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Spectroscopic analysis of the interaction between chromium (III) and apoovotransferrin
Journal of Luminescence 130 (2010) 2339–2345
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Spectroscopic analysis of the interaction between chromium (III) and apoovotransferrin Yingqi Li a,b, Bin Liu b, Binsheng Yang b,n a b
Department of Chemistry, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, PR China
a r t i c l e in fo
abstract
Article history: Received 19 March 2010 Received in revised form 27 May 2010 Accepted 19 July 2010 Available online 23 July 2010
Ovotransferrin (OTf) is a main member of the transferrin family that functions both as an iron transporter and an antibacterial agent. In this study, the thermodynamic property of the interaction between chromium (III) and ovotransferrin was investigated. The conditional binding constants for Cr3 + binding to the protein were determined by difference UV spectroscopy and were found to be log KC ¼13.087 0.24 and log KN ¼ 5.65 7 0.12. It was found that Cr3 + preferentially binds to the C-terminal site over the N-terminal site under these experimental conditions. The conformational changes in apoovotransferrin (apoOTf) during Cr3 + binding were studied by fluorescence spectroscopy using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as the fluorescence probe and by circular dichroism (CD) spectroscopy. The results show that a large conformational change in apoOTf can be attributed to binding of Cr3 + to the N-terminal site, instead of the C-terminal site. In addition, the binding of Cr3 + to apoOTf stabilizes the structure of OTf as determined by guanidine hydrochloride denaturation studies. These findings help advance our understanding of the biological effects of Cr3 + . & 2010 Elsevier B.V. All rights reserved.
Keywords: Apoovotransferrin Cr3 + Spectra Conformation change
1. Introduction Transferrins are a family of iron-binding proteins that require a synergistic anion in order for the proteins to interact with metals [1]. Although these proteins consist of a single chain of about 700 amino acid residues, they fold into two distinct homologous lobes connected by a short polypeptide [1,2]. Each lobe contains one high-affinity iron-binding site, which are designated as the C-terminal and the N-terminal sites. Transferrins (Tf) are mainly synthesized by hepatocytes and are present in blood plasma at a concentration of about 2.5 mg/mL. The major role of serum Tf is to carry iron from the sites of intake into the systemic circulation and to the cells and tissues [3]. Chromium is one of the most common elements in the earth’s crust and seawater and primarily exists in the environment in several oxidation states including metallic, trivalent and hexavalent chromium. Hexavalent chromium is highly toxic and is largely synthesized by the oxidation of the more common and naturally occurring trivalent chromium. Trivalent chromium is an essential nutrient with very low toxicity, which is found in most foods and nutrient supplements [4]. The appearance of chromic ions in transferrin can be found after oral or intravenous administration of chromic ions to mammals. Moreover, in vitro
n
Corresponding author. Tel.: +86 351 7016358. E-mail address: [email protected] (B. Yang).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.07.015
studies have shown that chromium binds to transferrin [5–7]; it has been proposed that transferrin is involved in chromium transport. The thermodynamics and kinetics between apotransferrin and metal ions have been widely studied [8–14]; however, the properties of chromium binding to apoovotransferrin (apoOTf) remain uncertain. Ovotransferrin (OTf) is a main member of the transferrin family that functions in the dual role as an iron transport and as an antibacterial agent [15]. We report herein a detailed characterization of Cr3 + binding to apoOTf using fluorescence, difference UV and CD spectroscopy.
2. Materials and methods 2.1. Materials Egg apoovotransferrin (apoOTf) was purchased from Sigma. (NH4)2Fe(SO4)2, CrCl3 6H2O, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), HEPES and guanidine hydrochloride (GdnHCl) were analytical grade. All containers were rinsed with 1.0 M HNO3 to diminish the influence of metal ions. A stock solution of CrCl3 in 0.1 M HCl was freshly prepared for each measurement. ApoOTf was prepared as previously described [13]. Protein concentration was determined using UV–vis spectroscopy by measuring the absorbance at 280 nm and calculating the
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concentration using the extinction coefficient e280 ¼91 200 cm 1 M 1 [16]. N-terminal monoferric ovotransferrin (FeN-OTf) was prepared by adding one equivalent of ferrous ammonium sulfate to the intact apoOTf proteins in 0.1 M HEPES, pH 7.4. Exposure to air for 30 min enabled the reaction. The solution was used without further purification.
2.2.1. Difference UV spectra The spectra of Cr3 + binding to apoOTf were recorded using an HP8453 UV–vis spectrophotometer at intervals of at least 30 min; this gave a sufficient time for the hydrolysis of Cr3 + to reach equilibrium. To correct for dilution during each titration and normalize the results from different titrations, the absorbance data were converted to absorptivities (De) by dividing the absorbance by the analytical concentration of apoOTf. The titration curves were then constructed by plotting De versus r (the mole ratio of total metal to total ligand).
6 5 4
0.15
Absorbance
2.2. Methods
0.20
3 0.10 2
0.05 1 0 0.00 245
2.2.3. Circular dichroism (CD) The CD of Cr3 + binding to apoOTf in 0.01 M HEPES at pH 7.4 was monitored on a Jasco J-810 at room temperature. The spectra were recorded from 190 to 260 nm at intervals of at least 30 min. 2.2.4. Interaction with hydrophobic probes TNS The Cr3 + -dependent changes in the exposed hydrophobic surface of OTf were followed by monitoring the fluorescence properties of TNS as described previously [17].
3. Results 3.1. Difference UV spectra of Cr3 + binding to apoOTf Chromium (III) solutions containing various mole ratios of IDA:Cr were used to titrate apoOTf. The apoOTf solutions were prepared in 0.1 M HEPES buffer (pH 7.4) in the air. A typical series of UV spectra is shown in Fig. 1, which shows that upon addition of Cr3 + to apoOTf, two absorbance bands appeared at 257 and 295 nm. These bands are characteristic of a metal ion binding to phenolate groups of tyrosine residues at the specific iron bindsites of apoOTf and indicative of induced deprotonation. Values of De were calculated from the absorbance at 257 nm and plotted as
350
0:1 2.5 a 2.0
0.5:1 b
1.5:1 c
1.5
10:1
d 1.0
0.5
0.0 0.0
2.2.5. Determination of guanidine hydrochloride (GdnHCl) denaturation Solutions of apoOTf, Cr–OTf and Cr2–OTf in 0.1 M HEPES at pH 7.4 were mixed with increasing concentrations of GdnHCl. The fluorescence spectra of each solution were monitored on a Hitachi 850 fluorescence spectrophotometer at room temperature. The spectra were recorded at intervals of at least 15 min.
280 315 Wavelength/nm
3.0
Absorptivity/104 cm-1M-1
2.2.2. Fluorescence spectroscopy The fluorescence spectra of Cr3 + binding to apoOTf or FeN-OTf in 0.1 M HEPES at pH 7.4 were monitored on a Hitachi 850 fluorescence spectrophotometer at room temperature. The spectra were recorded at intervals of at least 30 min. To correct for dilution during each titration and normalize the results from different titrations, the fluorescence intensity at the maximum emission peak measured after each addition of Cr3 + was divided by the total ligand concentration to give molar fluorescence intensity (FM). The titration curves were then constructed by plotting FM versus r.
