Electrostatic effects control the stability and iron release kinetics of ovotransferrin

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Metadata of the article that will be visualized in OnlineFirst

ArticleTitle Electrostatic effects control the stability and iron release kinetics of ovotransferrin

Article Sub-Title

Article CopyRight SBIC(This will be the copyright line in the final PDF)

Journal Name JBIC Journal of Biological Inorganic Chemistry

Corresponding Author Family Name KumarParticle

Given Name RajeshSuffix

Division School of Chemistry and Biochemistry

Organization Thapar University

Address Patiala, 147004, India

Email [email protected]

Author Family Name KumarParticle

Given Name SandeepSuffix




Email

Author Family Name SharmaParticle

Given Name DeepakSuffix

Division

Organization Institute of Microbial Technology, Council of Scientific and IndustrialResearch

Address Sector 39A, Chandigarh, India

Email

Author Family Name KumarParticle

Given Name RajeshSuffix




Email

Schedule

Received 4 August 2013

Revised

Accepted 29 April 2014

Abstract The contribution of electrostatic interactions to the stability of ovotransferrin-Fe3+ (oTf-Fe3+) complex hasbeen assessed by equilibrium experiments that measure iron retention level of diferric-ovotransferrin(Fe2oTf) as a function of pH and urea in the presence of salts (NaCl, Na2SO4, NaBr, NaNO3) and sucrose at25 °C. As [salt] is increased, the pH-midpoint for iron release increases monoexponentially and plateau at~0.4(±0.05) M NaCl/NaBr/NaNO3 or ~0.15(±0.03) M Na2SO4. However, at pH 7.4, the urea-midpoints foriron release (based on fluorescence emission at 340 nm) and for unfolding of Fe2oTf and apo-ovotransferrin(based on ellipticity values at 222 and 282 nm) decrease at low salt concentrations [≤0.1(±0.02) M Na2SO4

or ≤0.35(±0.15) M NaCl], but increase at higher salt concentrations. Furthermore, Na2SO4 has a greater effectthan NaCl in increasing the urea-midpoints for iron release and unfolding. These results indicate that at lowsalt concentrations, the electrostatic effects destabilize the oTf-Fe3+ complex and also decrease the structuralstability of the proteins. In contrast, at higher concentrations, salt ions behave according to Hofmeister series.At pH 5.6, as [salt] is increased, the rate constants for reductive iron release (Fe2+ release) and ureadenaturation-induced iron release (Fe3+ release) from the N-lobe of oTf (FeNoTf) increase monoexponentiallyand plateau at ~0.4(±0.1) M NaNO3/NaCl or ~0.2(±0.05) M Na2SO4. These results suggest that the anion-binding-induced conformational change as well as the electrostatic screening of surface Coulombicinteractions plays important role in accelerating the iron release from FeNoTf under endosomal pH conditions.

Keywords (separated by '-') Iron release - Electrostatic interactions - Anion-binding - Conformational change - Structural stability

Footnote Information Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1145-2)contains supplementary material, which is available to authorized users.

Metadata of the article that will be visualized in OnlineAlone

Electronic supplementarymaterial

Below is the link to the electronic supplementary material.MOESM1: Supplementary material 1 (PDF 57 kb).

UNCORRECTEDPROOF

ORIGINAL PAPER1

2 Electrostatic effects control the stability and iron release kinetics

3 of ovotransferrin

4 Sandeep Kumar • Deepak Sharma •

5 Rajesh Kumar • Rajesh Kumar

6 Received: 4 August 2013 /Accepted: 29 April 20147 � SBIC 2014

8 Abstract The contribution of electrostatic interactions to

9 the stability of ovotransferrin-Fe3? (oTf-Fe3?) complex

10 has been assessed by equilibrium experiments that measure

11 iron retention level of diferric-ovotransferrin (Fe2oTf) as a

12 function of pH and urea in the presence of salts (NaCl,

13 Na2SO4, NaBr, NaNO3) and sucrose at 25 �C. As [salt] is

14 increased, the pH-midpoint for iron release increases mo-

15 noexponentially and plateau at *0.4(±0.05) M NaCl/

16 NaBr/NaNO3 or *0.15(±0.03) M Na2SO4. However, at

17 pH 7.4, the urea-midpoints for iron release (based on

18 fluorescence emission at 340 nm) and for unfolding of

19 Fe2oTf and apo-ovotransferrin (based on ellipticity values

20 at 222 and 282 nm) decrease at low salt concentrations

21 [B0.1(±0.02) M Na2SO4 or B0.35(±0.15) M NaCl], but

22 increase at higher salt concentrations. Furthermore, Na2-23 SO4 has a greater effect than NaCl in increasing the urea-

24 midpoints for iron release and unfolding. These results

25 indicate that at low salt concentrations, the electrostatic

26 effects destabilize the oTf-Fe3? complex and also decrease

27 the structural stability of the proteins. In contrast, at higher

28 concentrations, salt ions behave according to Hofmeister

29 series. At pH 5.6, as [salt] is increased, the rate constants

30 for reductive iron release (Fe2? release) and urea dena-

31 turation-induced iron release (Fe3? release) from the

32N-lobe of oTf (FeNoTf) increase monoexponentially and

33plateau at*0.4(±0.1) M NaNO3/NaCl or*0.2(±0.05) M

34Na2SO4. These results suggest that the anion-binding-

35induced conformational change as well as the electrostatic

36screening of surface Coulombic interactions plays impor-

37tant role in accelerating the iron release from FeNoTf under

38endosomal pH conditions. 39

40Keywords Iron release � Electrostatic interactions �

41Anion-binding � Conformational change � Structural

42stability

43Introduction

44The stability of proteins is generally governed by nonco-

45valent interactions such as hydrophobic [1, 2], electrostatic

46[3–7], and hydrogen bonding [8, 9]. However, the relative

47contributions of these interactions to protein stability are

48not fully resolved. In particular, the role of ionic interac-

49tions in protein stability is relatively more complex [10].

50Buffer conditions such as pH and salt can have dramatic

51effect on the stability and biological functions of proteins.

52For example, the low pH triggers a large conformational

53change in transferrins (Tfs), which is a critical step for iron

54release in endosome [11–25]. In general, pH modulates the

55protein stability by altering the charges on ionizable groups

56in the proteins through protonation or deprotonation [26].

57Salt ions also modulate the stability of proteins [27, 28]. At

58low concentrations, salt ions affect the stability of proteins

59by altering the electrostatic (Debye-Huckel) screening of

60Coulombic interactions [9, 29, 30]. At relatively higher

61concentrations, salt ions follow the Hofmeister effect,

62which eventually depends on the nature of added ions and

63modulates the stability of proteins by increasing the surface

A1 Electronic supplementary material The online version of thisA2 article (doi:10.1007/s00775-014-1145-2) contains supplementaryA3 material, which is available to authorized users.

A4 S. Kumar � R. Kumar � R. Kumar (&)

A5 School of Chemistry and Biochemistry, Thapar University,

A6 Patiala 147004, India

A7 e-mail: [email protected]

A8 D. Sharma

A9 Institute of Microbial Technology, Council of Scientific

A10 and Industrial Research, Sector 39A, Chandigarh, India

AQ1

123Journal : Large 775 Dispatch : 12-5-2014 Pages : 16

Article No. : 1145h LE h TYPESET

MS Code : JBIC-13-08-00139 h CP h DISK4 4

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DOI 10.1007/s00775-014-1145-2

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64 tension of solvent that ultimately alters the hydrophobic

65 interactions [28, 31–34]. According to Hofmeister series, the

66 anions typically follow the order F-&HPO42-[ SO4

2-[

67 ICH3COO-[Cl-[NO3

-[Br-[ClO3

-[I-[ClO4

-[SCN-.

68 The species at the left of Cl- in this sequence are referred to

69 as kosmotropes (ordering of water structures and thus sta-

70 bilize the protein) and produce salting-out effect while those

71 at the right of Cl- are known as chaotropes (breaking of

72 water structure and thus destabilize the proteins) and produce

73 salting-in effects [28, 31–34].

74 The contributions of electrostatic and hydrophobic

75 interactions to the stability of proteins are generally

76 determined by measuring the unfolding transitions of pro-

77 teins as a function of salt concentration [5, 7, 35, 36].

78 Although the effect of salt concentrations on the stability of

79 proteins has been studied extensively [30, 35, 36], only few

80 studies measured the relative contributions of the electro-

81 static and hydrophobic interactions to the stability and

82 functional properties of proteins [30, 35]. The current study

83 provides this analysis for hen ovotransferrin (oTf), the iron

84 binding protein from egg white.

85 oTf is a member of the transferrin family of iron-binding

86 glycoproteins that also includes serum transferrin (sTf) and

87 milk lactoferrin (Lf) [14]. The affinity of Tfs for Fe3? is

88 extremely high (Ka * 1020 M-1) [37–40]. This high

89 affinity for Fe3? gives protection against iron-catalyzed

90 free radical formation and limits the bacterial growth. Tfs

91 can also bind non-ferrous metal ions [38, 41–46], few of

92 them are of therapeutic and diagnostic interest [38, 41–43].

