Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies.
Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit.
Martsev
S P
SP
Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Italy.
Vlasov
A P
AP
Arosio
P
P
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001052:Apoferritins; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D005293:Ferritins; D001052:Apoferritins