Báo cáo Y học: Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M-type homopolymer

Eur. J. Biochem. 269, 1600–1606 (2002) Ó FEBS 2002 Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M-type homopolymer Guiseppina Mignogna1, Roberta Chiaraluce1, Valerio Consalvi1, Stefano Cavallo1, Simonetta Stefanini1 and Emilia Chiancone1,2 1Department of Biochemical Sciences and 2CNR, Center of Molecular Biology, Department of Biochemical Sciences ‘A. Rossi Fanelli’, University of Rome ‘La Sapienza’, Italy FerritinfromthespleenoftheAntarcticteleostTrematomus bernacchiiiscomposedofasinglesubunitthatcontainsboth the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleationsite,typicalofmammalianL chains.Comparison of the amino-acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M-type homopolymer. Interestingly, the T. bernacchiiferritinchainshows85.7%identitywithacold-inducibleferritinchainoftherainbowtroutSalmogairdneri. The structural and functional properties indicate that cold acclimation and functional adaptation to low temperatures Several molecular adaptation mechanisms have been devel-oped by living organisms under extreme environmental conditions[1].Inmanycases,coldadaptationisachievedby modification of the structural and functional properties of proteins [2]. It follows that the correlation between the physicochemicalpropertiesofproteinsandcoldacclimation is particularly attractive for molecules that are highly thermostable [3]. This is the case for ferritin, the ubiquitous iron-storage protein, which is characterized by high thermal and chemical stability in all mesophilic species [4]. Ferritins areabletosequesterandstoreironinasolubleandavailable formtherebyprotectingtheorganismagainstthetoxiceffect of ÔfreeÕ iron. The extremely stable quaternary structure of the ferritin molecule is highly conserved. It consists of a hollow 24-mer protein shell, apoferritin (molecular mass 480 kDa), the cavity of which can accommodate up to 4500 iron atoms as an inorganic micellar core [4]. Mammalian ferritins are heteropolymers of two genetic-ally distinct subunits, L and H, of similar sequence, molecular mass (19–21 kDa, respectively) and with the same four-helix-bundle tertiary conformation. The ferritin subunits are expressed in different proportions in various cellsandtissues[5].Thus,L-richcopolymerspredominatein spleen and liver, which have an iron-storage function, whereas H-rich ferritins are found in other tissues such as Correspondence to E. Chiancone, CNR Center of Molecular Biology, Department of Biochemical Sciences ÔA. Rossi FanelliÕ, University of Rome ÔLa SapienzaÕ, P.le A. Moro, 5, 00185 Roma, Italy. Fax: + 39 06 4440062, Tel.: + 39 06 49910761, E-mail: emilia.chiancone@uniroma1.it Abbreviation: CHCA, a-cyano-4-hydroxycinnamic acid. (Received 28 September 2001, revised 30 November 2001, accepted 3 January 2002) are achieved without significant modification of the protein stability. In fact, the stability of T. bernacchii ferritin to de-naturationinducedbyacidortemperaturecloselyresembles that of mesophilic mammalian ferritins. Moreover iron is taken up eciently and the activation energy ofthereaction is 74.9 kJÆmol)1, a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJÆmol)1). Keywords: amino-acid sequence; cold adaptation; iron incorporation;stability;TrematomusbernachiiAntarcticfish ferritin. heart and kidney, which do not [6]. Accordingly, the H and L subunits have distinct and complementary functions. The H chains contain in the four-helix bundle a dinuclear ferroxidasecenter,whichpromotestheoxidationofFe2+ in the presence of molecular oxygen [7]. The iron ligands are highly conserved and are provided by residues E27, E61, E62, H65, E107 and Q141 [7]. The L chains lack such a center, but contain specific carboxylic groups (E57, E60, and E64 using the H-chain numbering) facing the inner surface of the apoferritin shell, that provide efficient nucleation sites for iron accumulation [8]. Ferritins from lower vertebrates have received relatively little attention. In amphibians, specifically in bullfrog tadpole erythrocytes, the occurrence ofthree distinct ferritin cDNAsandtheircell-specificexpressionhasbeendescribed. The