93 sTf is primarily responsible for iron transport from bio-

94 logical fluids to cytosol by receptor-mediated endocytosis

95 [47] while oTf and Lf act as bacteriostatic agents [48]. Few

96 earlier in vitro studies have revealed that oTf also possesses

97 the cellular iron transport functions [49–51].

98 oTf has structural characteristics similar to that of hen

99 sTf [52]. This is presumably because these two proteins

100 derived from same gene differ only in the linked carbo-

101 hydrate groups [52]. Structurally, Tfs are bi-lobed proteins

102 with N- and C-terminal domains of approximately similar

103 size, and each domain can bind one iron atom synergisti-

104 cally with a carbonate anion [14, 48, 53]. These two

105 domains are connected by linker sequence [54, 55]. Upon

106 complex formation with iron, each domain of Tfs under-

107 goes a large-scale conformational change [56–60]. Car-

108 bonate (or bicarbonate) is the dominant synergistic anion

109 in vivo that plays a critical role in physiological release and

110 uptake of iron by Tfs [47, 61–67]. Increasing evidence

111 suggests that many non-synergistic anions (SO42-, Cl-,

112 NO3-, ClO4

-, etc.) which do not bind directly to iron but

113 bind to the protein [17, 68–73] also play an important role

114 in chelator-mediated iron release from Tfs [17, 72, 74–88].

115 However, the molecular mechanism by which these non-

116 synergistic anions modulate the chelator-mediated iron

117release from monoferric- and diferric-Tfs remains contro-

118versial [72, and reference there in].

119In the absence of chelators, iron release from monofer-

120ric- and diferric-Tfs can be stimulated by a number of

121factors that can operate individually or together. In vitro,

122these include the acid lability of the two sites resulting in

123iron release at low pH [78, 89], reduction of Fe3? to the

124much more weakly bound Fe2? [80, 90, 91], thermal

125denaturation of holoprotein that results in iron release

126above melting temperature [30], and chemical denaturation

127of holoprotein resulting in iron release above denaturation

128midpoint [92]. To assess the role of molecular interactions

129responsible for the oTf-Fe3? complex stabilization and

130structural stability of Fe2oTf, we have carried out the pH-

131and urea-denaturation equilibrium experiments for Fe2oTf

132in the absence of chelators as a function of salts (NaCl,

133NaNO3, NaBr, and Na2SO4) and sucrose concentration at

13425 �C. Interestingly, the low concentrations of salt

135[B0.1(±0.02) M Na2SO4 or B0.35(±0.15) M NaCl/

136NaNO3/NaBr] destabilize the oTf-Fe3? complex and also

137decrease the structural stability of Fe2oTf. The present

138work also reveals that the salt-mediated decreased stability

139of Fe2oTf appears to result from (i) the destabilization of

140oTf-Fe3? complex and (ii) the ionic screening of electro-

141static interactions.

142To understand the underlying mechanism by which

143noncovalent molecular interactions control the dynamics of

144iron release from Tfs, we have also studied the kinetics of

145Fe2? and Fe3? release from FeNoTf at pH 7.4 and pH 5.6 in

146the absence of chelators as a function of salt (NaCl,

147NaNO3, and Na2SO4) and sucrose concentration. At pH

1487.4, with increase in the salt concentration from 0.0 to

1491.0 M NaCl/NaNO3 or 0.5 M Na2SO4, the rate constants

150for reductive iron release (i.e., Fe2? release) and urea

151denaturation-induced iron release (i.e., Fe3? release) from

152FeNoTf increase initially when the salt concentration is low

153[B0.12(±0.05) M NaCl or Na2SO4], but decrease at higher

154salt concentrations. As salt concentration is increased, the

155rate constants for Fe2? and Fe3? release from FeNoTf

156increase mono-exponentially and plateau at*0.4(±0.1) M

157NaNO3/NaCl or *0.2(±0.05) M Na2SO4 at pH 5.6, indi-

158cating that the anion-binding-induced conformational

159change and electrostatic destabilization of FeNoTf complex

160by salt regulate the kinetics of iron release from FeNoTf

161under endosomal pH conditions.

162Materials and methods

163Egg white apo-oTf (C0755), salts (NaCl, NaNO3, NaBr,

164and Na2SO4), sucrose, NaOH, and salts of buffers 4-(2-

165Hydroxyethyl)-1-piperazineethanesulfonate (HEPES) and

1662-(N-mopholino) ethanesulfonate (MES) were purchased

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167 from Sigma. pH of the buffers and protein samples were

168 adjusted using concentrated NaOH or HCl solutions.

169 Preparation of Fe2oTf

170 Fe2oTf complex was prepared by well-known procedure

171 used earlier for diferric sTf [93–95]. Briefly, an iron ni-

172 trilotriacetate solution was prepared by dissolving nitrilo-

173 acetic acid (*100 lmol) and FeCl3�6H2O (*50 lmol) in

174 2 mL solution of 6 M HCl. pH of this solution was

175 adjusted to 4.0 with concentrated NaOH solution. This

176 solution was finally diluted up to 10 mL. Apo-oTf (80 mg,

177 1 lmol) was dissolved in 3 mL of buffer (50 mM HEPES,

178 pH 7.4, 20 mM NaHCO3). Protein solution was then

179 diluted up to 5 mL by adding the freshly prepared iron

180 nitrilotriacetate solution. Protein solution thus prepared

181 was incubated overnight at *37 �C. To remove the

182 unbound iron, protein solution was exchanged at least five

183 times with original buffer (50 mM HEPES, pH 7.4) using a

184 Centricon filter of 10 kDa molecular mass cut-off (Milli-

185 pore, Bedford, MA, USA). Iron saturation of oTf was

186 checked by urea-polyacrylamide gel electrophoresis. The

187 concentration of Fe2oTf was determined spectrophoto-

188 metrically on the basis of e465 nm = 5 9 103 M-1 cm-1

189 [37]. The percentage of protein iron saturation was above

190 95 %.

191 Preparation of FeNoTf

192 FeNoTf was prepared by a well-known procedure used

193 earlier for monoferric sTf [95, 96]. The perchlorate ion

194 enhances the rate of iron release from the C-lobe 260 times

195 more than the N-lobe of Tfs. Therefore, the concept of

196 selective release of iron from C-lobe of Fe2oTf is used to

197 prepare the FeNoTf. For this, Fe2oTf (250 lM) was incu-

198 bated at 37 �C for *2 h in 0.1 M HEPES buffer that also

199 contained 0.1 M EDTA and 2.7 M NaClO4. To remove the

200 EDTA and NaClO4 from protein solution, the protein

201 solution was exchanged at least five times with original

202 buffer (50 mM HEPES, pH 7.4) using a Centricon filter of

203 10 kDa molecular mass cut-off (Millipore, Bedford, MA,

204 USA). Iron saturation of oTf was checked by urea–poly-

205 acrylamide gel electrophoresis. The concentration of FeN-206 oTf was determined spectrophotometrically on the basis of

207 e465 nm = 2,150 M-1 cm-1 [93].

208 Measurement of iron release from Fe2oTf as a function

209 of pH

210 Solutions of Fe2oTf (*10 lM) were prepared in mixture

211 of buffers (0.02 M HEPES ? 0.02 M MES, pH*8.0–3.0)

212 that contained different concentrations of salt (NaCl,

213 NaNO3, NaBr, and Na2SO4) or sucrose. Before the data

214collections, the protein samples were incubated for*4 h at

21525 �C. pH of the protein samples were measured both

216before and after the experiments. The reported pH values

217are those determined after taking the data. The decrease in

218absorbance maximum at 465 nm and increase in intrinsic

219fluorescence emission intensity at 340 nm (excitation at

220280 nm) were used as the reliable markers for iron release

221from diferric-Tfs [21, 97–99]. Fluorescence emission

222spectra (320–410 nm) were collected on Perkin Elmer LS-

22355 spectrofluorometer, and visible absorbance spectra

224(390–710 nm) were collected on Shimadzu 2450-UV

225spectrophotometer. The pH equilibrium profiles for Fe2oTf

226were measured by monitoring changes in fluorescence

227emission at 340 nm (kex = 280 nm) and absorbance at

228465 nm. On the basis of fluorescence emission at 340 nm

229and absorbance at 465 nm, the fraction of iron released was

230estimated.