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[Cr3+]/[apoOTf] Fig. 1. (A) Difference UV spectra generated by the titration approximately of 2.0 ml apoOTf (10 uM) at pH 7.4. The volume of Cr3 + (0.37 mM) (mL) (0)0; (1)16; (2)28; (3)46; (4)66; (5)92; (6)128. (B) The plot of Deobs vs [Cr3 + ]/[OTf] for the titration of apoOTf with Cr3 + solution that contained variation of Cr3 + :IDA as indicated in the figure. (a) 0:1; (b) 1:1; (c) 1:0.5 and (d) 1:10.
a function of r, the ratio of total Cr3 + concentration to total apoOTf concentration, as shown in curve a of Fig. 1B. The continued increase in De for points beyond r ¼1.0 is a strong indication that Cr3 + can occupy both specific metal binding sites of OTf. The curve then begins to decrease around r ¼1.5. The trend is indicative of the occupation of a second site with a lower affinity and that excess Cr3 + is needed to saturate both OTf binding sites. ApoOTf was titrated with [Cr(IDA)x] (x ¼0.5, 1.5 or 10), and the absorbance at 257 nm was monitored. A set of titration curves for IDA:Cr ratios ranging from 0.5:1 to 10:1 is shown in Fig. 1B. With increasing IDA:Cr ratios, the plots show more significant decrease at high r values due to the competition between OTf and IDA. Using the initial slope of curve a, the molecular absorption coefficient (De) of OTf saturated with one Cr3 + was determined as
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
DeCr ¼(1.75 70.04) 104 M 1 cm 1. It was assumed that both sites have the same molar absorptivity. Thus, complete saturation of the two OTf binding sites would produce an observed De of about 3.50 104 M 1 cm 1. It is very clear from the curves in Fig. 1B that no reasonable amount of Cr3 + , with or without IDA, will produce De of 3.50 104 M 1 cm 1. In systems with IDA present, the accumulation of IDA is the limiting factor. In systems with no IDA, OH ions appear to be the limiting factor. The absorbance data obtained at different molar ratios of IDA:Cr were used to calculate the effective Cr3 + –OTf binding constants by using the methods described previously [18]. Briefly, the system is described by the mass balance equations for Cr3 + , OTf and IDA. (1)
Cr+Cr–apoOTf"Cr–apoOTf–Cr According to Eqs. (1) and (2), the effective Cr constants could be obtained:
(2) 3+
–OTf binding
K1 ¼
½CrapoOTf ½Cr½apoOTf
ð3Þ
K2 ¼
½CrapoOTfCr ½Cr½CrapoOTf
ð4Þ
where K1 and K2 are apparent binding constants dependent on the experimental conditions. [Cr] and [apoOTf] refer to the molarities of free Cr3 + and free apoOTf; and [Cr–apoOTf] and [Cr–apoOTf–Cr] refer to the molarities of proteins bound to one or two Cr3 + ions, respectively. If it is assumed that the two binding sites have the same molar absorptivity (DeCr) in the Cr–OTf complex, then it is possible to estimate the [Cr] and [apoOTf] terms in the mass balance equations describing the titration of apoOTf solution with free Cr3 + without added IDA. This can be done using the Cr3 + hydrolysis constants, the effective conditional constant of Ga(IDA) [19], and initial estimates of K1 and K2. Subsequently, values of DeCal at any point in the titration curve can be calculated using the above concentrations and the value of DeCr from
DeCal ¼
DeCr K1 ½Cr½apoOTf þ2DeCr K1 K2 ½Cr2 ½apoOTf ½apoOTftot
The average values for the two conditional binding constants, log K1 ¼13.08 70.24 and log K2 ¼5.6570.12, were determined by nonlinear least-squares fits of the titration curves at pH 7.4 in air (Fig. 1B). At this pH and with a buffer concentration of 0.1 M HEPES, the ambient bicarbonate concentration is 0.14 mM [20].
3.2. Site selectivity for Cr3 + binding to ApoOTf Since this high ratio of K1/K2 indicates that there does not appear to be significant cooperativity in the binding of Cr3 + ion to the two ovotransferrin binding sites [21], the equilibration of r1 equiv of Cr3 + ion with apoOTf should result in preferential binding to the more stable site. The stronger binding site can be identified by titrating with monoferric OTf [21,22]. A sample of N-terminal monoferric OTf (FeNOTf) was titrated with Cr3 + (aq) ion. The Cr3 + ion does not displace the more tightly bound Fe3+ from the FeN-OTf, so its binding to the vacant binding site can be monitored using fluorescence spectroscopy. The result is shown in Fig. 2. Curve a illustrates that Cr3 + does not appear to bind to the same extent to the two OTf binding sites, while curve b levels off after the addition of almost 1.0 equiv of Cr3 + . Moreover, the slopes of the two titration curves are similar. This implies that, in the early stages of the titrations, essentially all of the added Cr3 + was binding to the protein. Thus, it is apparent that Cr3 + ion will preferentially bind to the C-terminal site when r1 equiv of Cr3 + was added. These results suggest that log K1 and log K2 represent log KC and log KN, respectively.
3.3. The conformational change Fig. 3 shows that two minimum peaks appeared at 208 and 220 nm. It is evident that Cr3 + addition causes some significant CD spectral modifications in the intrinsic region, consistent with a small decrease of alpha helix content. TNS can be used as a fluorescence probe to investigate the hydrophobic environment of proteins. TNS interacts with protein through a non-covalent bond. The structure of TNS is shown in Scheme 1.
ð5Þ
where [apoOTf]tot refers to the analytical concentration of apoOTf, and DeCr is the molar absorptivity per bound Cr3 + for the Cr–OTf difference spectrum at 257 nm. Nonlinear least squares minimization is used to adjust the values of K1 and K2 to minimize the squares of the residuals between observed and calculated absorptivities, so that reliable estimates of the binding constants of Cr3 + to OTf can be obtained. In principle, the titration curve a in Fig. 1B can be fitted using the nonlinear least squares method, where K1 and K2 are the only quantities allowed to vary. In practice, the use of chelating agents to complete the removal of the metal ions can decrease the correlation between K1 and K2, thereby leading to more accurate K1 and K2 estimates, as described previously [18,20]. Thus, the titrations with Cr3 + were repeated using metal ion solutions containing a range of IDA concentrations. The chelating agent competes with apoOTf for Cr3 + and so at any given point in the titration, there is a distribution of Cr3 + between apoOTf and the IDA chelating agent; therefore, the observed absorptivity decreases as the IDA:Cr3 + ratio increases. A series of titration curves for Cr3 + –IDA solutions is shown in Fig. 1B. The titration data in Fig. 1B can be fitted using Eq. (5), in which K1 and K2 are the only adjustable parameters. For the 10:1 IDA:Cr titration curve, the maximum De observed ( 14 000 cm 1 M 1) does not exceed the calculated molar absorptivity of 17 500 cm 1 M 1, even at r ¼3.0. This suggests that only one Cr3 + is bound to apoOTf under these conditions, and so these data were used to calculate log K1.
9 Cr-OTf-FeN
Cr2-apoOTf 8
F/104M-1
Cr+apoOTf"Cr–apoOTf
2341
7 b 6
a
5
4 0
1
2 [Cr]/[OTf]
3
4
5
Fig. 2. Titration curves for the addition of Cr3 + (65 mM) to apoOTf (2.5 mM of 2.0 mL) and Cr3 + (17 mM) to N-terminal monoferric OTf (8.5 mM) in saturated air and 0.01 M HEPES at pH 7.4.