corresponding subunits were named H (heavy), M (middle) and L (light) as they show distinct mobilities in denaturing gels [9]. With respect to the sequence elements of functional importance, the L chain contains the three negatively charged residues (E57, E60, and E64) responsible for iron nucleation and mineralization in the mammalian protein. The H and M chains, although differing in sequence and molecular mass, contain all the ligands of the ferroxidase center and, in addition, two out of the three carboxylic residues typical of mammalian L chains (E57 and E64, E60 is replaced by a histidine). Infishferritins,evidencefortwosubunitswasobtainedby screening of a liver cDNA library in the Atlantic salmon Salmosalar[10].Asintadpoleferritin,theHandMsubunits contain both the ligands typical of the H chain ferroxidase center and the canonical L chain carboxylate residues in positions 60 and 64. The canonical L chain glutamate residue in position 57 is present only in the M chains and is substituted by an asparagine in the H chains [8]. It is noteworthy that S. salar ferritin displays a different pattern Ó FEBS 2002 Homopolymeric M-type T. bernacchii ferritin (Eur. J. Biochem. 269) 1601 of subunit expression relative to mammalian ferritins. Thus, H chains predominate in spleen and liver at variance with thepresenceofL chainsinthesameorgansofmammals[6]. Interestingly, a study of cold-inducible gene expression of rainbow trout cells (Salmo gairdneri) revealed that the transcription and accumulation of the mRNA correspond-ing to three ferritin H isoforms H1, H2 and H3 is enhanced [11]. In turn, the induction of ferritin H expression during cold acclimation may suggest that this ferritin is particularly apt to function at low temperatures. This study was undertaken to characterize ferritin from an Antarctic fish and thereby establish whether cold adaptation affects the structural–functional properties of this protein. Ferritin extracted from the spleen of the Antarctic teleost Trematomus bernacchii, which lives at a constant temperature of )1.9 °C, was chosen. To our knowledge only spleen ferritin from another Antarctic teleost, Gymnodraco acuticeps, has been partially character-ized; it is an H-type homopolymer, as indicated by the N-terminal amino-acid sequence, that is able to accumulate iron as an L-rich mammalian ferritin molecule [12]. The results show that native T. bernacchii ferritin is a homopolymer with a high iron content ( 2500 iron atoms per molecule) and a high ferroxidase activity. The amino-acid sequence of the constitutive subunit shows a high similarity to one of the cold-inducible chains of S. gairdneri ferritin; like this chain, it contains the functional residues characteristic of both mammalian L and H chains. The molecular adaptation essential to function at low tempera-ture is not accompanied by a significant modification of the protein stability to chemical and physical denaturants with respect to the mesophilic proteins. MATERIALS AND METHODS Enzymes and chemicals were purchased from the following suppliers: Asp-N endoproteinase and trypsin from Roche Diagnostics Corporation; pepsin and 4-vinylpyridine from Sigma; CNBr from Fluka; guanidinium chloride (recrystal-lized from methanol) from Merck; the liquid chromatogra-phy solvents, HPLC-grade, from Carlo Erba Reagenti; sequence-grade chemicals from Applied Biosystems. Purification and characterization of T. bernacchii ferritin Specimens of T. bernacchii were sampled from Terra Nova Bay Station, Ross Sea; the spleens were immediately removed and frozen at )80 °C until use. Spleen ferritin was purified following the procedure described previously [12]. Iron was removed from the native protein, which containsabout2500ironatomspermolecule,byincubation for 24 h in 0.5 M acetate buffer, pH 4.8, containing 1% (w/v) sodium dithionite and subsequent chelation of Fe2+ with 2,2¢-bipyridyl. The concentration of apoferritin was determined from the A280 using an absorption coefficient (e1%,1 cm ˆ 6.5) calculated as described by Gill & Von Hippel [13]. Analysis of amino-acid sequence Theproteinsample(1.5 mg)wassuspendedin0.5 mL0.5 M Tris/HCl, pH 7.5, containing 2 mM EDTA, 