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

Abstract

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 ...

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

“…The dimer is predicted to occur at s 2 ~ 24.4 S based on the relationship s 2 / s 1 = ( M 2 / M 1 ) 2/3 for same shaped molecules and using the average s 1 = 15.4 S for the monomer ( vide supra ). This type of sedimentation pattern has been reported previously for solutions of apoHoSF in which the higher molecular weight species at ~ 24 S have been determined to be apoHoSF dimers [56,57,59]. The Gaussian linewidth σ obs = σ shell = 6.5 G for the monomeric species in Fig.…”

“…In our work, s values ranged from 14.3 to 16.7 S for various apoprotein samples prepared from commercially supplied horse spleen ferritin and from protein isolated in our laboratory. This large range reflects oxidative damage to the protein, the tendency of the protein to cross-link to form dimers and higher order oligomers and the inherent heterogeneity associated with the variable subunit composition of the protein itself [49,53,56,57,59,65,76–78]. From s 20, w = 17.0 S, M = 484,120 g/mol and R = 8.3145 × 10 7 erg-K −1 mol −1 , we calculate through $$D_{20,w}=\frac{s_{20,w}·RT}{M·(1-\overline{\nu }{\rho }_{20,w})}$$ Eq.…”

A proteasomal stress response: pre‐treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

“…Apoferritin (Sigma) was run as a standard. Apoferritin ‘monomer’ migrates at 450 kDa with a ‘dimer’ at 900 kDa; Stokes radius of the monomer has been calculated at 16.6S (Bjork 1973; de Haen 1987), consistent with the elution profile of monomer after the proteasome activity peak at 20–26S.…”

Redox Capacity of APO Mammalian Ferritin

“…The heavier band (3-5% of the total) moved at about twice the velocity as the major band (>95%) indicating the presence of a contaminating dimer (18). The sedimentation value of the major band was 15S, a value smaller that typically found (18) for apo MF (16-18s at zero protein concentration), but perhaps is due to the non ideality of these solutions at the high protein concentrations required to monitor protein movement at 400 nm. Taken together, the results strongly indicate that the yellow color is an integral property of apo MF.…”

“…Sedimentation velocity measurements at 48,000 rpm at protein concentrations of 15-25 mg/mI, following protein movement at 400 nm, established two protein sedimentation bands. The heavier band (3-5% of the total) moved at about twice the velocity as the major band (>95%) indicating the presence of a contaminating dimer (18). The sedimentation value of the major band was 15S, a value smaller that typically found (18) for apo MF (16-18s at zero protein concentration), but perhaps is due to the non ideality of these solutions at the high protein concentrations required to monitor protein movement at 400 nm.…”