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addition of apoOTf, Cr–OTf, and Cr2–OTf to TNS all caused a blue shift in the fluorescence emission maximum to 441 nm. Addition of Cr2–OTf complex to TNS led to a further 20% decrease in the fluorescence intensity as compared with Cr–OTf addition. These data strongly suggest that the extent of exposed hydrophobic region is different for the different conformations of OTf. The results also indicate that the second Cr3+ binding to the N-terminus induced a large conformational change in apoOTf, whereas initial binding to the C-terminus only resulted in a minor conformational change.
20 15
Cr2-Tf
10
apoTf
CD/106 M-1mdeg
5 0
3.4. Determination of guanidine hydrochloride (GdnHCl) denaturation
-5 -10
Fig. 5A shows a typical GdnHCl denaturation curve, where the reaction reached equilibrium in 15 min. The fluorescence
-15 -20
190
200
210 220 230 240 Wavelength/nm
250
260
Fig. 3. CD spectra of apotransferrin in the far UV region in the absence and presence of Cr3 + , HEPES 0.1 M, pH 7.4.
5 4
60
270
3 Fluorescent intensity/a.u.
-25 180
70
2 50
1
40 30 20
Scheme 1. The structure of TNS.
10 350 300
0 300
apoOTf
330
360
390
420
Cr-OTf
250
120 200
Cr2-OTf
150
100
100
F356nm/104M-1
Fluorescence intencity/a.u.
Wavelength/nm
50 TNS alone 0
300
350
400
500 450 Wavelength/nm
550
600
80
apoOTf Cr-OTf Cr2-OTf
60
a b
650
40 c
3+
Fig. 4. Cr -dependent exposure of hydrophobic side chains. Hydrophobic exposure was monitored at room temperature in 0.1 M HEPES with the hydrophobic TNS. The fluorescence was measured at fixed lex ¼ 320 nm, slit¼ 10 nm.
Fig. 4 shows the fluorescence spectra of TNS in the absence and presence of different forms of ovotransferrin (i.e. apoOTf, Cr–OTf, and Cr2–OTf). For Cr2–OTf samples, the Cr:OTf molar ratio used was 2.5:1 in order to ensure an excess of Cr3+ to saturate the two ovotransferrin binding sites. As shown in Fig. 4, TNS emits fluorescence very weakly at 498 nm when excited at 320 nm in 0.1 M HEPES (pH 7.4). The
20 0
2
4 [Gu-HCl]/M
6
8
Fig. 5. (A) Fluorescence spectra of apoOTf (4.5 mM of 1.5 mL) with the addition of GdnHCl denaturant in 0.1 M HEPES at pH 7.4. The mass of Gdn–HCl (mg): (1)0 mg, (2)269 mg, (3) 307 mg, (4) 678 mg and (5) 1311 mg. (B) Plots denatured percentage with the concentration of GdnHCl. a: apoOTf; b: Cr–OTf; c: Cr2–OTf.
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
energy of unfolding (DGD) can be calculated.
intensity of apoOTf increased and shifted red from 336 to 356 nm with increasing concentration of GdnHCl, until 7.0 M was reached. Complete denaturatization results from insolubility or aggregation of the unfolded protein, indicating that the conformation of apoOTf was changed. GdnHCl denaturations of Cr–OTf and Cr2–OTf were also carried out under the same conditions and the fluorescence results were similar to that of apoOTf. Fig. 5B shows a typical solvent denaturation curve. A two-state mechanism was used for the analysis of GdnHCl denaturation curves [23]. The mechanism of the denaturation is shown in the following equation: N$D
2343
KD ¼ fD =fN ¼ ðyyN Þ=ðyD yÞ
ð7Þ
DGD ¼ RT ln KD
ð8Þ
DGD ¼ DGHD2 O þ m½GdnHCl
ð9Þ
DGHD2 O is DGD at zero concentration of denaturant, which represents an estimate of the conformational stability in the native states. Term m is defined as the apparent molar DGD, which indicates the conformational stability of the denatured states. The dependence of DGD on the concentration of GdnHCl is shown in Fig. 6. The parameters obtained are listed in Table 1.
ð6Þ 4. Discussion There is increasing interest of using chromium in biology [4–7]. It has been reported that Cr3 + can bind to the two specific metal-binding sites of transferrin and that there is a difference between the binding sites [6]; however, property studies on the interaction of Cr3 + binding to apoovotransferrin are scant. Complexation of metal ions to the phenolic group of the tyrosine residues of OTf in the specific iron site perturbs the J–J* transitions of the aromatic group and leads to two absorption bands at E257 and 295 nm in the difference UV spectrum. The changes in the UV spectra of OTf after Cr3 + binding are similar to
8
10
6
8
Free energies transfer (Kcal/mole)
Free energies of transfer (Kcal/mole)
in which only the native state (N) and the denatured state (D) are present at significant concentrations in the transition region. Values of y characteristic of the native state (yN) and of the denatured state (yD) can be obtained in the transition region by extrapolating the linear portions of the denaturation curve at low and high denaturant concentrations. These linear changes generally result from solvent affecting the properties of the native and denatured states. For a two-state mechanism, fD + fN ¼1, and y¼yDfD + yNfN ¼1 where fN and fD represent the fraction of protein present in the native and denatured states, respectively. Combining these equations, fD ¼(y yN)/(yD yN) and fN ¼(yD y)/(yD yN) followed by Eq. (8), the equilibrium constant (KD) and the free
4 2 0 -2 -4 -6 -8
6 4 2 0 -2 -4 -6 -8 -10 -12
-10 0
1
2
5 3 4 [GdnHCl]/M
6
7
8
0
2
1
3 5 4 [GdnHCl]/M
6
7
8
Free energies of transfer (Kcal/mole)
15
10
5
0
-5
-10 0
1
2
3 4 [GdnHCl]/M
5
6
Fig. 6. DGD as a function of GdnHCl molarity. DGD was calculated from the data in Fig. 5B using Eq. (11). (A) apoOTf; (B) Cr–OTf; (C) Cr2–OTf.
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Table 1 The parameters of unfolding of OTf. Sample
DGHD2 O a
mb
[D]1/2c
apoOTf CrC–OTf Cr2–OTf
7.12 70.600 9.97 70.620 11.1 70.448
2.08 70.130 2.85 70.160 3.46 70.130
2.00 2.50 3.50
a DGHD2 O represents estimating the conformational stability in the native states in kcal/mol. b Slopes of the free energy plots in Fig. 8, in kcal/mol M. c The [GdnHCl]1/2 values, in M.