4 M guanidi-niumchlorideand12 lmoldithiothreitol,andincubatedfor 3 h at 55 °C. Thereafter, 4-vinylpyridine (90 lmol, 10 lL) was added, and, after 10 min incubation, the protein was desalted by HPLC using a guard cartridge (C8, 4.6 mm · 30 mm). An aliquot (0.5 mg) of the denaturated pyridylethylated protein was dissolved in 0.2 mL 80% (v/v) trifluoroacetic acid, incubated in the dark with 4 mg CNBr for 24 h at room temperature, and lyophilized. A second aliquotofprotein(0.5 mg)wassuspendedin0.5 mL10 mM Tris/HCl, pH 7.5, containing 10% acetonitrile, and incuba-ted at 37 °C overnight after the addition of 4 lg Asp-N endoproteinase. A third aliquot (0.3 mg) was dissolved in 0.2 mL 5% (v/v) formic acid, and incubated with 6 lg pepsin at 25 °C for 5 min. The peptide mixtures obtained after enzymatic digestions were purified immediately after the incubation with proteases, without lyophilization. The peptide mixtures were purified by HPLC using a BeckmanSystemGoldchromatographeronamacroporous reversed-phase column (C8208TP52; 4.6 mm · 250 mm; 5 lm Vydac; Esperia, CA, USA). They were eluted with a linear gradient from 0 to 35% acetonitrile in 0.2% (v/v) trifluoroacetic acid at a flow rate of 1.0 mLÆmin)1. Elution of the peptides was monitored using a diode array detector (Beckman model 168) at 220 and 280 nm. The amino-acid sequence of peptide samples was deter-mined by automated Edman degradation using an Applied Biosystems model 476A sequencer. Samples (0.1–0.5 nmol) were loaded on to poly(vinylidene difluoride) membranes (ProBlott;AppliedBiosystems),coatedwith2 lLpolybrene (100 mgÆmL)1; 50% methanol), and run with a Blott cartridge using an optimized gas-phase fast program. N-Terminal sequence analysis of the protein was per-formed on samples (5 lg) electrotransferred on ProBlott membranes after SDS/PAGE [14], using a liquid-phase fast program. Peptides were numbered retrospectively according to their location in the sequence, starting from the N-terminus. CNBr peptides were designated with B, Asp-N peptides with A, and peptic peptides with P. MS analysis Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) measurements were performed using a Voyager-DE (Applied Biosystems) mass spectrometer. Solutions(1 lL)containingpeptides(1–5 pmol)weremixed with 1 lL of the matrix solution: 30% aqueous solution of acetonitrile and 0.1% trifluoroacetic acid saturated with a-cyano-4-hydroxycinnamicacid(CHCA)or2,5-dihydroxy-benzoic acid dissolved in water. The mixture of peptide and matrix was placed on the MALDI stainless-steel plate and allowed to dry spontaneously. Ions were generated by irradiating the sample area with a nitrogen laser at a wavelengthof337 nm.Calibrationswerecarriedoutusinga mixture of angiotensin I (1297.51 MH+), adrenocortico-tropic hormone ACTH (clip 1–17) (2094.46 MH+), ACTH (clip 18–39) (2466.72 MH+), ACTH (clip 7–38) (3660.19 MH+) and bovine insulin (5734.59 MH+) (SequazimeTM Peptide Mass Standards kit; Applied Biosystems). Mass analysis of the N-terminal-blocked peptide Analiquot(10 lg)ofpyridylethylatedproteinwasdissolved in 50 lL 50 mM NH4HCO3, pH 8.5, and incubated at 1602 G. Mignogna et al. (Eur. J. Biochem. 269) 37 °C overnight after addition of 1 lg trypsin. The peptide mixture was desalted using the ZipTipC18 (Millipore) and then mixed with the matrix solution (a-cyano-4-hydroxy-cinnamic acid) for MALDI-TOF MS analysis. Structure comparison A search of the SwissProt-TrEMBLE database, pairwise and multiple sequence alignments, and prediction of secon-dary structures were carried out with the programs from EXPASY (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). Iron incorporation experiments Ironincorporationexperimentswereperformedbyaddition of a freshly prepared anaerobic solution of ferrous ammo-nium sulfate to an air-equilibrated solution of apoferritin. T. bernacchii and human recombinant apoferritins were used in parallel experiments. Human recombinant H homopolymer (100% H subunit) was overexpressed in Escherichia coli and purified essentially as described by Levi et al. [15]. The kinetics of iron oxidation and uptake were followed at the desired temperature measuring the absorbance of the ferric