231Measurement of iron release from Fe2oTf as a function

232of urea

233Solutions of Fe2oTf (10 lM) were prepared in a buffer

234(0.05 M HEPES, pH 7.4 or 0.05 M MES, pH 5.8–5.4) that

235contained different concentrations of urea and a desired

236concentration of salt (NaCl, and Na2SO4) or sucrose. The

237denaturant concentration of Fe2oTf solution was measured

238before and after data acquisition using Abbe refractometer

239and found not to change within the uncertainty of the

240measurement. Before the data collection, the protein sam-

241ples were incubated for*5 h at 25 �C. The fraction of iron

242released from Fe2oTf by urea was estimated by their

243fluorescence emission (ex: 280 nm) at 340 nm or absor-

244bance at 465 nm.

245Unfolding of Fe2oTf and apo-oTf by urea

246Solutions of Fe2oTf or apo-oTf were prepared in a buffer

247[0.05 M HEPES (pH 7.4) or 0.05 M MES (pH 5.8–5.4)]

248that contained different concentrations of urea and a

249desired concentration of salt (NaCl, and Na2SO4) or

250sucrose. Prior to data collection, the protein samples were

251incubated for *5 h at 25 �C. To determine the effect of

252salts on the structural stability of Fe2oTf and apo-oTf, we

253have collected the urea dependent (0.0–11.0 M) far-UV

254(205–250, 1 mm pathlength) and near-UV (250–310,

2555 mm pathlength) CD spectra for Fe2oTf and apo-oTf in

256the presence of a desired concentration of salt (NaCl,

257Na2SO4) or sucrose at 25 �C. The final concentrations of

258Fe2oTf or apoTf for far-UV and near-UV CD were*5 and

259*10 lM, respectively. The fraction of unfolded protein

260(fD) was estimated from the far-UV (222 nm) and near-UV

261(282 nm) CD data.

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262 Kinetics of iron release from FeNoTf by urea

263 denaturation

264 When FeNoTf was incubated with 9.0 M urea at pH 7.4 or

265 6.0 M urea at pH 5.6, the absorbance at 465 nm decreased

266 rapidly [92]. The kinetics of urea denaturation-induced iron

267 release were was measured by monitoring the change in

268 absorbance at 465 nm, 37 �C. For kinetic experiments at

269 pH 7.4, protein solution (40 lL, 110 lM, pH 7.4) was

270 mixed rapidly to HEPES buffer (0.05 M, 0.8 mL, pH 7.4)

271 that contained 9.0 M urea and a desired concentration of

272 salt (NaCl or Na2SO4) or sucrose. At pH 5.6, iron release

273 reaction is relatively faster; therefore, the kinetics of iron

274 release at this pH were measured by a Shimadzu 2450

275 spectrophotometer coupled with rapid kinetics stopped-

276 flow mixing accessory (applied photophysics RX 2000).

277 FeNoTf (pH 7.4) was placed in one syringe and the MES

278 buffer (0.05 M, pH 5.6) that contained 6.0 M urea and a

279 desired concentration of salt (NaCl or Na2SO4) or sucrose

280 was placed in other stopped flow syringe. For kinetic

281 experiments at pH 7.4 and pH 5.6, the final concentrations

282 of protein were *5.5 and *10 lM, respectively.

283 Kinetics of reductive release of iron from FeNoTf

284 by sodium dithionite

285 For kinetic experiments at pH 7.4, a small volume of

286 FeNoTf solution (40 lL, 110 lM, pH 7.4) was mixed

287 rapidly to a degassed HEPES buffer (0.05 M, 0.8 mL, pH

288 7.4) that contained 10 mM sodium dithionite, 0.25 mM

289 BPS, and a desired concentration of salt (NaNO3 or Na2-290 SO4) or sucrose. The kinetics of reductive iron release were

291 measured by Fe2?-BPS complex absorbance at 538 nm

292 [80, 91]. The kinetics of reductive iron release at pH 5.6

293 were measured by a Shimadzu 2450 spectrophotometer

294 coupled with rapid kinetics stopped-flow mixing accessory

295 because the reductive iron release reaction is relatively

296 faster at this pH. FeNoTf (pH 7.4) was placed in one syr-

297 inge and MES buffer (0.05 M, pH 5.5) that contained

298 sodium dithionite, BPS, and a desired concentration of salt

299 (NaNO3 or Na2SO4) or sucrose was placed in other stopped

300 flow syringe. The final concentrations of protein, sodium

301 dithionite, and BPS were *5 lM, *10 mM, and

302 *0.25 mM, respectively.

303 Results

304 pH dependence of the absorbance and fluorescence

305 emission spectra of Fe2oTf

306 At pH 7.4, the electronic absorption spectrum of Fe2oTf

307 shows a maximum at 465 nm, which indicates that iron is

308fully associated with protein (Fig. 1a). When pH is lowered

309from 7.4 to 3.5, the absorbance maximum at 465 nm is

310completely lost (Fig. 1a). This suggests that iron is fully

311released from both sites of Fe2oTf. Lowering the pH from

3127.4 to 3.5, the quenching of Trp fluorescence by bound

313Fe3? also diminishes (Fig. 1b), confirming that iron is fully

314released from both sites of Fe2oTf.

315Salt dependence of the pH-midpoint for iron release

316of Fe2oTf

317To determine the effect of salts or sucrose on the stability

318of oTf-Fe3? complex, the pH-titrations for Fe2oTf were

319obtained by monitoring fluorescence emission at 340 nm

320and absorbance at 465 nm in the presence of various

321concentrations of salt (NaCl, NaNO3, NaBr, Na2SO4) or

322sucrose at 25 �C. For brevity, we present the fluorescence-

323monitored pH titrations of Fe2oTf in the absence of addi-

324tive and in the presence of *0.15 and 0.5 M NaCl, 0.15

325and 0.5 M NaNO3, 0.15 M NaBr, 0.25 M Na2SO4, and 0.5

326and 1.0 M sucrose (Fig. 1c). As salt concentration is

327increased, the transition midpoint shifts towards the higher

328pH (Fig. 1c, d), which reveals that the salt reduces the

329stability of oTf-Fe3? complex. On the contrary, as sucrose

330concentration is increased, the transition midpoint shifts

331towards the lower pH (Fig. 1c), indicating that sucrose

332increases the stability of oTf-Fe3? complex.

333To determine the role of molecular interactions

334responsible for the stability of oTf-Fe3? complex, the pH

335profiles of Fe2oTf measured at various concentrations of

336salt (NaCl, NaNO3, NaBr, Na2SO4) or sucrose were fitted

337to Eq. (1) [30].

y ¼cf þ cu 10 Cm�pHð Þ

� �

1þ 10 Cm�pHð Þ

" #

ð1Þ

339339where cf and cu are the normalized absorbance or

340fluorescence signals for iron-loaded native state and

341iron-free acid-denatured state, respectively, and Cm is

342the pH midpoint where Fe2oTf complex lost its half of

343the metal ions. As salt concentration is increased, the

344Cm for iron release increases mono-exponentially and

345plateau at *0.4(±0.05) M NaCl/NaBr/NaNO3 or

346*0.15(±0.03) M Na2SO4. (Fig. 1d). On the contrary, as

347sucrose concentration is increased from 0.0 to 1.0 M,

348the Cm decreases by *0.4 unit (Fig. 1c). Figure 1d also

349shows that the increase in Cm for Fe2oTf is more

350pronounced for Na2SO4 and least for NaBr. The salt-

351induced increase in Cm for Fe2oTf typically follows the

352order Na2SO4[NaNO3[NaCl[NaBr. These findings

353indicate that Na2SO4 has a greater effect than that of

354NaBr/NaNO3/NaCl in destabilizing the oTf-Fe3?

355complex.

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356 Urea dependence of the absorbance and fluorescence

357 emission spectra of Fe2oTf

358 At pH 7.4, when urea concentration is increased from 0.0 to

359 10.0 M, the absorbance maximum at 465 nm in the

360 absorption spectrum of Fe2oTf is almost eliminated

361 (Fig. 2a), indicating that both sites of Fe2oTf have com-

362 pletely released their bound metal ions. At 10.0 M urea, the

363 quenching of Trp fluorescence by bound Fe3? is also

364 diminished (Fig. 2b), indicating that the iron is fully dis-

365 sociated from both sites of Fe2oTf complex.

366 Effects of salt on the secondary and tertiary structures

367 of Fe2oTf and apo-oTf

368 The far-UV CD spectra (200–250 nm) of native Fe2oTf

369 (pH 7.4) in the absence and presence of *0.3 M NaCl

370 shows the negative extrema at 208 nm and a shoulder

371 around 215–225 nm (Fig. 3a). These spectral features

372reflect the presence of secondary structural elements in the

373protein. Figure 3a also presents the far-UV CD spectra of

374Fe2oTf (pH 7.4) measured at *10.0 M urea without salt

375and at *7.5 M urea in the absence and presence of

376*0.3 M NaCl. Irrespective of the exclusion of salt, at

377*10.0 M urea, the negative extrema at 208 nm and a

378shoulder around 215–225 nm are substantially disrupted

379(Fig. 3a), indicating that the secondary structure of protein

380is significantly lost. However, in the absence of salt, at

381*7.5 M urea, the negative extrema at 208 nm and a

382shoulder around 215–225 nm are only partially disrupted

383(Fig. 3a). Interestingly, in the presence of *0.3 M NaCl,

384these partially disrupted peptide bands are substantially

385disrupted (Fig. 3a), indicating that the low concentrations

386of salt significantly destabilize the secondary structure of

387the partially denatured state of protein.