those observed previously for the binding of other metal ions to the specific Fe3 + binding sites. Therefore, the observed absorption bands at 257 and 295 nm are attributed to binding to tyrosine ligands (J–J* transitions). Both UV and fluorescent spectral data suggest that Cr3 + can bind to both iron specific binding sites of apoOTf; however, Cr3 + binds more tightly to the C-terminal site. The binding constants for Cr3 + binding to apoOTf at the C- and N-terminus were determined to be log KC ¼13.0870.24, log KN ¼5.6570.12, respectively. CD spectroscopy is commonly used for general structural characterization of soluble proteins; the technique is particularly suited for monitoring the conformational state of proteins in solution. Specifically, CD provides direct information on the secondary structure of proteins [21,23]. Our data show that Cr3 + caused some significant CD spectral modifications in the intrinsic region, indicating a small decrease in alpha helix content, which is consistent with that induced by the addition of Al (V) [21]. These results suggest Cr3 + binding contributed to conformational changes. Conformational change was also determined by using TNS as a hydrophobic fluorescent probe. TNS does not fluoresce in water but fluoresces in organic solvents or hydrophobic environment [17]. It was found that the addition of apoOTf to TNS enhanced fluorescence intensity and blue-shifted from 494 to 441 nm, indicating that a hydrophobic patch was exposed on apoOTf surface. Addition of Cr–OTf to TNS decreased the fluorescence intensity at 441 nm, which suggests the exposed hydrophobic patch on apoOTf was changed (see Fig. 4). Furthermore, the results show that a decrease in exposed hydrophobic surface corresponds to an increase in Cr3 + concentration. It is believed that the exposed hydrophobic patch seems to be a specific property of the N-terminal site of apoOTf. Fig. 4 shows that while Cr3 + bound to the C-terminal binding site induces a small conformational change, the greatest conformational change could be attributed to the binding of Cr3 + to the N-terminal site of the protein. This result is consistent with previous reports [14,20]. The structural stability of the protein also exhibits a significant sensitivity to the interaction between apoOTf and Cr3 + . The protein denaturation with GdnHCl provides an estimate of stability dependent on the contributions from hydrophobic interactions [24]. Denaturation of the protein with GdnHCl was found to closely approach a two-state mechanism. Since Gdn + and Cl ions mask the positively and negatively charged amino acid side chains that lead to destabilized electrostatic interactions, GdnHCl has no effect on the conformational change. As seen in Fig. 5B, the proteins remained stable at low concentrations of GdnHCl. However, at high concentrations, GdnHCl becomes a denaturant regardless of the type of electrostatic interactions present in the protein. The binding of the Gdn + ions to the proteins is presumed to predominate and drive the equilibrium towards the unfolded state. After the conformational change, the peak position of the tryptophan resides would shift from 336 to 356 nm and the fluorescence intensity would gradually increase.
2O The free energy of unfolding (DGH D ) in the absence of denaturant is the estimate of protein stability to GdnHCl denaturation. Results for the GdnHCl denaturation experiments are summarized 2O in Table 1. For apooTf, Cr–OTf and Cr2–OTf, the DGH values D increased from 7.1 to 11.1 kcal/mol. The slope (m) for the plots of DGDH2 O vs [denaturant] is a measure of the ability of the denaturant to unfold a protein. The m values for apooTf, Cr–OTf and Cr2–OTf decreased from 2.08 to 3.46 kcal/mol M. The data also show that [GdnHCl]1/2 increased from 2.00 to 3.50 M. The results indicate that the interaction between Cr3 + and apoOTf stabilized the structure of apoOTf and the stabilization was further strengthened by increasing the concentration of Cr3 + .
5. Conclusions The interactions of Cr3 + with ovotransferrins were investigated by various spectroscopic studies. The comparison between Cr3 + titrations with apoOTf and monoferric OTf revealed that Cr3 + binds more tightly to the C-terminal site, which is different than other metal ions [11,12]. The interaction between TNS and the protein indicated a decrease in the extent of exposed hydrophobic surface is correlated to an increase in Cr3 + concentration. The large conformational change observed for apoOTf was mainly attributed to Cr3 + binding to the N-terminus site, while binding to the C-terminus would only result in minor conformational change. Also, the addition of Cr3 + stabilized the structure of apoOTf as indicated by the guanidine hydrochloride (GdnHCl) denaturation results. Transferrin has been reported to facilitate the cellular uptake of gallium and aluminum via the normal transferrin receptormediated mechanism for iron uptake [21,25]. Based on these in vitro property studies, it is suggested that transferrin might also be involved in Cr3 + transport. Further work is in progress to investigate the role of the receptors at the cell surface of Crsaturated transferrin. Such information may enhance our understanding of the fate of Cr3 + in the bloodstream as well as cell Cr3 + metabolism.
Acknowledgements We are thankful to the National Natural Science Foundation of China (Grant no. 20771068), the Shanxi Provincial Natural Science Foundation (Grant no. 2009011012-3 and 2010011011-1) and the Shanxi Provincial Natural Science Foundation for Youth (Grant no. 20051006). References [1] E.N. Baker, P.F. Lindley, J. Inorg. Biochem. 47 (1992) 147–160. [2] R. Sarra, R. Garratt, B. Gorinsky, H. Jhoti, P. Lindley, Acta Crystallogr. B. 46 (1990) 763–771. [3] N.D. Chasteen, Coord. Chem. Rev. 22 (1977) 1–36. [4] W.T. Cefalu, F.B. Hu, Diabetes Care 27 (2004) 2741–2751. [5] F. Borguet, R. Cornelis, N. Lameire, Biol. Trace Elem. Res. 27 (1990) 449–460. [6] P. Aisen, R. Aasa, A.G. Redfield, J. Biol. Chem. 244 (1969) 4628–4633. [7] Y. Sun, J. Ramirez, S.A. Woski, J.B. Vincent, J. Biol. Inorg. Chem. 5 (2000) 129–136. [8] R.E. Cowert, N. Kojima, G.W. Bates, J. Biol. Chem. 257 (1982) 7560–7565. [9] W.R. Harris, B. Gang, Polyhedron 16 (1997) 1069–1079. [10] B.S. Yang, J.Y. Feng, Y.Q. Li, F. Gao, Y.Q. Zhao, J.L. Wang, J. Inorg. Bicochem. 96 (2003) 416–424. [11] B.S. Yang, Y.Q. Li, Chem. Res. Chinese U 17 (2001) 6. [12] Y.Q. Li, B.S. Yang, Chin. J. Inorg. Chem. 16 (2000) 939–944. [13] Y.Q. Li, B.S. Yang, Chin. J. Chem. 22 (2004) 1153–1157. [14] E.N. Baker, Adv. Inorg. Chem. 11 (1994) 389–463. [15] J. Williams, T.C. Elleman, I.B. Kingston, A.G. Wilkins, K.A. Kuhn, Eur. J. Biochem. 122 (1982) 297–303. [16] M.A. James, J.V. Hans, J. Am. Chem. Soc. 115 (1993) 245–252. [17] W.O. McClure, G.M. Edelman, Biochemistry 5 (1966) 1908–1919.
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
[18] Y.Q. Li, B. Liu, Z.G. Ge, B.S. Yang, J. Photochem. Photobiol. B: Biol. 91 (2008) 137–142. [19] A.E. Martell, R.M. Smith, in: Critical Stability Constants, Plenum Press, New York, 1974, pp. 204. [20] W.R. Harris, V.L. Pecoraro, Biochemistry 22 (1983) 292–299. [21] W.R. Harris, Z.P. Wang, C. Brook, B.S. Yang, A. Islam, Inorg. Chem. 42 (2003) 5880–5889.
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[22] R. Hovius, P. Vallotton, T. Wohland, H. Vogel, Trends Pharmacol. Sci. 21 (2000) 266–273. [23] A. Rodger, B. Norden, in: Circular dichroism and linear dichroism, Oxford University Press, Oxford, New York, 1997. [24] O.D. Monera, C.M. Kay, R.S. Hodges, Protein Sci. 3 (1994) 1984–1991. [25] Y.Q. Li, B. Liu, C.G. Zhao, W. Zhang, B.S. Yang, Clin. Chim. Acta 366 (2006) 225–232.