oxide hydrate micelle at 310 nm using the absorption coefficient of ferritin iron, e1%,1 cm ˆ 450 [16]. As a control, the rate of Fe autoxidation was measured in parallel. To assess iron incorporation, at the end of the reaction the sampleswereanalyzedbynondenaturinggelelectrophoresis (stainingwithPrussianblueforironandCoomassiebluefor protein) and by sedimentation velocity in a Beckman Optima XL-A analytical ultracentrifuge at 49 000 g and 10 °C. The sedimentation coefficients were reduced to s20, w by standard procedures. Analysis of the state of association The state of association was analysed by size-exclusion chromatography experiments at 20 °C on a Superose 12 column (Pharmacia) eluted with 20 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl at a flow rate of 0.5 mLÆmin)1 controlled by a Dionex gradient pump. After 24 h incubation at pH 1.5–4.0, the samples were diluted 20-fold into the column injection loop. The Superose column was calibrated with horse spleen apoferritin (440 kDa, elution volume Ve ˆ 7.8 mL), rabbit muscle aldolase (161 kDa, elution volume Ve ˆ 9.2 mL), horse liver alcohol dehydrogenase (80 kDa, elution volume Ve ˆ 9.8 mL), BSA (66 kDa, elution volume Ve ˆ 10.0 mL), ovalbumin (45 kDa, elution volume Ve ˆ 10.3 mL), and cytochrome c (12 kDa, elution volume Ve ˆ 12.4 mL). pH-dependence experiments T. bernacchii ferritin (0.03–1.2 mgÆmL)1) was incubated for 24 h at 20 °C at pH 1.5 (31.6 mM HCl), pH 2.0 (10.0 mM HCl), pH 2.5 (3.2 mM HCl), pH 3.0 (1.0 mM HCl) and pH 2.0 in the presence of 31.6 mM NaCl (pCl 1.5). The pH of the solutions was measured with an InLab 422 electrode (Mettler–Toledo AG) connected to a Corning P507 ion meter before and after the addition of the protein. After 24 hincubationat20 °C,thesampleswereanalyzedbyCD, Ó FEBS 2002 fluorescence spectroscopy, and size exclusion chromato-graphy. Spectroscopic methods Intrinsicfluorescenceemissionandlight-scatteringmeasure-ments were carried out with an LS50B PerkinElmer spectrofluorimeter using a 1-cm pathlength quartz cuvette. Intrinsic fluorescence emission spectra were recorded at 300–400 nm (1 nm sampling interval) with the excitation wavelength set at 295 nm. Light scattering was measured withbothexcitationandemissionwavelengthsetat480 nm. CD spectra were recorded on a Jasco J-720 spectropolari-meter. Far-UV (190–250 nm) and near-UV CD (250– 310 nm) measurements were performed in a 0.1-cm and 1.0-cm pathlength quartz cuvette, respectively. The results are expressed as mean residue ellipticity ([Q]) assuming a mean residue weight of 110 per amino-acid residue. All the spectroscopic measurements were performed at 20 °C. Thermal denaturation For thermal scans, the protein samples (0.06 mgÆmL)1) in 20 mM sodium phosphate at pH 7.0 and in 40 mM glycine/ HCl at pH 4.0 were heated from 10 to 95 °C and subsequently cooled to 10 °C with a heating/cooling rate of 1 degreeÆmin)1 controlled by a Jasco programmable Peltier element. Far-UV CD spectra were recorded every 5 or 2.5 °C, and the dichroic activity at 222 nm was monit-ored continuously every 0.5 °C with 4 s averaging time. All thespectrawerecorrectedforthesolventcontributionatthe different temperatures and pH values examined. The melting temperatures were determined by taking the first derivative of the ellipticity signal at 222 nm with respect to temperature. RESULTS Determination of amino-acid sequence The complete sequence of the single subunit that gives rise to T. bernacchii ferritin is reported in Fig. 1. The subunit contains 176 amino-acid residues. The sequence was deduced after the isolation and identification of an almost completesetofCNBrpeptides,whichwereorderedwiththe helpofoverlappingpeptidesproducedbyAsp-Nandpepsin cleavage. The sequence of each peptide was confirmed by MS analysis. The automated Edman degradation of the native protein was unsuccessful for the possible presence of a blocked N-terminus. MALDI-TOF MS analysis of a tryptic digest of the protein indicated for the N-terminal peptide MDSQVR a value of m/z 777, which points to the presence of acetylmethionine. This residue is commonly found in the N-terminus of eukaryotic proteins together with N-acetyl Ala, Ser, Gly and Thr [17]. Secondary-structure prediction, performed as described by Rost [18] shows the presence of four ahelices in the regions corresponding to positions 8–40, 45–75, 94–120 and 128–158 (Fig. 2). This four-helix pattern is analogous to the four-helix-bundle characteristic of mammalian ferritin [4]. A search in the SwissProt-TrEMBLE Database with the T. bernacchii ferritin as a probe retrieved many ferritin sequences. The alignment, obtained using the program Ó FEBS 2002 Homopolymeric M-type T. bernacchii ferritin (Eur. J. Biochem. 