388The near-UV CD spectra (250–300 nm) of native Fe2-389oTf (Fig. 3b) and apo-oTf (Fig. 3c) in the absence and

390presence of*0.3 M NaCl show the three aromatic residue

pH

3 4 5 6 7 8

% I

ron

Re

ma

inin

g

0

50

100

[Salts] or [Sucrose] (M)

0.0 0.3 0.6 0.9 1.2

Cm

4.5

4.8

5.1

5.4

5.7

Wavelength (nm)

400 450 500 550 600 650 700

Ab

so

rba

nc

e

0.00

0.02

0.04

0.06

0.08

Wavelength ( nm)

310 335 360 385 410

Flu

ore

sc

en

ce

0

200

400

600

(a)

(b)

(c)

(d)

Fig. 1 pH dependence of iron release from Fe2oTf in the presence of

salts and sucrose. a Absorbance and b fluorescence emission spectra

of Fe2oTf [(solid line) 0.05 M HEPES, pH 7.4; (dotted line) 0.05 M

MES, pH 3.5]. c pH equilibrium profiles of Fe2oTf monitored by

fluorescence (ex: 280 nm). All experiments were performed at 25 �C:

(filled circle) no salt; (filled square) 0.15 M NaBr; (unfilled inverted

triangle) 0.15 M NaCl; (filled inverted triangle) 0.15 M NaNO3;

(filled triangle) 0. 5 M NaCl; (unfilled square) 0.5 M NaNO3;

(unfilled triangle) 0.25 M Na2SO4; (unfilled circle) 0.5 M and

(unfilled diamond) 1.0 M sucrose. The solid lines in panel c were

fitted according to Eq. (1). d The dependence of the pH-midpoint at

which half of the iron is released from Fe2oTf on [Salts] [(filled

circle) NaBr; (unfilled circle) NaCl; (filled square) NaNO3; (unfilled

square) Na2SO4] and [Sucrose] [(unfilled diamond) sucrose]. The

solid lines in panel d represent non-linear least squares fits of the data

to a single-exponential function

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391 bands at 255–270 nm (phenylalanine), 282 nm (tyrosine),

392 and 291 nm (tryptophan). These spectral features reflect

393 the tertiary structures of Fe2oTf and apo-oTf. The near-UV

394 CD spectroscopy can also be used to probe the structural

395 changes induced by metal binding to Tfs [100]. Upon iron

396 binding, the shapes and intensities of the aromatic bands of

397 apo-oTf alter significantly (Fig. 3b, c), indicating that the

398 metal binding to apo-oTf produces significant changes in

399 the tertiary structure of protein. Figure 3b also presents the

400 near-UV CD spectra of Fe2oTf (pH 7.4) measured at 10 M

401 urea without salt and at *7.5 M urea both in the absence

402 and presence of*0.3 M NaCl. The near-UV CD spectra of

403 apo-oTf measured at *10.0 M urea without salt and at

404 *3.5 M urea both in the absence and presence of*0.3 M

405 NaCl are shown in Fig. 3c. Irrespective of the exclusion of

406 salt, at *10.0 M urea, the aromatic bands at 282 and

407 291 nm are substantially disrupted for both apo-oTf and

408 Fe2oTf (Fig. 3b, c), indicating that the tertiary structures of

409 proteins are significantly lost. However, in the absence of

410 salt, at *7.5 M urea for Fe2oTf and at *3.5 M urea for

411 apo-oTf, the aromatic bands at 282 and 291 nm are only

412 partially disrupted (Fig. 3b, c). Interestingly, in the pre-

413 sence of *0.3 M NaCl, these partially disrupted aromatic

414bands are substantially lost (Fig. 3b, c), indicating that the

415low concentration of salts significantly destabilize the ter-

416tiary structure of the partially denatured state of protein.

417Effect of pH on the urea denaturation-induced iron

418release and unfolding of Fe2oTf

419Figures 4a and 5a show the urea-induced denaturation

420curves for iron release (based on fluorescence emission at

421340) and unfolding of Fe2oTf (based on CD at 222 nm),

Wavelength (nm)

400 450 500 550 600 650

Ab

so

rba

nc

e

0.00

0.02

0.04

0.06

0.08

Wavelength ( nm)

325 350 375 400

Flu

ore

sc

en

ce

0

250

500

750

(a)

(b)

Fig. 2 Urea dependence of absorbance and fluorescence emission

spectra of Fe2oTf. Panels a, and b show the absorbance and

fluorescence emission spectra of Fe2oTf, respectively, in the absence

(solid line) and presence of 10.0 M urea (dotted line) at pH 7.4, 25 �C

Wavelength (nm)

250 260 270 280 290 300

-6

-4

-2

0

2

-4

-2

0

2

4

Wavelength (nm)

210 220 230 240 250

Ell

ipti

cit

y (

md

eg

)

E

llip

tic

ity

(m

de

g)

Ell

ipti

cit

y (

md

eg

)

-20

-15

-10

-5

0

(a)

(b)

(c)

Fig. 3 Panels a and b present the far-UV and near-UV CD spectra of

Fe2oTf, respectively, measured at *0.05 M HEPES (solid line) and

0.05 M HEPES with 0.3 M NaCl (dotted line); 0.05 M HEPES with

10.0 M urea (short dash line); 0.05 M HEPES with 7.5 M urea (dash

dot line); and 0.05 M HEPES with 7.5 M urea and 0.3 M NaCl (dash

double dot line). Panel c presents the near-UV CD spectra of apo-oTf

measured at *0.05 M HEPES (solid line) and 0.05 M HEPES with

0.3 M NaCl (dotted line); 0.05 M HEPES with 10.0 M urea (short

dash line); 0.05 M HEPES with 3.5 M urea (dash dot line); and

0.05 M HEPES with 3.5 M urea and 0.3 M NaCl (dash double dot

line)

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422 respectively, at pH 7.4, 5.8, 5.6, and 5.4. The normalized

423 urea-induced denaturation curves for the iron release and

424 unfolding of Fe2oTf collected at pH 7.4, 5.8, 5.6, and 5.4

425 (Figs. 4a, 5a) were fitted to Eq. (2) [101]:

Sobs ¼ðcf þ mf ½D�Þ þ ðcu þ mu½D�Þ exp

�DGoDþmg½D�

RT

� �

1þ exp�DGo

Dþmg½D�

RT

� � ;

ð2Þ

427427 where Sobs is the fluorescence or CD signals at given urea

428 concentration (D), cf and cu, and mf and mu represent

429 intercepts and slopes of iron loaded native and iron free

430 urea-denatured baselines, respectively, DGoD is the dena-

431 turation free energy in the absence of denaturant, and mg

432 is the surface area exposed by the solvent. The resulting

433 mg and DGoD for iron release and unfolding of Fe2oTf are

434 provided in Table S1 (Supporting Information). Although

435 the physical importance of the factor mg is not clearly

436understood, it is suggested to reflect the difference

437between the accessibility of surface areas of native and

438denatured states of the polypeptide chain for a given

439denaturant [102]. Decreasing the pH from 7.4 to 5.4, the

440mg values for iron release and unfolding of Fe2oTf

441become larger [Table S1 (Supporting Information)], sug-

442gesting that solvent exposed surface area increases upon

443iron release and unfolding at low pH. At each pH, the

444DGoD determined by far-UV CD is larger than the DGo

D

445measured by fluorescence [Table S1 (Supporting Infor-

446mation)]. This is presumably because Fe2oTf responds to

447increased urea concentration by means of a coupled pro-

448cess in which the urea-induced iron release precedes the

449unfolding reaction (Fe2oTf ? 2Fe3? ? apo-

450oTf ? unfolding). The far-UV CD monitored urea-dena-

451turation of Fe2oTf reflects this coupled process (iron

452release and unfolding) while the fluorescence or absor-

453bance (465 nm) monitored urea-denaturation of Fe2oTf

[Urea] (M)

Fra

cti

on

Iro

n R

ele

ased

0.0

0.5

1.0

[Urea] (M)

Fra

cti

on

Iro

n R

ele

ased

0.0

0.5

1.0

(a)

pH

5678

Cm

(M)

2.5

3.5

4.5

5.5

6.5


0 2 4 6 8 10

0 2 4 6 8 10

0.0 0.4 0.8 1.2

Cm

(M)

4.5

5.5

6.5

7.5

(c)

(d) (b)