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Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Spectroscopic analysis of the interaction between chromium (III) and apoovotransferrin Yingqi Li a,b, Bin Liu b, Binsheng Yang b,n a b
Department of Chemistry, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, PR China
a r t i c l e in fo
abstract
Article history: Received 19 March 2010 Received in revised form 27 May 2010 Accepted 19 July 2010 Available online 23 July 2010
Ovotransferrin (OTf) is a main member of the transferrin family that functions both as an iron transporter and an antibacterial agent. In this study, the thermodynamic property of the interaction between chromium (III) and ovotransferrin was investigated. The conditional binding constants for Cr3 + binding to the protein were determined by difference UV spectroscopy and were found to be log KC ¼13.087 0.24 and log KN ¼ 5.65 7 0.12. It was found that Cr3 + preferentially binds to the C-terminal site over the N-terminal site under these experimental conditions. The conformational changes in apoovotransferrin (apoOTf) during Cr3 + binding were studied by fluorescence spectroscopy using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as the fluorescence probe and by circular dichroism (CD) spectroscopy. The results show that a large conformational change in apoOTf can be attributed to binding of Cr3 + to the N-terminal site, instead of the C-terminal site. In addition, the binding of Cr3 + to apoOTf stabilizes the structure of OTf as determined by guanidine hydrochloride denaturation studies. These findings help advance our understanding of the biological effects of Cr3 + . & 2010 Elsevier B.V. All rights reserved.
Keywords: Apoovotransferrin Cr3 + Spectra Conformation change
1. Introduction Transferrins are a family of iron-binding proteins that require a synergistic anion in order for the proteins to interact with metals [1]. Although these proteins consist of a single chain of about 700 amino acid residues, they fold into two distinct homologous lobes connected by a short polypeptide [1,2]. Each lobe contains one high-affinity iron-binding site, which are designated as the C-terminal and the N-terminal sites. Transferrins (Tf) are mainly synthesized by hepatocytes and are present in blood plasma at a concentration of about 2.5 mg/mL. The major role of serum Tf is to carry iron from the sites of intake into the systemic circulation and to the cells and tissues [3]. Chromium is one of the most common elements in the earth’s crust and seawater and primarily exists in the environment in several oxidation states including metallic, trivalent and hexavalent chromium. Hexavalent chromium is highly toxic and is largely synthesized by the oxidation of the more common and naturally occurring trivalent chromium. Trivalent chromium is an essential nutrient with very low toxicity, which is found in most foods and nutrient supplements [4]. The appearance of chromic ions in transferrin can be found after oral or intravenous administration of chromic ions to mammals. Moreover, in vitro
n
Corresponding author. Tel.: +86 351 7016358. E-mail address: [email protected] (B. Yang).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.07.015
studies have shown that chromium binds to transferrin [5–7]; it has been proposed that transferrin is involved in chromium transport. The thermodynamics and kinetics between apotransferrin and metal ions have been widely studied [8–14]; however, the properties of chromium binding to apoovotransferrin (apoOTf) remain uncertain. Ovotransferrin (OTf) is a main member of the transferrin family that functions in the dual role as an iron transport and as an antibacterial agent [15]. We report herein a detailed characterization of Cr3 + binding to apoOTf using fluorescence, difference UV and CD spectroscopy.
2. Materials and methods 2.1. Materials Egg apoovotransferrin (apoOTf) was purchased from Sigma. (NH4)2Fe(SO4)2, CrCl3 6H2O, 2-p-toluidinylnaphthalene-6-sulfonate (TNS), HEPES and guanidine hydrochloride (GdnHCl) were analytical grade. All containers were rinsed with 1.0 M HNO3 to diminish the influence of metal ions. A stock solution of CrCl3 in 0.1 M HCl was freshly prepared for each measurement. ApoOTf was prepared as previously described [13]. Protein concentration was determined using UV–vis spectroscopy by measuring the absorbance at 280 nm and calculating the
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concentration using the extinction coefficient e280 ¼91 200 cm 1 M 1 [16]. N-terminal monoferric ovotransferrin (FeN-OTf) was prepared by adding one equivalent of ferrous ammonium sulfate to the intact apoOTf proteins in 0.1 M HEPES, pH 7.4. Exposure to air for 30 min enabled the reaction. The solution was used without further purification.
2.2.1. Difference UV spectra The spectra of Cr3 + binding to apoOTf were recorded using an HP8453 UV–vis spectrophotometer at intervals of at least 30 min; this gave a sufficient time for the hydrolysis of Cr3 + to reach equilibrium. To correct for dilution during each titration and normalize the results from different titrations, the absorbance data were converted to absorptivities (De) by dividing the absorbance by the analytical concentration of apoOTf. The titration curves were then constructed by plotting De versus r (the mole ratio of total metal to total ligand).
6 5 4
0.15
Absorbance
2.2. Methods
0.20
3 0.10 2
0.05 1 0 0.00 245
2.2.3. Circular dichroism (CD) The CD of Cr3 + binding to apoOTf in 0.01 M HEPES at pH 7.4 was monitored on a Jasco J-810 at room temperature. The spectra were recorded from 190 to 260 nm at intervals of at least 30 min. 2.2.4. Interaction with hydrophobic probes TNS The Cr3 + -dependent changes in the exposed hydrophobic surface of OTf were followed by monitoring the fluorescence properties of TNS as described previously [17].
3. Results 3.1. Difference UV spectra of Cr3 + binding to apoOTf Chromium (III) solutions containing various mole ratios of IDA:Cr were used to titrate apoOTf. The apoOTf solutions were prepared in 0.1 M HEPES buffer (pH 7.4) in the air. A typical series of UV spectra is shown in Fig. 1, which shows that upon addition of Cr3 + to apoOTf, two absorbance bands appeared at 257 and 295 nm. These bands are characteristic of a metal ion binding to phenolate groups of tyrosine residues at the specific iron bindsites of apoOTf and indicative of induced deprotonation. Values of De were calculated from the absorbance at 257 nm and plotted as
350
0:1 2.5 a 2.0
0.5:1 b
1.5:1 c
1.5
10:1
d 1.0
0.5
0.0 0.0
2.2.5. Determination of guanidine hydrochloride (GdnHCl) denaturation Solutions of apoOTf, Cr–OTf and Cr2–OTf in 0.1 M HEPES at pH 7.4 were mixed with increasing concentrations of GdnHCl. The fluorescence spectra of each solution were monitored on a Hitachi 850 fluorescence spectrophotometer at room temperature. The spectra were recorded at intervals of at least 15 min.
280 315 Wavelength/nm
3.0
Absorptivity/104 cm-1M-1
2.2.2. Fluorescence spectroscopy The fluorescence spectra of Cr3 + binding to apoOTf or FeN-OTf in 0.1 M HEPES at pH 7.4 were monitored on a Hitachi 850 fluorescence spectrophotometer at room temperature. The spectra were recorded at intervals of at least 30 min. To correct for dilution during each titration and normalize the results from different titrations, the fluorescence intensity at the maximum emission peak measured after each addition of Cr3 + was divided by the total ligand concentration to give molar fluorescence intensity (FM). The titration curves were then constructed by plotting FM versus r.