269) 1603 Fig. 1. Complete amino-acid sequence of T. bernacchii ferritin. The extent of the various fragments used to reconstruct the sequence is shown. B, CNBr peptides; A, Asp-N peptides; P, peptic peptides. Ac-M, acetylmethionine. CLUSTALW, is reported in Fig. 2 where, for the sake of simplicity, only the human L and H chains are shown to represent mammalian ferritins. The percentage identity among the various sequences ranges from 87.5 to 59, the latter value pertaining to human L chains. The most similar totheT. bernacchiiconstitutivechainaretheH2chainfrom S. gairdneri (87.5%) and the M chains from S. salar (86.9%) and Gillichthys mirabilis (78.7%). The percentage identity for the H chains from S. salar and Oncorhynchus nerka is significantly lower (70.5 and 70.4, respectively).Despitethepaucityofavailablesequencedata, it appears that T. bernacchii ferritin can be classified as an M homopolymer and that the H2 chain from S. gairdneri should be likewise considered an M chain. The amino-acid residues of functional relevance in mammalian L and H chains are all conserved in the T. bernacchii spleen ferritin chain. More specifically, E27, E61, E62, H65, E107 and Q141, correspond to amino acids characteristic of the H-chain ferroxidase site, while E57, D60 and E64 correspond to sites of iron nucleation in L chains. This characteristic, first described for the poly-peptide chains of bullfrog ferritin [9], is common to fish ferritins on the basis of the available sequences. A further distinctive property of the fish H chains known to date relative to those of mammals appears to be the lack of the four-amino-acid extension at the N-terminus. An exception is the H chain of G. acuticeps spleen ferritin, the N-terminal amino-acid sequence of which, TTASTSQVRQNYHQDSE, shows the typical four-ami-no-acid extension of mammalian ferritin H chains [12]. Iron incorporation Iron uptake by T. bernacchii apoferritin was studied at different temperatures in 50 mM Mops/NaOH buffer at pH 6.5 after the aerobic addition of 500 iron atoms per molecule.Inparallel,therecombinanthuman Hhomopoly-mer was examined. As shown in Fig. 3, at 20 °C the time course of Fe2+ oxidation by T. bernacchii apoferritin is characterized by a half-time of about 120 s, which is higher than that measured under similar conditions for the human H homopolymer (t1/2 ˆ 55 s) and significantly lower than that of the L-rich apoferritin of horse spleen (t1/2 ˆ 600 s) [19]. The iron-oxidation capacity is maintained by Fig. 2. Amino-acid sequence comparison among T. bernacchii ferritin and M, H and L chain of ferritins. The alignment was obtained using ClustalW. TbS_M, M chain from T. bernacchii spleen; SgG_H2, H2 chain from S. gairdneri gonadal fibroblast (TrEMBL accession num-ber: P79822); SaL_M, M chain from S. salar liver (SwissProt acces-sion number: P49947); GmL_M, M chain from G. mirabilis liver (TrEMBL accession number: Q9DFP0); SaL_H, H chain from S. salar liver (SwissProt accession number: P49946); OnB_H, H chain from O. nerka brain (TrEMBL accession number: Q98TT0); HuL_H, H chain from human liver (SwissProt accession number: P02794); HuL_L, L chain from human liver (SwissProt accession number: P02792). Residues conserved in all sequences are in boldface type. Amino acids that constitute the H-chain ferroxidase center are in blue; those formingthe L chainiron micellenucleationsitearein red.Green arrows indicate the four predicted ahelices (A, B, C, D). Yellow boxes indicate the ahelix (A,B,C, Dand E)identified inthe crystallographic structure of human H chain. The human H chain numbering has been adopted. T. bernacchii apoferritin at low