Fig. 4 a The pH dependence of the urea-induced release of iron from

Fe2oTf as evaluated from the change in fluorescence emission at

340 nm [(filled circle) pH 5.4; (unfilled triangle) pH 5.6; (filled

triangle) pH 5.8; and (unfilled circle) pH 7.4]. Panel b shows the

fluorescence-monitored (ex: 280 nm, em: 340 nm) urea dependence

of iron release from Fe2oTf at pH 7.4, in the absence (unfilled circle),

and presence of *0.1 (unfilled triangle), 0.5 (filled square), and

1.0 M NaCl (unfilled square); *0.1 (filled triangle), and 0.25 M

Na2SO4 (unfilled diamond); and *0.5 M sucrose (filled circle). The

solid lines in panels a and b represent a non-linear least squares fit of

the data to Eq. (2). The urea-induced iron release mid-point, Cm

(=DGoD=mg) was calculated as a function of pH, salt (NaCl, and

Na2SO4) and sucrose concentration. Panel c shows pH dependence

(filled square) of urea-induced iron release midpoint. Panel d shows

salt [NaCl (unfilled square), and Na2SO4 (filled triangle)] or sucrose

(filled circle) dependence of urea-induced iron release midpoint at pH

7.4

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454 reflects the iron release process of Fe2oTf. The urea mid-

455 points for iron release and unfolding (Cm = DGoD

�

mg)

456 were calculated as a function of pH. The plots of Cm for

457 iron release and unfolding of Fe2oTf as function of pH are

458 shown in Figs. 4d and 5d, respectively. Lowering the pH

459 from 7.4 to 5.4, the Cm values for both iron release and

460 unfolding decrease significantly (Figs. 4d, 5d), indicating

461 that the electrostatic interactions destabilize the oTf-Fe3?

462 complex and also decrease the structural stability of

463 Fe2oTf.

464Effects of salt and sucrose on urea denaturation-induced

465iron release from Fe2oTf and unfolding of Fe2oTf

466and apo-oTf

467Figure 4b shows the urea-denaturation curves for iron release

468(based on fluorescence emission at 340) from Fe2oTf at

469various concentrations of salt (NaCl and Na2SO4) and sucrose

470at pH 7.4. The far-UV CD (222 nm) monitored urea-induced

471unfolding curves of Fe2oTf and apo-oTf measured at various

472concentrations of salt (NaCl and Na2SO4) and sucrose at pH

[Urea] (M)

0.0

0.5

1.0 (a)

Fra

cti

on

Un

fold

ed

0.0

0.5

1.0

[Urea] (M)

Fra

cti

on

Un

fold

ed

0.0

0.5

1.0

(b)

(c)

pH

Cm

(M)

4.5

5.5

6.5

7.5

Cm

(M)

5

6

7

8


0 2 4 6 8 10

0 2 4 6 8 10

5678

0.0 0.3 0.6 0.9 1.2

Cm

(M)

3.0

3.5

4.0

4.5

(d)

(e)

(f)

Fig. 5 a The pH dependence of the urea-induced unfolding of Fe2oTf

as evaluated from the change in far-UV CD at 222 nm [(filled circle)

pH 5.4; (unfilled triangle) pH 5.6; (filled triangle) pH 5.8; and

(unfilled circle) pH 7.4]. Panels b and c show the far-UV CD

monitored (222 nm) unfolding of Fe2oTf and apo-oTf, respectively, at

pH 7.4 in the absence (unfilled circle) and presence of*0.1 (unfilled

triangle), 0.5 (filled square), and 1.0 M NaCl (unfilled square);*0.1

(filled triangle), 0.25 (unfilled diamond), and 0.35 M Na2SO4 (filled

inverted triangle); and*0.5 M sucrose (filled circle). The solid lines

in panels a, b and c represent a non-linear least squares fit of the data

to Eq. (2). The urea-induced unfolding midpoint, Cm (=DGoD=mg) was

calculated as a function of pH, salt (NaCl, and Na2SO4) and sucrose

concentrations. Panel d shows pH dependence (filled square) of Cm.

Panels e and f show salt [NaCl (unfilled square), and Na2SO4 (filled

triangle)] and sucrose (filled circle) dependence of Cm of Fe2oTf and

apo-oTf, respectively, at pH 7.4

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473 7.4 are shown in Fig. 5b and c, respectively. Figure 6 shows

474 the near-UV CD (282 nm) monitored urea-induced unfolding

475 curves of Fe2oTf (Fig. 6a) and apo-oTf (Fig. 6b) measured at

476 various concentrations of salt (NaCl and Na2SO4) and sucrose

477 at pH 7.4. To determine the molecular interactions respon-

478 sible for the stabilization of oTf-Fe3? complex and structural

479 stability of Fe2oTf and apo-oTf, the urea-denaturation curves

480 collected at various concentrations of salts and sucrose for

481 iron release from Fe2oTf and unfolding of Fe2oTf and apo-

482 oTf were analyzed using Eq. (2) [101]. The resulting values

483 of DGoD and mg for iron release and unfolding are shown in

484 Table S2 and Table S3 (Supporting Information), respec-

485 tively. At each salt concentration, the DGoD for unfolding is

486 larger for Fe2oTf than the apo-oTf [Table S3 (Supporting

487 Information)], which suggests that the conformational sta-

488 bility of apo-oTf increases upon complex formation with iron.

489 At each salt concentration, the DGoD for unfolding of Fe2oTf

490 (based on ellipticity values at 222 and 282 nm) is also larger

491 than the DGoD measured for iron release (based on fluores-

492 cence emission at 340 nm). This finding further confirms that

493 the Fe2oTf responds to increased urea concentration by means

494 of a coupled process in which the urea-induced iron release

495 reaction occurs prior to the unfolding reaction. The urea mid-

496 points for iron release and unfolding (Cm = DGoD

�

mg) were

497 calculated as a function of salt (NaCl and Na2SO4) and

498 sucrose concentration [Table S2 and Table S3 (Supporting

499 Information)]. The plots of Cm for iron release of Fe2oTf as

500 function of salt and sucrose concentration are shown in

501 Fig. 4d. Figure 5e and f shows the plots of Cm for unfolding

502 of secondary structures of Fe2oTf and apo-Tf, respectively, as

503 function of salt and sucrose concentration. The plots of Cm for

504 unfolding of tertiary structures of Fe2oTf and apo-Tf as

505 function of salt and sucrose concentration are shown in

506 Fig. 6c and d, respectively. As salt concentration is increased,

507 the Cm values for iron release from Fe2oTf and unfolding of

508 Fe2oTf and apo-oTf decrease at low salt concentrations

509 [B0.1(±0.02) M Na2SO4 or B0.35(±0.15) M NaCl] but

510 increase at higher salt concentrations (Figs. 4d; 5e, f; 6c, d).

511 At higher concentrations, Na2SO4 has a greater effect than

512 that of NaCl in increasing the Cm for both iron release from

513 Fe2oTf and unfolding of Fe2oTf and apo-oTf (Figs. 4d; 5e, f;

514 6a, d), which indicates that at higher concentrations, salt ions

515 stabilize the oTf-Fe3? complex and also increase the struc-

516 tural stability of the proteins according to Hofmeister series

517 [28, 31–34].

518 Effects of salts and sucrose on the pH-linked kinetics

519 of urea denaturation induced iron release from FeNoTf

520 complex

521 To gain insight into the linkage of protein and active site

522 stability to the dynamics of Fe3? release, the kinetics of

523urea denaturation-induced Fe3? release from FeNoTf were

524studied as a function of salt (NaCl and Na2SO4) and

525sucrose concentration at pH 7.4 (Fig. 7a, c) and 5.6

526(Fig. 7b, d). Figure 7a and b shows the representative urea

527denaturation induced iron release kinetic traces of FeNoTf

528measured at pH 7.4 and 5.6, respectively, in the absence of

529salt at 37 �C. Both kinetic traces were fitted well in a

530mono-exponential decay function with rate constant, kobs,

531of *3.8 9 10-3 and 5.8 9 10-1 s-1 at pH 7.4 and 5.6,

532respectively. Clearly by decreasing the pH from 7.4 to 5.6,

533the kobs is increased by *150 fold [Table S4 (Supporting

534Information)]. At pH *7.4, with raising the concentration

535of NaCl from 0.0 to 0.25 M, kobs is increased by*1.5 fold

536[Fig. 7c and Table S4 (Supporting Information)]. On the

537contrary, with increasing the concentration of sucrose from

5380.0 to 0.5 M, kobs is decreased by*1.3 fold [Fig. 7c; Table

539S4 (Supporting Information)].

540Figure 7c and d shows the kobs vs [salts] or [sucrose]

541plots for the Fe3? release from FeNoTf complex at pH 7.4

542and 5.6, respectively. At pH 7.4, as salt concentration is

543increased, the kobs value increases at low salt concentration

544[B0.12(±0.03) M NaCl or Na2SO4] but decreases at higher

545salt concentrations [Fig. 7c; Table S4 (Supporting Infor-

546mation)]. This result is consistent with an earlier report that

547showed that the rate constant for iron release from the

548isolated N-lobe of human sTf by EDTA in the absence of

549urea increases at low salt concentrations (B0.3 M KCl) but

550decreases at higher salt concentrations [18]. Figure 7c also

551indicates that at higher salt concentrations, the extent of

552decrease in the kobs is more for Na2SO4 than that of NaCl.