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[Cr3+]/[apoOTf] Fig. 1. (A) Difference UV spectra generated by the titration approximately of 2.0 ml apoOTf (10 uM) at pH 7.4. The volume of Cr3 + (0.37 mM) (mL) (0)0; (1)16; (2)28; (3)46; (4)66; (5)92; (6)128. (B) The plot of Deobs vs [Cr3 + ]/[OTf] for the titration of apoOTf with Cr3 + solution that contained variation of Cr3 + :IDA as indicated in the figure. (a) 0:1; (b) 1:1; (c) 1:0.5 and (d) 1:10.
a function of r, the ratio of total Cr3 + concentration to total apoOTf concentration, as shown in curve a of Fig. 1B. The continued increase in De for points beyond r ¼1.0 is a strong indication that Cr3 + can occupy both specific metal binding sites of OTf. The curve then begins to decrease around r ¼1.5. The trend is indicative of the occupation of a second site with a lower affinity and that excess Cr3 + is needed to saturate both OTf binding sites. ApoOTf was titrated with [Cr(IDA)x] (x ¼0.5, 1.5 or 10), and the absorbance at 257 nm was monitored. A set of titration curves for IDA:Cr ratios ranging from 0.5:1 to 10:1 is shown in Fig. 1B. With increasing IDA:Cr ratios, the plots show more significant decrease at high r values due to the competition between OTf and IDA. Using the initial slope of curve a, the molecular absorption coefficient (De) of OTf saturated with one Cr3 + was determined as
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
DeCr ¼(1.75 70.04) 104 M 1 cm 1. It was assumed that both sites have the same molar absorptivity. Thus, complete saturation of the two OTf binding sites would produce an observed De of about 3.50 104 M 1 cm 1. It is very clear from the curves in Fig. 1B that no reasonable amount of Cr3 + , with or without IDA, will produce De of 3.50 104 M 1 cm 1. In systems with IDA present, the accumulation of IDA is the limiting factor. In systems with no IDA, OH ions appear to be the limiting factor. The absorbance data obtained at different molar ratios of IDA:Cr were used to calculate the effective Cr3 + –OTf binding constants by using the methods described previously [18]. Briefly, the system is described by the mass balance equations for Cr3 + , OTf and IDA. (1)
Cr+Cr–apoOTf"Cr–apoOTf–Cr According to Eqs. (1) and (2), the effective Cr constants could be obtained:
(2) 3+
–OTf binding
K1 ¼
½CrapoOTf ½Cr½apoOTf
ð3Þ
K2 ¼
½CrapoOTfCr ½Cr½CrapoOTf
ð4Þ
where K1 and K2 are apparent binding constants dependent on the experimental conditions. [Cr] and [apoOTf] refer to the molarities of free Cr3 + and free apoOTf; and [Cr–apoOTf] and [Cr–apoOTf–Cr] refer to the molarities of proteins bound to one or two Cr3 + ions, respectively. If it is assumed that the two binding sites have the same molar absorptivity (DeCr) in the Cr–OTf complex, then it is possible to estimate the [Cr] and [apoOTf] terms in the mass balance equations describing the titration of apoOTf solution with free Cr3 + without added IDA. This can be done using the Cr3 + hydrolysis constants, the effective conditional constant of Ga(IDA) [19], and initial estimates of K1 and K2. Subsequently, values of DeCal at any point in the titration curve can be calculated using the above concentrations and the value of DeCr from
DeCal ¼
DeCr K1 ½Cr½apoOTf þ2DeCr K1 K2 ½Cr2 ½apoOTf ½apoOTftot
The average values for the two conditional binding constants, log K1 ¼13.08 70.24 and log K2 ¼5.6570.12, were determined by nonlinear least-squares fits of the titration curves at pH 7.4 in air (Fig. 1B). At this pH and with a buffer concentration of 0.1 M HEPES, the ambient bicarbonate concentration is 0.14 mM [20].
3.2. Site selectivity for Cr3 + binding to ApoOTf Since this high ratio of K1/K2 indicates that there does not appear to be significant cooperativity in the binding of Cr3 + ion to the two ovotransferrin binding sites [21], the equilibration of r1 equiv of Cr3 + ion with apoOTf should result in preferential binding to the more stable site. The stronger binding site can be identified by titrating with monoferric OTf [21,22]. A sample of N-terminal monoferric OTf (FeNOTf) was titrated with Cr3 + (aq) ion. The Cr3 + ion does not displace the more tightly bound Fe3+ from the FeN-OTf, so its binding to the vacant binding site can be monitored using fluorescence spectroscopy. The result is shown in Fig. 2. Curve a illustrates that Cr3 + does not appear to bind to the same extent to the two OTf binding sites, while curve b levels off after the addition of almost 1.0 equiv of Cr3 + . Moreover, the slopes of the two titration curves are similar. This implies that, in the early stages of the titrations, essentially all of the added Cr3 + was binding to the protein. Thus, it is apparent that Cr3 + ion will preferentially bind to the C-terminal site when r1 equiv of Cr3 + was added. These results suggest that log K1 and log K2 represent log KC and log KN, respectively.
3.3. The conformational change Fig. 3 shows that two minimum peaks appeared at 208 and 220 nm. It is evident that Cr3 + addition causes some significant CD spectral modifications in the intrinsic region, consistent with a small decrease of alpha helix content. TNS can be used as a fluorescence probe to investigate the hydrophobic environment of proteins. TNS interacts with protein through a non-covalent bond. The structure of TNS is shown in Scheme 1.
ð5Þ
where [apoOTf]tot refers to the analytical concentration of apoOTf, and DeCr is the molar absorptivity per bound Cr3 + for the Cr–OTf difference spectrum at 257 nm. Nonlinear least squares minimization is used to adjust the values of K1 and K2 to minimize the squares of the residuals between observed and calculated absorptivities, so that reliable estimates of the binding constants of Cr3 + to OTf can be obtained. In principle, the titration curve a in Fig. 1B can be fitted using the nonlinear least squares method, where K1 and K2 are the only quantities allowed to vary. In practice, the use of chelating agents to complete the removal of the metal ions can decrease the correlation between K1 and K2, thereby leading to more accurate K1 and K2 estimates, as described previously [18,20]. Thus, the titrations with Cr3 + were repeated using metal ion solutions containing a range of IDA concentrations. The chelating agent competes with apoOTf for Cr3 + and so at any given point in the titration, there is a distribution of Cr3 + between apoOTf and the IDA chelating agent; therefore, the observed absorptivity decreases as the IDA:Cr3 + ratio increases. A series of titration curves for Cr3 + –IDA solutions is shown in Fig. 1B. The titration data in Fig. 1B can be fitted using Eq. (5), in which K1 and K2 are the only adjustable parameters. For the 10:1 IDA:Cr titration curve, the maximum De observed ( 14 000 cm 1 M 1) does not exceed the calculated molar absorptivity of 17 500 cm 1 M 1, even at r ¼3.0. This suggests that only one Cr3 + is bound to apoOTf under these conditions, and so these data were used to calculate log K1.
9 Cr-OTf-FeN
Cr2-apoOTf 8
F/104M-1
Cr+apoOTf"Cr–apoOTf
2341
7 b 6
a
5
4 0
1
2 [Cr]/[OTf]
3
4
5
Fig. 2. Titration curves for the addition of Cr3 + (65 mM) to apoOTf (2.5 mM of 2.0 mL) and Cr3 + (17 mM) to N-terminal monoferric OTf (8.5 mM) in saturated air and 0.01 M HEPES at pH 7.4.