temperature; the rate of the reaction is reduced sixfold (t1/2 ˆ 715 s) when the temperature is decreased from 20 °C to 4 °C. The human recombinant H homopolymer shows a similar decrease in the catalytic activity (t1/2 ˆ 360 s) at 4 °C. The effect of temperature on the half-time of the iron-oxidation reaction, measured between 4 °C and 50 °C, was analysed using the Arrhenius equation. The activation energy, E , of T. bernacchii apoferritin is 74.9 kJÆmol)1, a value only slightly lower than that measured for the recombinant H protein (80.8 kJÆmol)1). All the added iron is incorporated inside the apoferritin shell as indicated by native gel electrophoresis and sedimen-tation velocity experiments. The reconstitution products obtained on incubation of apoferritin with 2500 iron atoms 1604 G. Mignogna et al. (Eur. J. Biochem. 269) Fig. 3. Progress curves of iron oxidation uptake by T. bernacchii and humanrecombinantHapoferritinsonadditionof500Featoms/molecule as ferrous ammonium sulfate at 20 and 4 °C. T. bernacchii apoferritin (– ) – 20 °C, –ÆÆ– 4 °C); human recombinant H homopolymer (–20 °C, ÆÆÆÆ 4 °C). Protein concentration: 0.2 lM. Buffer: 50 mM Mops/NaOH, pH 6.5. Inset: effect of temperature on t1/2 value in T. bernacchii (d) and human recombinant H homopolymer (j) (Arrhenius plot). per molecule in Mops/NaOH buffer, pH 6.5, sediment as a heterogeneous peak with an average sedimentation coeffi-cientofabout43 S.Thedistributionofironmicellesandthe value of the sedimentation coefficient are very similar to those measured for the native protein. Ó FEBS 2002 Structure of T. bernacchii ferritin as a function of pH The effect of low pH on the association state of the protein was investigated to compare the stability of T. bernacchii ferritinwiththatofL-typeandH-typemammalianferritins, which are known to dissociate at pH 2.5 and 2.8–3.0, respectively [20]. The stability of T. bernacchii ferritin at acid pH values was studied after incubation of the apoprotein in the pH range 3.0–1.5 at 20 °C for 24 h, a time established to be sufficient to reach equilibrium. T. bernacchii apoferritin maintains its quaternary assembly when incubated at pH 3.0 and at pH 2.5, as indicated by the corresponding elution volumes from a Superose 12 column, which are decreased only slightly (Ve ˆ 7.7 mL) compared with that of the native protein at pH 7.0 (Ve ˆ 7.8 mL). On incubation at pH 3.0, the secondary structure of native apoferritin is almost completely preserved, as indicated by the far-UV CD spectrum (Fig. 4A). Likewise, the near-UV CD spectrum resembles that measured at pH 7.0 with minor differences (Fig. 4B). Consistently with the modest changes observed in the near-UV and far-UV CD spectra compared with the protein at pH 7.0, the fluorescence emission of apoferritin at pH 3.0 is decreased by only 20%, and is not red-shifted relative to the protein at pH 7.0, which shows a kmax ˆ 333 nm on excitation at 295 nm (Fig. 4C). IncubationofT. bernacchiiapoferritinatpH 2.5(3.2 mM HCl) does not induce any change in the Superose 12 elution profile, but alters significantly the protein spectral proper-ties. The near-UV CD spectrum displays a consistent decrease in all the aromatic residue contributions. Interest-ingly, the 262 nm phenylalanine band is of opposite sign to the protein at pH 7.0 (Fig. 4B). The far-UV CD spectrum of T. bernacchii apoferritin at pH 2.5 shows a modest blue shift of the zero intercept and an overall decrease in the ellipticity relative to the protein at pH 7.0 (Fig. 4A). The fluorescence spectrum concomitantly shows a 47% quench- Fig. 4. Effect of pH on the spectral properties of T. bernacchii ferritin. (A) Far-UV CD (0.1 cm quartz cuvette) and (C) fluorescence (295 nm excitation wavelength) spectra were recorded at 0.05 mgÆmL)1 protein concentration. (B) Near-UV CD spectra were recorded in a 1-cm quartz cuvette at 1.20 mgÆmL)1 protein concentration. All the spectra were recorded at 20 °C after 24 h incubation of the protein at pH 7.0 (20 mM sodium phosphate, –––), pH 3.0 (1.0 mM HCl, – Æ –), pH 2.0 (10.0 mM HCl, —–), pH 2.5 (3.2 mM HCl, – Æ Æ –), pH 2.0 pCl 1.5 (31.6 mM NaCl – ) –), and pH 1.5 (31.6 mM HCl, ÆÆÆÆÆÆ). ... - tailieumienphi.vn