553This result suggests that at higher concentrations (pH 7.4),

554the salt ions behave according to Hofmeister series [28, 31–

55534].

556At pH 5.6, as salt concentration is increased, the kobs557increases mono-exponentially and plateau at

558*0.4(±0.1) M NaCl or*0.2(±0.05) M Na2SO4 [Fig. 7d;

559Table S4 (Supporting Information)]. This result is consis-

560tent with an earlier report that showed that the rate constant

561for iron release from the N-lobe of human sTf at pH 5.6

562increases mono-exponentially with salt concentration [72].

563Figure 7d also indicates that at pH 5.6, the extent of

564increase in kobs is higher for Na2SO4 than that of NaCl.

565This suggests that Na2SO4 as compared to NaCl is more

566effective in accelerating iron release from FeNoTf.

567We have recently shown that at low to intermediate

568concentrations of salt (B0.35 M), an extended Debye-

569Huckel model can be used to explain the effect of ionic

570strength (I) on the rate of iron release from human serum

571diferric transferrin [30]. Inset of Fig. 7d shows the log kobs572vs I1/2/(1 ? I1/2) plot for FeNoTf at pH 5.6. Clearly, the log

573kobs increase linearly with I1/2/(1 ? I1/2), which indicates

574that at low to intermediate concentrations of salt, the

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575 Debye-Huckel screening of diffusive counterions acceler-

576 ates Fe3? release from FeNoTf at pH 5.6.

577 Salt dependence of the apparent activation enthalpy

578 (DH�) of Fe3? release from FeNoTf

579 The effects of salts on the kinetics of Fe3? release were

580 further evaluated by studying the effect of temperature on

581 the urea denaturation-induced iron release rate constant,

582 kobs of FeNoTf both in the absence and presence of salt

583 (NaCl and Na2SO4). Figure 8a and b show the Arrhenius

584 plots (ln kobs vs 1,000/T) for Fe3? release at pH 7.4 and 5.6,

585 respectively, in the absence of additive and in the presence

586 of *0.25 M NaCl and 0.1 M Na2SO4. To determine the

587 salt dependence of apparent activation energy or activation

588 enthalpy, DH� (Ea & DH�) for Fe3? release, the Arrhenius

589 plots in Fig. 8a and b were fitted linearly to Arrhenius

590 equation [30, 35]. The resulting DH� for iron release are

591provided in Table 1. As salt concentration is increased

592from 0.0 to 0.25 M NaCl or 0.1 M Na2SO4, the value of

593DH� for iron release decreases significantly (Table 1). This

594indicates that at low to intermediate concentrations of salt,

595the salt-induced acceleration of Fe3? release is accompa-

596nied by substantial decrease in DH�.

597Effects of salts on the pH-linked kinetics of reductive

598release of iron from FeNoTf complex by sodium

599dithionite using BPS

600To understand how electrostatic interactions influence the

601dynamics of reductive release of iron (i.e., Fe2? release)

602from FeNoTf complex, the kinetics of reductive release of

603iron from FeNoTf complex by sodium dithionite in the

604presence of BPS were studied as a function of salt (NaNO3

605and Na2SO4) and sucrose concentration at pH 7.4 and 5.6.

606Figure 9a and b shows the representative reductive iron

[Urea] (M)

0 2 4 6 8 10

Fra

cti

on

Un

fold

ed

0.0

0.5

1.0

(a)

[Salts] (M)

0.0 0.3 0.6 0.9 1.2

0.0 0.3 0.6 0.9 1.2

4.8

5.8

6.8

7.8


2.5

3.5

4.5

5.5

(c)

(d)

[Urea] (M)

0.0 1.5 3.0 4.5 6.0 7.5

Fra

cti

on

Un

fold

ed

0.0

0.5

1.0

(b)

Cm

(M)

Cm

(M)

Fig. 6 Panel a shows the near-UV CD monitored (282 nm) unfolding

curves of Fe2oTf measured at pH 7.4 in the absence (unfilled circle)

and presence of *0.1 (unfilled triangle), 0.25 (filled diamond), 0.5

(filled square), and 1.0 M NaCl (unfilled square); *0.1 (filled

triangle), and 0.35 M Na2SO4 (filled inverted triangle). The near-UV

CD monitored (282 nm) unfolding curves of apo-oTf measured at pH

7.4 in the absence (unfilled circle) and presence of *0.1 (unfilled

triangle), 0.25 (filled diamond), 0.5 (filled square), and 1.0 M NaCl

(unfilled square); *0.1 (filled triangle), 0.35 M Na2SO4 (filled

inverted triangle) and *0.5 M sucrose (filled circle) are shown in

panel b. The solid lines in panel a and panel b represent a non-linear

least squares fit of the data to Eq. (2). The urea-induced unfolding

midpoint, Cm (=DGoD=mg) was calculated as a function of salt (NaCl,

and Na2SO4) and sucrose concentration. Panel c shows the salt [NaCl

(unfilled square), and Na2SO4 (filled triangle)] dependence of Cm of

Fe2oTf at pH 7.4. The salt [NaCl (unfilled square), and Na2SO4 (filled

triangle)] and sucrose (filled circle) dependence of Cm of apo-oTf at

pH 7.4 is shown in panel d

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607 release kinetic traces of FeNoTf measured at pH 7.4 and

608 5.6, respectively, in the absence of salt at 25 �C. The

609 kinetics results of reductive iron release were well fitted to

610 a mono-exponential rise function with rate constants, kobs,

611 of *1.1 9 10-2 and 9.0 9 10-2 s-1 at pH 7.4 and 5.6,

612 respectively. Clearly, decreasing the pH from 7.4 to 5.6,

613 the kobs is increased by *8.2 fold [Table S5 (Supporting

614 Information)]. At pH 7.4, with increasing salt concentration

615 from 0.0 to 0.1 M NaNO3 or Na2SO4, kobs is increased by

616 *1.3 fold [Fig. 9c; Table S5 (Supporting Information)].

617 Figure 9c and d shows the kobs vs [salts] plots for Fe2?

618 release from FeNoTf complex at pH 7.4, and 5.6, respec-

619 tively. At pH 7.4, as salt concentration is increased from

620 0.0 to 1.0 M NaNO3 or 0.5 M Na2SO4, the kobs initially

621 increases at low salt concentrations [B0.1(±0.03) M

622 NaNO3 or Na2SO4] but decreases at higher salt concen-

623 trations [Fig. 9c; Table S5 (Supporting Information)]. The

624extent of decrease in kobs at higher salt concentrations is

625more for Na2SO4 than that of NaNO3. This finding suggests

626that at higher concentrations (pH 7.4), the salt ions behave

627according to Hofmeister series [28, 31–34].

628At pH 5.6, as salt concentration is increased, the kobs629increases mono-exponentially and level off at

630*0.4(±0.1) M NaNO3 or *0.2(±0.05) M Na2SO4

631[Fig. 9d; Table S5 (Supporting Information)]. Figure 9d

632also shows that at low to intermediate concentrations of salt

633(B0.2 M NaNO3 or Na2SO4), Na2SO4 as compared to

634NaNO3 is more effective in accelerating Fe2? release from

635FeNoTf. Inset of Fig. 9d represents the log kobs vs I1/2/

636(1 ? I1/2) plot for FeNoTf at pH 5.6. The log kobs increases

637linearly with I1/2/(1 ? I1/2), indicating that at low to

638intermediate concentrations of salt, the Debye-Huckel

639screening of diffusive counterions promotes the reductive

640release of iron from FeNoTf at pH 5.6.

Time (sec)

Ab

so

rba

nc

e (

46

5 n

m)

0.005

0.010

0.015

0.020


ko

bs

(sec

-1)

0.002

0.004

0.006

(a) (c)

Time (sec)

Ab

so

rba

nc

e (

46

5 n

m)

0.00

0.02

0.04

0.06


ko

bs

(sec

-1)

0.5

1.0

1.5

(b) (d)

I1/2/(1+I

1/2)

0.00.20.40.6

log

ko

bs (

sec

-1)

-0.2

0.0

0.2

0.4

0 400 800 1200 1600 0.0 0.4 0.8 1.2 1.6

0 10 20 30 40 50 0.0 0.4 0.8 1.2 1.6

Fig. 7 Kinetics of urea-induced iron release from FeNoTf at*37 �C

as monitored by the change in absorbance at 465 nm. Panels a and

b present single-phase iron release kinetic traces of FeNoTf in the

absence of salt at pH 7.4 and 5.6, respectively. The solid lines in

panels a and b show least-squares fit of the data to a single

exponential function. The resulting rate constants for iron release,

kobs, are plotted as a function of salt [NaCl (unfilled square), and

Na2SO4 (filled triangle)] and sucrose (filled circle) concentration in

panels c and d, respectively. The solid lines in panel c through data

have been drawn to guide the eye only. The solid lines in panel

d represent non-linear least squares fit of the data to a single-

exponential function. The dependences of log kobs on I1/2/(1 ? I1/2) at

pH 5.6 are also shown in the inset of panel d [NaCl (unfilled square),

and Na2SO4 (filled triangle)]. The solid line in the inset of panel

d represents linear least squares fits to the data

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641 Discussion

642 How could low concentrations of salt destabilize

643 the Fe2oTf and apo-oTf?