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addition of apoOTf, Cr–OTf, and Cr2–OTf to TNS all caused a blue shift in the fluorescence emission maximum to 441 nm. Addition of Cr2–OTf complex to TNS led to a further 20% decrease in the fluorescence intensity as compared with Cr–OTf addition. These data strongly suggest that the extent of exposed hydrophobic region is different for the different conformations of OTf. The results also indicate that the second Cr3+ binding to the N-terminus induced a large conformational change in apoOTf, whereas initial binding to the C-terminus only resulted in a minor conformational change.
20 15
Cr2-Tf
10
apoTf
CD/106 M-1mdeg
5 0
3.4. Determination of guanidine hydrochloride (GdnHCl) denaturation
-5 -10
Fig. 5A shows a typical GdnHCl denaturation curve, where the reaction reached equilibrium in 15 min. The fluorescence
-15 -20
190
200
210 220 230 240 Wavelength/nm
250
260
Fig. 3. CD spectra of apotransferrin in the far UV region in the absence and presence of Cr3 + , HEPES 0.1 M, pH 7.4.
5 4
60
270
3 Fluorescent intensity/a.u.
-25 180
70
2 50
1
40 30 20
Scheme 1. The structure of TNS.
10 350 300
0 300
apoOTf
330
360
390
420
Cr-OTf
250
120 200
Cr2-OTf
150
100
100
F356nm/104M-1
Fluorescence intencity/a.u.
Wavelength/nm
50 TNS alone 0
300
350
400
500 450 Wavelength/nm
550
600
80
apoOTf Cr-OTf Cr2-OTf
60
a b
650
40 c
3+
Fig. 4. Cr -dependent exposure of hydrophobic side chains. Hydrophobic exposure was monitored at room temperature in 0.1 M HEPES with the hydrophobic TNS. The fluorescence was measured at fixed lex ¼ 320 nm, slit¼ 10 nm.
Fig. 4 shows the fluorescence spectra of TNS in the absence and presence of different forms of ovotransferrin (i.e. apoOTf, Cr–OTf, and Cr2–OTf). For Cr2–OTf samples, the Cr:OTf molar ratio used was 2.5:1 in order to ensure an excess of Cr3+ to saturate the two ovotransferrin binding sites. As shown in Fig. 4, TNS emits fluorescence very weakly at 498 nm when excited at 320 nm in 0.1 M HEPES (pH 7.4). The
20 0
2
4 [Gu-HCl]/M
6
8
Fig. 5. (A) Fluorescence spectra of apoOTf (4.5 mM of 1.5 mL) with the addition of GdnHCl denaturant in 0.1 M HEPES at pH 7.4. The mass of Gdn–HCl (mg): (1)0 mg, (2)269 mg, (3) 307 mg, (4) 678 mg and (5) 1311 mg. (B) Plots denatured percentage with the concentration of GdnHCl. a: apoOTf; b: Cr–OTf; c: Cr2–OTf.
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
energy of unfolding (DGD) can be calculated.
intensity of apoOTf increased and shifted red from 336 to 356 nm with increasing concentration of GdnHCl, until 7.0 M was reached. Complete denaturatization results from insolubility or aggregation of the unfolded protein, indicating that the conformation of apoOTf was changed. GdnHCl denaturations of Cr–OTf and Cr2–OTf were also carried out under the same conditions and the fluorescence results were similar to that of apoOTf. Fig. 5B shows a typical solvent denaturation curve. A two-state mechanism was used for the analysis of GdnHCl denaturation curves [23]. The mechanism of the denaturation is shown in the following equation: N$D
2343
KD ¼ fD =fN ¼ ðyyN Þ=ðyD yÞ
ð7Þ
DGD ¼ RT ln KD
ð8Þ
DGD ¼ DGHD2 O þ m½GdnHCl
ð9Þ
DGHD2 O is DGD at zero concentration of denaturant, which represents an estimate of the conformational stability in the native states. Term m is defined as the apparent molar DGD, which indicates the conformational stability of the denatured states. The dependence of DGD on the concentration of GdnHCl is shown in Fig. 6. The parameters obtained are listed in Table 1.
ð6Þ 4. Discussion There is increasing interest of using chromium in biology [4–7]. It has been reported that Cr3 + can bind to the two specific metal-binding sites of transferrin and that there is a difference between the binding sites [6]; however, property studies on the interaction of Cr3 + binding to apoovotransferrin are scant. Complexation of metal ions to the phenolic group of the tyrosine residues of OTf in the specific iron site perturbs the J–J* transitions of the aromatic group and leads to two absorption bands at E257 and 295 nm in the difference UV spectrum. The changes in the UV spectra of OTf after Cr3 + binding are similar to
8
10
6
8
Free energies transfer (Kcal/mole)
Free energies of transfer (Kcal/mole)
in which only the native state (N) and the denatured state (D) are present at significant concentrations in the transition region. Values of y characteristic of the native state (yN) and of the denatured state (yD) can be obtained in the transition region by extrapolating the linear portions of the denaturation curve at low and high denaturant concentrations. These linear changes generally result from solvent affecting the properties of the native and denatured states. For a two-state mechanism, fD + fN ¼1, and y¼yDfD + yNfN ¼1 where fN and fD represent the fraction of protein present in the native and denatured states, respectively. Combining these equations, fD ¼(y yN)/(yD yN) and fN ¼(yD y)/(yD yN) followed by Eq. (8), the equilibrium constant (KD) and the free
4 2 0 -2 -4 -6 -8
6 4 2 0 -2 -4 -6 -8 -10 -12
-10 0
1
2
5 3 4 [GdnHCl]/M
6
7
8
0
2
1
3 5 4 [GdnHCl]/M
6
7
8
Free energies of transfer (Kcal/mole)
15
10
5
0
-5
-10 0
1
2
3 4 [GdnHCl]/M
5
6
Fig. 6. DGD as a function of GdnHCl molarity. DGD was calculated from the data in Fig. 5B using Eq. (11). (A) apoOTf; (B) Cr–OTf; (C) Cr2–OTf.
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Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
Table 1 The parameters of unfolding of OTf. Sample
DGHD2 O a
mb
[D]1/2c
apoOTf CrC–OTf Cr2–OTf
7.12 70.600 9.97 70.620 11.1 70.448
2.08 70.130 2.85 70.160 3.46 70.130
2.00 2.50 3.50
a DGHD2 O represents estimating the conformational stability in the native states in kcal/mol. b Slopes of the free energy plots in Fig. 8, in kcal/mol M. c The [GdnHCl]1/2 values, in M.
