Röntgenkleinwinkeluntersuchung von Apoferritin in Lösung

Bielig, Hans‐Joachim; Kratky, O.; Rohns, G.; Wawra, H.

doi:10.1016/s0040-4039(00)71717-5

Cited by 15 publications

(9 citation statements)

References 14 publications

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“…It is obvious that if apoferritin has true spherical shape, the solvent inside the shell can account for only a small part of this hydration and that a larger amount of water bound to the surface of the protein must be assumed. This has been suggested by Fishbach and Anderegg [32] and by Bielig et al [33] on the basis of low-angle X-ray scattering data, although these two papers disagree somewhat on the amount of water bound. Alternatively, apoferritin may not be truly spherical, but may have a slightly extended shape not detected by the methods referred to earlier.…”

Section: Discussionmentioning

confidence: 75%

“…These solvent molecules trapped inside the shell will of course be part of the hydration contributing to the hydrodynamic properties of the protein. An estimation from the size of the hollow center (about 7.0 nm in diameter, see [32,33]) of the maximal amount of solvent trapped in this way gives a value of 0.23 g/g protein. The amount of hydration calculated from either the frictional ratio or the intrinsic viscosity of apoferritin, assuming a spherical shape of the protein, is about 0.8 g/g protein.…”

Section: Discussionmentioning

confidence: 99%

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Association‐Dissociation Behaviour and Hydrodynamic Properties of Apoferritin Monomer and Dimer

Björk

1973

European Journal of Biochemistry

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The association behaviour of a homogeneous preparation of apoferritin has been investigated. Contrary to studies by other authors on less pure material, no evidence for a reversible association of the protein was detected in sedimentation equilibrium, light scattering or sedimentation velocity experiments. Correspondingly, no dissociation of an isolated dimer of apoferritin was observed. Hydrodynamic parameters, such as frictional ratio and intrinsic viscosity, indicate that both apoferritin monomer and dimer have a somewhat larger degree of hydration or a slightly more extended or irregular shape than most typical globular proteins. The solvent, presumably trapped inside the protein shell, cannot alone account for this behaviour.Apoferritin, which is the iron-free form of ferritin, has been shown t o be a multi-subunit protein, consisting of 24 identical subunits Recent observations indicated that the conclusion drawn by Richter and Walker [3] may be erroneous [2], and for this reason it was decided to investigate the phenomenon more closely. Careful studies of a homogeneous preparation of apoferritin monomer (or-apoferritin) revealed no indications of a reversible association behaviour, i.e. the protein behaved normally throughout the pH range (5.5-8.0) and concentration range (0.1 -10 mg/ml) studied. Moreover, it was possible to isolate and characterize the apoferritin dimer supposedly involved in the equilibrium and it was found that this protein behaved as a stable entity, showing no detectable signs of dissociation. In addition to these investigations a hydrodynamic characterization of apo ferritin monomer and dimer is presented. MATERIALS AND METHODSHorse spleen ferritin (2 x crystallized) was obtained from Miles laboratories Inc. (Kankakee, Ill., U.S.A.) or was prepared essentially by the method developed by Behrens and Taubert [ 121. Apoferritin was prepared by a modification of method 2 described by Granick and Michaelis [13]. 0.5-0.8 g ferritin in 50 ml 0.1 M acetate buffer pH 4.6, was reduced with 1.6-2.0 g sodium dithionite for 1 h a t room temperature. Excess dithionite was removed by rapid gel chromatography on a column (5 ~4 5 cm) of Sephadex 6-25 (Pharmacia Ltd, Uppsala, Sweden) a t 4 "C. The protein peak was concentrated to 50 ml by ultrafiltration and reduction was repeated. This time, 50 mg a,&'-bipyridyl were added after 1 h and excess reagents were again removed by gel chromatography after another 30-60 min. The apoferritin peak was concentrated to 6-7 ml by ultrafiltration and centrifuged for 1 h a t 110000 x g in rotor 40.2 in a Spinco ModelL preparative ultracentrifuge (Beckman Instruments, Palo Alto, Calif., U.S.A.).The supernatant was further purified by gel chromatography on 60/, agarose. About 100mg of apoferritin in 3 to 5 ml solution was applied to a column (2.5 x 95 cm) of Sepharose 6-B (Pharmacia Ltd, Uppsala, Sweden) in 0.02 M acetate buffer pH 5.5, containing 0.1 M NaC1. The column was eluted a t a rate of 20 ml/h and 5-ml fractions were collected.Analytical polyacrylamide-gel ...