644 Salt ions could affect the stability of natively folded pro-

645 teins in a complex way. The Hofmeister series is often used

646 to define the effectiveness of salt ions in stabilizing the

647 proteins at higher concentrations ([0.5 M) [103]. How-

648 ever, at low concentrations (\0.3 M), salt ions can exhibit

649 either stabilizing or destabilizing effect which varies with

650 different proteins. For example, SO42- stabilizes native

651 Ribonuclease A [104], but destabilizes human sTf [30].

652 There is growing evidence that the specific interactions

653 between anions and protein surface affect protein stability

654 [30, 105, 106]. The current study presents direct

655experimental evidence that at low salt concentrations

656[B0.1(±0.02) M Na2SO4 or B0.35(±0.15) M NaCl], both

657NaCl and Na2SO4 destabilize the oTf-Fe3? complex and

658also decrease the structural stability of Fe2oTf and apo-oTf

659(Figs. 4d; 5e, f; 6c, d). Since Na2SO4 is a kosmotropic salt

660which generally has a stabilizing effect on proteins [104,

661106, 107], the destabilization of the oTf-Fe3? complex and

662decreased structural stability of Fe2oTf and apo-Tf by

663Na2SO4 are highly unusual. Few other examples are also

664available which showed that at low to intermediate con-

665centrations of Na2SO4 (\0.15 M), the ionic screening of

666electrostatic interactions can destabilize human sTf [30],

667human plasma lipoproteins [5, 6], human prion protein

668[36], and archaebacterial carboxypeptidase [35]. At higher

669salt concentrations [C0.1(±0.02) M Na2SO4 or

6700.35(±0.15) M NaCl], the destabilizing effect was pro-

671gressively reversed and salt ions acted as according to their

672ranking in the Hofmeister series (Figs. 4d, 5e, 6c).

673The decrease in the structural stability of apo-oTf

674[Figs. 5f, 6d; Table S3 (Supporting Information)] at low

675salt concentrations [B0.1(±0.02) M Na2SO4 or

6760.25(±0.05) M NaCl], suggests that the destabilization of

677Fe2oTf complex by salt is attributed to the salt-induced

678destabilization of oTf-Fe3? complex and weakening of

679Coulombic interactions at the protein surface. The current

680study provides several experimental supports that the salt-

681induced destabilization of Fe2oTf complex and apo-oTf are

682due to the ionic screening of electrostatic interactions.

683First, the salt concentration range (0.01–0.35 M) that

684destabilizes the Fe2oTf complex and apo-oTf (Figs. 4d; 5e,

685f; 6c, d), the effects of salt on macromolecular stability are

686governed by ionic screening. On the other hand, the spe-

687cific ion binding effects of salt often occurs at less than one

688millimolar concentrations [108] while the hydrophobic

689effect dominates at molar concentrations ([0.5 M) [4, 5, 9,

69029, 107]. The single exponential character of salt effect on

691the stability of oTf-Fe3? complex (Fig. 1d) is typical of the

692electrostatic screening by diffusive counterions (e.g., co-

693solute screening) [5, 29, 30]. Finally, the opposite effects of

694salt (destabilizing) and sucrose (stabilizing) on the stability

695of Fe2oTf and apo-oTf confirm that the hydrophobic effect

696is not the factor for the salt-induced destabilization of these

697proteins [Figs. 4d; 5e, f; 6d; Table S2, Table S3 (Sup-

698porting Information)].

699pH and anion-binding-induced conformational changes

700play a vital role in promoting iron release from Fe2oTf

701complex

702During the past few decades, several investigators have

703spurred great interest in understanding the role of pH and

704anion-binding-induced conformational changes that prompt

705iron release from the monoferric- and diferric-Tfs [11–25,

ln k

ob

s(s

ec

-1)

-6

-5

-4

[1000/T] (K-1)

3.15 3.20 3.25 3.30 3.35 3.40

ln k

ob

s(s

ec

-1)

-1.0

-0.5

0.0

0.5

(a)

(b)

Fig. 8 Arrhenius plots derived from the temperature dependence of

rate constant of urea-induced iron release from FeNoTf at pH 7.4 and

5.6 are shown in panels a and b, respectively [(unfilled circle), no

addition; (unfilled square), 0.25 M NaCl; and (filled triangle), 0.1 M

Na2SO4]. The activation enthalpy derived from linear least-squires

fitting to Arrhenius plots for kobs is summarized in Table 1

Table 1 Salt dependence of the activation enthalpy DH� (Ea = DH�)

of Fe3? release from FeNoTf.*

pH *7.4 pH *5.6

DH� DH�

Control 18.0 12.0

0.25 M NaCl 14.5 9.3

0.1 M Na2SO4 15.5 8.3

* The uncertainty of DH� values reported here is ±0.5 kcal mol-1

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706 30, 72, 74, 77, 80, 83]. Dewan et al. [15] suggested that the

707 charge repulsion and a conformational change resulting

708 from the protonation of the dilysine pair (Lys209-Lys301

709 located on the opposite domains of N-lobe) appear to be the

710 main driving force to trigger cleft opening of N-lobe at low

711 pH. A recent hydrogen/deuterium exchange study of

712 N-terminal lobe of human sTf in combination with mass

713 spectrometry suggested that at slightly acidic pH condi-

714 tions, holoprotein exists in open conformation [13]. Few

715 earlier site-directed mutagenesis studies of human sTf with

716 iron bound only to the N-lobe indicate that charge repul-

717 sion and a conformational change resulting from the pro-

718 tonation of the so-called dilysine pair (Lys206-Lys296) are

719 the main driving forces for anion-induced iron release

720 acceleration upon decrease in pH [17, 72, 83].

721 Kretchmar et al. [77] reported that the anion requirement

722 is a prerequisite for iron release from monoferric-Tf. The

723 anions requirement is also necessary for iron release from

724Fe3?-Tf-receptor complex [81], signifying the implications

725of some anions for the cellular iron release. Few earlier

726kinetic and spectroscopic studies revealed that the anion-

727binding-induced conformational change involves in salt-

728induced release of iron from the monoferric- and differic-

729Tfs [30, 68, 72, 80]. Recent anion-dependent fluorescence

730studies of diferric human sTf at low pH also provided

731direct evidence that the anion binding to the protein

732induces a structural or conformational change around one

733or more Trp residues near the iron binding site [30]. In an

734earlier study, Baldwin et al. [80] have argued that both

735conformational change and electrostatic interactions

736induced by ion binding to the protein prompt iron release

737from monoferric-Tfs. The current study showed that in the

738presence of salt, there is a considerable change in the slope

739of the Arrhenius plot (Fig. 8a, b), insofar this is due to

740anion-induced conformational change in the protein. Fur-

741thermore, the single exponential saturation effect of salt on

Time (sec)

0 100 200 300 400 500

Ab

so

rba

nc

e (

53

8 n

m)

0.00

0.02

0.04

[Salts] (M)

0.00 0.25 0.50 0.75 1.00

ko

bs (

sec

-1)

0.002

0.008

0.014

(a) (c)

Time (sec)

0 50 100

Ab

so

rba

nc

e (

53

8 n

m)

0.00

0.02

0.04

[Salts] (M)

0.0 0.2 0.4 0.6

ko

bs (

sec

-1)

0.05

0.10

0.15

0.20(b) (d)

I1/2/(1+I

1/2)

0.0 0.2 0.4 0.6

log

ko

bs (

se

c-1)

-1.0

-0.8

-0.6

Fig. 9 Kinetics of reductive release of iron from the FeNoTf complex

by sodium dithionite using BPS as monitored by the change in

absorbance at 538 nm. Panels a and b present single-phase reductive

iron release kinetic traces of FeNoTf in the absence of salt at pH 7.4

and 5.6, respectively. The solid lines in panels a and b show least-

squares fit of the data to a single exponential function. The resulting

rate constants for reductive iron release, kobs, are plotted as a function

of salt [NaNO3 (unfilled square), and Na2SO4 (filled triangle)]

concentration in panels c and d, respectively. The solid lines in panel

c through data have been drawn to guide the eye only. The solid lines

in panel d represent non-linear least squares fit of the data to a single-

exponential function. The dependences of log kobs on I1/2/(1 ? I1/2) at

pH 5.6 is also shown in the inset of panel d [NaNO3 (unfilled square)

and Na2SO4 (filled square)]. The solid line in the inset of panel

d represents linear least squares fits to the data

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742 the iron release rate constant at pH 5.6 (Figs. 7d, 9d)

743 confirms that anion-binding-induced conformational

744 change promotes Fe2? and Fe3? release from FeNoTf.