those observed previously for the binding of other metal ions to the specific Fe3 + binding sites. Therefore, the observed absorption bands at 257 and 295 nm are attributed to binding to tyrosine ligands (J–J* transitions). Both UV and fluorescent spectral data suggest that Cr3 + can bind to both iron specific binding sites of apoOTf; however, Cr3 + binds more tightly to the C-terminal site. The binding constants for Cr3 + binding to apoOTf at the C- and N-terminus were determined to be log KC ¼13.0870.24, log KN ¼5.6570.12, respectively. CD spectroscopy is commonly used for general structural characterization of soluble proteins; the technique is particularly suited for monitoring the conformational state of proteins in solution. Specifically, CD provides direct information on the secondary structure of proteins [21,23]. Our data show that Cr3 + caused some significant CD spectral modifications in the intrinsic region, indicating a small decrease in alpha helix content, which is consistent with that induced by the addition of Al (V) [21]. These results suggest Cr3 + binding contributed to conformational changes. Conformational change was also determined by using TNS as a hydrophobic fluorescent probe. TNS does not fluoresce in water but fluoresces in organic solvents or hydrophobic environment [17]. It was found that the addition of apoOTf to TNS enhanced fluorescence intensity and blue-shifted from 494 to 441 nm, indicating that a hydrophobic patch was exposed on apoOTf surface. Addition of Cr–OTf to TNS decreased the fluorescence intensity at 441 nm, which suggests the exposed hydrophobic patch on apoOTf was changed (see Fig. 4). Furthermore, the results show that a decrease in exposed hydrophobic surface corresponds to an increase in Cr3 + concentration. It is believed that the exposed hydrophobic patch seems to be a specific property of the N-terminal site of apoOTf. Fig. 4 shows that while Cr3 + bound to the C-terminal binding site induces a small conformational change, the greatest conformational change could be attributed to the binding of Cr3 + to the N-terminal site of the protein. This result is consistent with previous reports [14,20]. The structural stability of the protein also exhibits a significant sensitivity to the interaction between apoOTf and Cr3 + . The protein denaturation with GdnHCl provides an estimate of stability dependent on the contributions from hydrophobic interactions [24]. Denaturation of the protein with GdnHCl was found to closely approach a two-state mechanism. Since Gdn + and Cl ions mask the positively and negatively charged amino acid side chains that lead to destabilized electrostatic interactions, GdnHCl has no effect on the conformational change. As seen in Fig. 5B, the proteins remained stable at low concentrations of GdnHCl. However, at high concentrations, GdnHCl becomes a denaturant regardless of the type of electrostatic interactions present in the protein. The binding of the Gdn + ions to the proteins is presumed to predominate and drive the equilibrium towards the unfolded state. After the conformational change, the peak position of the tryptophan resides would shift from 336 to 356 nm and the fluorescence intensity would gradually increase.
2O The free energy of unfolding (DGH D ) in the absence of denaturant is the estimate of protein stability to GdnHCl denaturation. Results for the GdnHCl denaturation experiments are summarized 2O in Table 1. For apooTf, Cr–OTf and Cr2–OTf, the DGH values D increased from 7.1 to 11.1 kcal/mol. The slope (m) for the plots of DGDH2 O vs [denaturant] is a measure of the ability of the denaturant to unfold a protein. The m values for apooTf, Cr–OTf and Cr2–OTf decreased from 2.08 to 3.46 kcal/mol M. The data also show that [GdnHCl]1/2 increased from 2.00 to 3.50 M. The results indicate that the interaction between Cr3 + and apoOTf stabilized the structure of apoOTf and the stabilization was further strengthened by increasing the concentration of Cr3 + .
5. Conclusions The interactions of Cr3 + with ovotransferrins were investigated by various spectroscopic studies. The comparison between Cr3 + titrations with apoOTf and monoferric OTf revealed that Cr3 + binds more tightly to the C-terminal site, which is different than other metal ions [11,12]. The interaction between TNS and the protein indicated a decrease in the extent of exposed hydrophobic surface is correlated to an increase in Cr3 + concentration. The large conformational change observed for apoOTf was mainly attributed to Cr3 + binding to the N-terminus site, while binding to the C-terminus would only result in minor conformational change. Also, the addition of Cr3 + stabilized the structure of apoOTf as indicated by the guanidine hydrochloride (GdnHCl) denaturation results. Transferrin has been reported to facilitate the cellular uptake of gallium and aluminum via the normal transferrin receptormediated mechanism for iron uptake [21,25]. Based on these in vitro property studies, it is suggested that transferrin might also be involved in Cr3 + transport. Further work is in progress to investigate the role of the receptors at the cell surface of Crsaturated transferrin. Such information may enhance our understanding of the fate of Cr3 + in the bloodstream as well as cell Cr3 + metabolism.
Acknowledgements We are thankful to the National Natural Science Foundation of China (Grant no. 20771068), the Shanxi Provincial Natural Science Foundation (Grant no. 2009011012-3 and 2010011011-1) and the Shanxi Provincial Natural Science Foundation for Youth (Grant no. 20051006). References [1] E.N. Baker, P.F. Lindley, J. Inorg. Biochem. 47 (1992) 147–160. [2] R. Sarra, R. Garratt, B. Gorinsky, H. Jhoti, P. Lindley, Acta Crystallogr. B. 46 (1990) 763–771. [3] N.D. Chasteen, Coord. Chem. Rev. 22 (1977) 1–36. [4] W.T. Cefalu, F.B. Hu, Diabetes Care 27 (2004) 2741–2751. [5] F. Borguet, R. Cornelis, N. Lameire, Biol. Trace Elem. Res. 27 (1990) 449–460. [6] P. Aisen, R. Aasa, A.G. Redfield, J. Biol. Chem. 244 (1969) 4628–4633. [7] Y. Sun, J. Ramirez, S.A. Woski, J.B. Vincent, J. Biol. Inorg. Chem. 5 (2000) 129–136. [8] R.E. Cowert, N. Kojima, G.W. Bates, J. Biol. Chem. 257 (1982) 7560–7565. [9] W.R. Harris, B. Gang, Polyhedron 16 (1997) 1069–1079. [10] B.S. Yang, J.Y. Feng, Y.Q. Li, F. Gao, Y.Q. Zhao, J.L. Wang, J. Inorg. Bicochem. 96 (2003) 416–424. [11] B.S. Yang, Y.Q. Li, Chem. Res. Chinese U 17 (2001) 6. [12] Y.Q. Li, B.S. Yang, Chin. J. Inorg. Chem. 16 (2000) 939–944. [13] Y.Q. Li, B.S. Yang, Chin. J. Chem. 22 (2004) 1153–1157. [14] E.N. Baker, Adv. Inorg. Chem. 11 (1994) 389–463. [15] J. Williams, T.C. Elleman, I.B. Kingston, A.G. Wilkins, K.A. Kuhn, Eur. J. Biochem. 122 (1982) 297–303. [16] M.A. James, J.V. Hans, J. Am. Chem. Soc. 115 (1993) 245–252. [17] W.O. McClure, G.M. Edelman, Biochemistry 5 (1966) 1908–1919.
Y. Li et al. / Journal of Luminescence 130 (2010) 2339–2345
[18] Y.Q. Li, B. Liu, Z.G. Ge, B.S. Yang, J. Photochem. Photobiol. B: Biol. 91 (2008) 137–142. [19] A.E. Martell, R.M. Smith, in: Critical Stability Constants, Plenum Press, New York, 1974, pp. 204. [20] W.R. Harris, V.L. Pecoraro, Biochemistry 22 (1983) 292–299. [21] W.R. Harris, Z.P. Wang, C. Brook, B.S. Yang, A. Islam, Inorg. Chem. 42 (2003) 5880–5889.
2345
[22] R. Hovius, P. Vallotton, T. Wohland, H. Vogel, Trends Pharmacol. Sci. 21 (2000) 266–273. [23] A. Rodger, B. Norden, in: Circular dichroism and linear dichroism, Oxford University Press, Oxford, New York, 1997. [24] O.D. Monera, C.M. Kay, R.S. Hodges, Protein Sci. 3 (1994) 1984–1991. [25] Y.Q. Li, B. Liu, C.G. Zhao, W. Zhang, B.S. Yang, Clin. Chim. Acta 366 (2006) 225–232.