacrylamide gels can be observed in Fig. 5. Figure 5(a) shows photography of gel slabs with two kinds of stain. The gel slab in Fig. 5(a)-II corresponds to the pattern of pI standards, whose migration distance is superimposed in Fig. 5(b)-II on the gradient course, as plotted for pH reading with a surface electrode. "Isoferritins" appear with a pI between 4.5 and 4.9. These isoferritins exist due to charged amino acids, and the presence of carbohydrates (overall sialic acids). Incomplete equilibrium focusing in the system is not the cause of isoferritin existence because the pH calibration line is practically coincident with standard calibration lines. On the other hand, we think that isoferritins are not system artifacts, because in Fig. 5(a) lane I, the gel slab has been stained with an iron-specific stain and isoferritins appear in the same pH range and with the same number of bands. The observed pI values are in agreement with results obtained from dolphin spleen, whose ferritin has a pI between 4.2 and 4.6 (Kato, 1970), and frog with a pI around 4.9 (Brown and Theil, 1978). The observed pI is more acidic than those obtained for

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Fig. 5. IEF in thin-layer polyacrylamide gel of ferritin. IEF was performed in thin layers with 5% polyacrylamide and 5% ampholyte in the pH range 3.5-10. Gel was prefocused at 130 V/hr and was run at 3300 V/hr. The pH gradient course was measured with a surface electrode and the distance from anode of every proteins was determinated (ferritin and standards). Figure 5(a) shows in lane I IEF of ferritin after iron-specific stain (Prussian Blue), and in lane II IEF of standards with protein-specific stain (Coomassie Blue). The curves in Fig. 5(b) representing the pH course as evaluated by pH measures (continuous line) and for the focusing position of pI markers (break line) are completely superimposed.

other ferritins (for example, horse: Russell and Harrison 1978; human: Munro and Linder, 1978; and rat: Egachi, 1980) and which do not coincide with the molecule used in this study. Significant differences were not found in the isoelectrophoretic behaviour between iron-poor and iron-rich ferritin. The high percentage of aspartic and glutamic acid and the presence of sialic acids (Miguel et al., 1991)justify the acid isoelectric point. Regarding its isoelectrophoretic behaviour, it may be concluded that trout liver ferritin exhibits heterogeneity in polyacrylamide gel in a pH gradient. The heterogeneity demonstrated points to the existence of polymorphic forms but not of polymer forms. Polydispersions are considered to be due to molecules with different surface charges. The variations in ferritin surface charge may simply be accounted for by variations in side-chain pK values, resulting from conformational differences. Perhaps the heterogeneity of this molecules is also explained by the presence of a large amount of carbohydrates and by the presence of sialic acids (Miguel et al., 1991), an argument utilized to account for the heterogeneity of serum ferritin by Cragg et al. (1980). REFERENCES

Bottke W. (1985) Electrophoretic and immunologic studies on the structure of a mollusk ferritin. Comp. Biochem. Physiol. 81B,(2) 325-334. CBPB 106/4--L

Brown J. E. and Theil E. (1978) Red cells, ferritin, and iron storage during amphibian development. J. biol. Chem. 258,(8) 2673-2678. Cabezas J. A., Porto J. V., Frois M. D., Marino C. and Arzua J. (1964) Acide sialiquv dans les larmes humaines. Biochim. biophys. Acta 83, 318-325. Cragg S. J., Wagstaff M. and Worwood M. (1980) Sialic acid and the microheterogeneityof human serum ferritin. Clin. Sci. 58,(3) 259-262. Crichton R. R. and Bryce C. F. A. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 344-346. Davis D. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. Y. Acad. Sci. 121, 404--427. Diez J. M., Agapito M. T. and Recio J. M. (1985) Isolation and purification of duck liver ferritin. Rev. Esp. Fisiol. 41, 341-344. Egachi Y. (1980) Purification and microheterogeneities of rat tissue ferritins. Nippon Ika Daigaku Zasshi. 47,(3) 302-314. Hoy T. G., Harrison P. M. and Shabbir M. (1974) Uptake and release of ferritin iron. Biochem. J. 139, 603-610. Kato T. (1970) Isolation and properties of fcrritin from dolphin (Delphinus cetacea) spleen. J. Biochem. 68,(5) 681-687. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Laemmli U. K. and Ffivre M. (1973) Maturation of the head of bacteriophage T4. J. molec. Biol. 80, 575-599. Linder M. C. and Munro H. N. (1972) Assay of tissue ferritin. Analyt. Biochem. 48, 266-278. Miguel J. L., Pablos M. I., Agapito M. T. and Recio J. M. (1991) Isolation and characterization of ferritin from the

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liver of the rainbow trout (Salmo gairdneri R). Biochem. Cell Biol. 69, 735-742. Munro H. N. and Linder M. C. (1978) Ferritin: structure, biosynthesis and role in iron metabolism. Physiol. Rev. 58,(2) 317-396. Russell S. M. and Harrison P. M. (1978) Heterogeneity in horse ferritins. Biochem. J. 175, 91-104. Van der Mark F. and Van der Briel W. (1985) Purification and partial characterization of ferritin from normal and

iron-loaded leaves of Phaseolus vulgaris. Plant Sci. 39,(1) 55-60. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulphatepolyacrylamide gel electrophoresis. J. biol. Chem. 244,(16) 4406-4412. Zacharius R. M., Zell T. E., Morrison J. H. and Woodlock J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gel. Analyt. Biochem. 30, 148-153.