745 Electrostatic screening effect of electrolytes controls

746 the kinetics of iron release from FeNoTf

747 While the kinetics of iron release from the monoferric- and

748 diferric-Tfs have been studied as a function of salt con-

749 centration [17, 18, 30, 72, 74–88], few have been studied in

750 sufficient details to provide the mechanism by which the

751 salt ions influence the kinetics of iron release [30, 35].

752 Especially, the role played by electrostatic interactions in

753 controlling the kinetics of Fe2? and Fe3? release are not

754 clearly known. The current study gives several experi-

755 mental evidences which suggest that salt ions prompt Fe2?

756 and Fe3? release from FeNoTf complex through the ionic

757 screening effects (Figs. 7c, d; 9c, d). If salt-induced Fe2?

758 and Fe3? release from FeNoTf are due to the interference

759 with hydrophobic interactions, then kosmotropic salt (i.e.,

760 increase the macromolecular stability by strengthening the

761 hydrophobic interactions [31, 32, 34]) such as Na2SO4

762 should slow down the Fe2? and Fe3? release from FeNoTf

763 via strengthening the intramolecular hydrophobic interac-

764 tions. But, within the pH range of 7.4–5.6, the current

765 results reveal that at low salt concentrations

766 [B0.12(±0.05) M NaCl/NaNO3 or Na2SO4 (pH 7.4);

767 B0.4(±0.1) M NaNO3/NaCl or 0.2(±0.05) M Na2SO4 (pH

768 5.6)], all the salts examined (NaCl, NaNO3, and Na2SO4)

769 here promote Fe2? and Fe3? release from FeNoTf. The

770 current results also reveal that at higher salt concentrations

771 [C0.12(±0.05) M NaCl/NaNO3 or Na2SO4], the salt ions

772 retard the Fe2? and Fe3? release from FeNoTf at pH 7.4

773 (Figs. 7c, 9c), while they accelerate the Fe2? and Fe3?

774 release at pH 5.6 (Figs. 7d, 9d). At low to intermediate

775 concentrations (0.01–0.35 M), the effects of salt on protein

776 stability are generally attributed to ionic screening effect

777 [9, 29, 30]. On the other hand, the specific ion binding

778 effect of salt is generally apparent at millimolar concen-

779 trations [108] while hydrophobic effect occurs at molar

780 concentrations ([0.5 M) [4, 5, 9, 29, 30, 107]. In dilute to

781 moderately concentrated ionic solution, an extended

782 Debye-Huckel model can easily interpret the effect of ionic

783 strength on the rate of iron release from human serum

784 diferric transferrin [30]. At pH 5.6, the log kobs for Fe2?

785 and Fe3? release increases linearly with I1/2/(1 ? I

1/2)

786 (Insets of Figs. 7d and 9d), indicating that the Debye-

787 Huckel screening of diffusive counterions accelerates the

788 iron release from FeNoTf under endosomal pH conditions.

789 Finally, the opposite effects of salts and sucrose (i.e., salt

790 prompts iron release while sucrose inhibits it) confirm that

791 the acceleration of the iron release by salt is not due to the

792 hydrophobic effect.

793Both anion binding to KISAB sites and electrostatic

794screening effect of electrolytes control the kinetics

795of iron release from FeNoTf

796To emphasize the allosteric effect on iron release, Egan

797et al. [76, 77, 82, 86] proposed naming the sites to which

798nonsynergistic anions bind as ‘‘kinetically significant

799anion binding’’ or KISAB sites. It is widely accepted that

800the anions binding to KISAB sites influence the rate of

801iron release from Tfs [109, and references therein].

802However, the KISAB sites have not been well character-

803ized [109]. Few earlier studies have shown that multiple

804KISAB sites exist for each lobe of Tf [15, 17, 22, 72, 74,

80583, 109]. A recent report shows that Arg143 serves as an

806authentic KISAB site in the N-lobe of human sTf [74].

807While both electrostatic effect (Debye-Huckel screening

808of diffusive counterions) and anions binding to the KISAB

809sites influence the rates of iron release from FeNoTf, the

810extent of these two effects on rates of iron release will

811depend on the pH of reaction medium and the concen-

812tration of anions. The N-lobe is the first lobe to release

813iron [62], so it is to be noted that the concentration of

814anion has the greatest effect on iron release from the

815N-lobe [74]. At low salt concentrations and at pH 7.4,

816Debye-Huckel screening of diffusive counterions facili-

817tates the iron release from FeNoTf but under these con-

818ditions of pH and salt, anions binding to KISAB sites may

819have little effect on iron release because at neutral pH

820only weak interactions exist between the anions and KI-

821SAB sites [72]. At mildly acidic pH (pH *5.6), the

822anions’ binding strength to KISAB sites increases [72],

823which results in acceleration of iron release from N-lobe

824of Tfs [72]. The previous section has presented several

825lines of evidence which suggests that at pH 5.6, Debye-

826Huckel screening of diffusive counterions facilitates iron

827release from FeNoTf. So, at pH *5.6, it is likely that both

828anions binding to the KISAB sites and ionic screening of

829electrostatic interactions together facilitate the iron release

830from FeNoTf.

831Conclusions

832To assess the effects of salts and sucrose on the stabiliza-

833tion of oTf-Fe3? complex and structural stability of Fe2oTf,

834we have studied the Fe2oTf as a function of pH and urea in

835the presence salt (NaCl, Na2SO4, NaBr, and NaNO3) and

836sucrose. Interestingly, the low concentrations of salt

837destabilize the oTf-Fe3? complex and also decrease the

838structural stability of Fe2oTf. In contrast, the higher con-

839centrations of salt stabilize the oTf-Fe3? complex and also

840increase the structural stability of Fe2oTf. The current

841study reveals that the destabilization of Fe2oTf by salt

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842 results from both destabilization of oTf-Fe3? complex and

843 ionic screening of electrostatic interactions. At pH 7.4, the

844 low concentrations of salt (NaCl, NaNO3, and Na2SO4)

845 promote the Fe2? and Fe3? release from FeNoTf. At higher

846 concentrations, these salts inhibit the Fe2? and Fe3? release

847 and salt ions behave according to Hofmeister series. At pH

848 5.6, these salts also accelerate the Fe2? and Fe3? release

849 from FeNoTf. However, at pH 5.6, the salt-induced accel-

850 eration of Fe2? and Fe3? release is particularly pronounced

851 at low salt concentrations but is saturated at

852 *0.2(±0.05) M Na2SO4 or *0.4(±0.1) M NaNO3/NaCl,

853 which suggests that the Coulombic interactions play crucial

854 role in the accelerating the iron release from FeNoTf under

855 endosomal pH conditions. The current results also suggest

856 that there is a general effect from Debeye-Huckel screening

857 that operates in addition to the anion binding to KISAB or

858 any unrecognized sites in regulating the kinetics of iron

859 release from FeNoTf at pH *5.6.

860 Acknowledgments This work was supported by DST-SERC Fast861 Track Research Grant (to R.K., project No. SR/FT/CS-070/2009), a862 Ramalingaswami Re-entry Fellowship by DBT, India (to D.S., BT/863 RLF/RE-ENTRY-33-2010), and UGC major grant (to R.K., F. No.864 41-258/2012 (SR), Government of India.

865

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AQ1 Please check and confirm the edit in the following sentence: The contribution of electrostatic interactionsto the stability of ovotransferrin-Fe3? (oTf-Fe3?) complex has been assessed by equilibriumexperiments that measure iron retention level of diferric-ovotransferrin (Fe2oTf) as a function of pHand urea in the presence of salts (NaCl, Na2SO4, NaBr, NaNO3) and sucrose at 25 �C

AQ2 Please confirm if both terms "urea-induced denaturation", and "urea denaturation-induced’ signifydifferent processes

AQ3 Please check and confirm the edit in the following sentence: This finding further confirms that the Fe2oTfresponds to increased urea concentration by means of a coupled process in which the urea-induced ironrelease reaction occurs prior to the unfolding reaction

AQ4 Please check and confirm the edit in the following sentence: However, at pH 5.6, the salt-inducedacceleration of Fe2? and Fe3? release is particularly pronounced at low salt concentrations but issaturated at *0.2(±0.05) M Na2SO4 or *0.4(±0.1) M NaNO3/NaCl, which suggests that theCoulombic interactions play crucial role in the accelerating the iron release from FeNoTf underendosomal pH conditions

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Electrostatic effects control the stability and iron release kinetics of ovotransferrin

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