Role of Macromolecular Crowding on Stability and Iron Release

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Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin sandeep kumar, Deepak Sharma, and Rajesh Kumar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05702 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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The Journal of Physical Chemistry

Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin Sandeep Kumar‡, Deepak Sharma† and Rajesh Kumar*††

††

Centre for Chemical Sciences, School of Bassic and Applied Sciences, Central University of

Punjab, Bathinda, 151001, India ‡

School of Chemistry and Biochemistry, Thapar University, Patiala 147004, India

†

Council of Scientific and Industrial Research—Institute of Microbial Technology, Sector 39A, Chandigarh, India

*Correspondence:

Rajesh Kumar Email: [email protected] Phone: 91-164-286-4255

1 ACS Paragon Plus Environment


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ABSTRACT The macromolecular crowding influences the structural stability and functional properties of transferrin (Tf). The equilibrium as well as kinetic studies of Tf at different concentration of crowding agents (dextran 40, dextran 70, ficoll 70) and at fixed concentration of dextran 40 under different concentrations of NaCl at pH 7.4 and 5.6 (1) revealed that: (i) the crowder environment increases the diferric-Tf (Fe2Tf) stability against iron loss and overall denaturation of the protein, (ii) both in the absence and presence of crowder, the presence of salt promotes the loss of iron and overall denaturation of Fe2Tf which is due to ionic screening of electrostatic interactions, (iii) the crowder environment retards iron release from monoferric N-lobe of Tf (FeNTf) by increasing enthalpic barrier, (iv) the retardation of iron release by crowding is enthalpically dominated than the entropic one, (v) both in the absence and presence of crowder, the presence of salt accelerates the iron release from FeNTf due to ionic screening of electrostatic interactions and anion binding to KISAB sites, (vi) the crowders environment is unable to diminish: (a) the salt-induced destabilization of Fe2Tf against the loss of iron and overall denaturation, and (b) anion effect and ionic screening of diffusive counterions responsible to promote iron release from FeNTf.


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INTRODUCTION Structurally, transferrins (Tfs; serum transferrin (Tf), ovotransferrin (oTf) and lactoferrin (Lf)) are bi-lobed glyco-proteins with N- and C-terminal domains of approximately similar size, and each domain can bind one iron (Fe3+) synergistically with a carbonate (or bicarbonate) anion.1-4 These two domains are connected through a linker sequence.1-4 The synergistic binding of carbonate is an absolute necessary for the interaction of Tfs with the iron.5-6 In vivo, the carbonate is dominant synergistic anion that plays a vital role in physiological uptake and release of iron by Tfs.7-11 The affinity of Tfs for Fe3+ is very high (Ka 1020 M-1),2,12 which provides protection against iron-catalyzed free radical formation and limits the bacterial growth. Tf is rich in blood plasma (~2.5-3.5 mg ml-1) with only 30% saturated with Fe3+ and gives protein the potential ability to transport the other metals ions that enter the body and few of them are of therapeutic

and

diagnostic

interest13-15

(see

also

refs

12

and

16

references therein). Delivery of Fe3+ from plasma to tissue cells occurs via pH-dependent receptor-mediated endocytosis.17-19 At neutral pH, circulating Tf preferentially binds to the extracellular portion of the transferrin receptor (TFR) on the cell surface followed by rapid internalization into an acidic endocytic compartment.20 In endosome, acid induced conformational change releases the Fe3+ from serum transferrin-Fe3+ (Tf-Fe3+) complex.21-22 Now, there is prerequisite for reduction of Fe3+ because only Fe2+ is transported out of endosomal lumen to cytoplasm by divalent metal transporter (DMT1).7 Reduction of Fe3+ is mediated by an endosomal ferrireductase.20-21 After iron release, apo-sTf remains bound to the TFR and recycled back to the cell surface. Under ensosomal pH conditions, conformational change (from closed (holo) to open (apo) conformation) appears to be the main driving force during iron release.23-24 However, this



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acidification is not sufficient for iron release from Tf-Fe3+ complex. The release of iron from Tfs can be modulated by numerous factors including: (i) chelation and labilization of the Tf-Fe3+ bound by the TFR,25-26 (ii) reduction of Fe3+ to much weekly bound Fe2+,20,27-29 (iii) pH and anion-binding-induced conformational changes23-24,30-46 (see also ref 47 and references therein) and (iv) Debye-Hückel screening of diffusive counter ions.33-34 However, it is difficult to assess the impact of these factors individually or together. Factors affecting the stability and iron release from Tfs in dilute solution have been well established by different research groups5-6,89,23-50