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Association‐Dissociation Behaviour and Hydrodynamic Properties of Apoferritin Monomer and Dimer

Björk

1973

European Journal of Biochemistry

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“…Similar effective magnetic moments have been reported by Schoffa (1965), for ferritin, and by Schugar et al (1967) for solutions of the synthetic iron core polymer, and were interpreted by them as showing depressed magnetic moments due to dimer spin pairing. However, as Blaise et al (1965) have shown, such a measurement on a superparamagnetic material is of very little value unless combined with a thorough magnetic field dependence study of the magnetization. Thus, Blaise et al arrived at a moment of 5.08 BM/iron in their comprehensive study of ferritin, to be compared with only 3.85 BM obtained by Schoffa from a plot like that of Figure 4.…”

Section: I88mentioning

confidence: 99%

“…The nitrate ions are not coordinated directly to the iron, but act only as counterions to the positively F erritin is an iron-carrying protein known to consist of an inner micelle of hydrated ferric oxide-Fhosphate, having a maximum diameter of about 75 A which is surrounded by an outer proteinaceous sheath (apoferritin) having an inner diameter of 75 A and an outer diameter of approx;mately 120 A (Harrison, 1964;Bielig et a/., 1966). Though the apoferritin shell may be filled with varying amounts of the iron hydroxidephosphate, at maximum capacity, the core amounts to about 2000 iron atoms (Fischhach and Anderegg, 1965).…”

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confidence: 99%

The structure of an iron core analog of ferritin

Brady¹,

Kurkjian²,

Lyden³

et al. 1968

Biochemistry

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“…In fact, its structure is that of a hollow sphere with an inner diameter of 74A and an outer diameter of 122A (Bielig, Kratky, Rohns & Haward, 1966;Fischbach & Anderegg, 1965;Stuhrmann, Haas, Ibel, Koch & Crichton, 1976). In fact, its structure is that of a hollow sphere with an inner diameter of 74A and an outer diameter of 122A (Bielig, Kratky, Rohns & Haward, 1966;Fischbach & Anderegg, 1965;Stuhrmann, Haas, Ibel, Koch & Crichton, 1976).…”

Section: Introductionmentioning

confidence: 99%

A new polarized target for neutron scattering studies on biomolecules: first results from apoferritin and the deuterated 50S subunit of ribosomes

Knop¹,

Hirai²,

Schink³

et al. 1992

J Appl Cryst

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A new polarized target for neutron scattering has been designed by CERN and tested successfully using the reactor FRG‐1 at the GKSS Research Centre. The nuclear spins are aligned with respect to the external field – parallel or antiparallel – by dynamic nuclear polarization (DNP). To avoid absorption of neutrons by 3He, the frozen solutions of biomolecules are immersed in liquid 4He which in turn is thermally coupled to the cooling mixture of 3He/4He of the dilution refrigerator. Compared with earlier experiments where the sample had been cooled directly by 3He, the rate of detectable neutrons increased by a factor of 30. Another factor of 30 is due to the installation of the cold source and the beryllium reflector in FRG‐1. Polarized neutron scattering from apoferritin in deuterated solvent shows that the proton spin polarization is homogeneous in apoferritin molecules. After saturation of proton nuclear magnetic resonance (NMR), polarized neutron scattering is dominated by deuteron spin contrast. With the deuterated large subunit of E. coli ribosomes, three different basic scattering functions are derived from spin‐contrast variation, reflecting the known scattering‐length‐density distribution of the architecture of rRNA and ribosomal proteins. The planned in situ structure determination of a mRNA fragment is discussed in the light of the present results.

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Röntgenkleinwinkeluntersuchung von Apoferritin in Lösung

Cited by 15 publications

References 14 publications

Association‐Dissociation Behaviour and Hydrodynamic Properties of Apoferritin Monomer and Dimer

Association‐Dissociation Behaviour and Hydrodynamic Properties of Apoferritin Monomer and Dimer

The structure of an iron core analog of ferritin

A new polarized target for neutron scattering studies on biomolecules: first results from apoferritin and the deuterated 50S subunit of ribosomes

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