(see also refs 34 and 47 and references therein). However, the physiological environment is

poorly represented by such dilute solutions.51 The cellular interior contains a heterogeneous mixture of macromolecules including proteins, nucleic acids, ribosomes and carbohydrates.52-53 The estimated concentration of these macromolecules ranges between 80-400 mg ml-1,52-53 which corresponds to volume occupancy of 5-40%.54 The term “macromolecular crowding” coined by Minton implies the nonspecific influence of steric repulsions on specific reactions that occur in high volume occupied media,55 and is generally described as an excluded volume effect.56 Any reaction that amplifies the available volume will be stimulated by macromolecular crowding.57 According to excluded volume effects, crowding is a nonspecific force that favors processes resulting in the reduction of total volume, such as the folding of proteins.58 Macromolecular crowding has a significant impact on the protein conformational change, structure, folding, stability, activity, binding, mobility, protein-protein interactions, aggregation and protein allostry.55-56,58-69 The highly crowded and concentrated milieu of cytoplasm tends to alter the kinetics and thermodynamics of biochemical reactions.59-60,70-75 The macromolecular crowding alters the equilibrium constants and reaction rates through: (i) change in reactant molecules mobility,76 (ii) depletion interaction (excluded volume effect) that results in attraction between


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protein and crowder60,70,77 and (iii) non-specific chemical interaction between protein and crowder.73-74,78 Crowded environment is not confined to cellular interiors but also occurs in the extracellular matrix of tissues and takes place at membrane surfaces.59 The blood plasma contains ~80 mg ml-1 of protein, a concentration sufficient to cause significant crowding effect.59 The Tf-Fe3+-receptor complex releases its iron under a highly crowded cellular environment. Thus, the macromolecular crowding in the blood plasma, on the cell surface and in the endosome could play an important role in the conformational transition during iron release from Tf. In vivo, the environment around Tf is crowded with molecules of different shapes and sizes. To model the iron release from Tf in in vivo conditions, in vitro crowded environment can be achieved by using high concentrations of crowding agents to the system.59-60,75,79 To study the macromolecular crowding effect using different biophysical techniques, the crowders must60,80-81 (i) be inert and highly soluble, (ii) have a defined shape and size, (iii) not make attractive interactions with the protein studied, (iv) not interfere with the spectroscopic signals of the with the protein studied, and (v) not have phase transitions in the temperature regions to be studied. Commonly used crowding agents are dextrans, ficolls and polyethylene glycol (PEG). The use of PEG as a crowding agent is disfavored because in addition to inducing volume exclusion effect it also forms attractive interactions with proteins and the strength of which can vary significantly between different proteins of approximately equal size (see ref 60 and references therein). The use of in vivo natural crowders (like proteins) appears to be the best option because that would more closely resemble an in vivo situation.82-84 However, the protein crowders have some limitations because these are not soluble in high concentrations and may form charge-charge interactions because most proteins have charges distributed over their full surface.82 It is thus



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essential to screen these charges using high salt concentrations. The protein crowders itself may undergo folding/unfolding transition under the conditions used to induce folding/unfolding in the protein of interest. The spectroscopic techniques used to probe the protein of interest will be subject to interference from the other protein crowder. The molecular weight of Tf (80 kDa) is closer to the average molecular weight of crowding protein in the cell cytoplasm (67 kDa).85 In the present study, the used crowding agents are of 40 kDa (dextran 40) and 70 kDa (dextran 70 and ficoll 70). Dextran 40 and dextran 70 are polymers of D-glucopyranose while ficoll 70 is a copolymer of sucrose. Under solution conditions, dextran is regarded as rod-shaped while ficoll is regarded as spherical-shaped.80 Furthermore, at a given particular concentration, dextran 70 has always higher viscosity than the dextran 40.81 Thus, the relative studies employing these crowding agents allow us to examine how the nature and the geometric shape of the respective crowding agent affect the stability and iron release from Tf. To understand how Tf executes its biological function in a highly crowded cellular environment, the current studies evaluated the effects of selected crowding agents (dextran 70, dextran 40 and ficoll 70) on (i) diferric-Tf (Fe2Tf) stability against the loss of iron and overall denaturation, and (ii) kinetics of Fe2+ and Fe3+ release from monoferric N lobe of Tf (FeNTf) at pH 5.5 and 7.4. Previous studies revealed that anions (SO42–, Cl–, NO3–, ClO4–, etc.) play an important role in the stability and iron release dynamics of FeNTf and Fe2Tf.30-34,36,41-47 However, the effect of crowding agents on the salt dependence of the stability and iron release kinetics of FeNTf and Fe2Tf are not explored so far. In the present study we also report the effects of crowding agent on the salt-dependent: (i) Fe2Tf stability against the loss of iron and overall denaturation, and (ii) kinetics of Fe2+ and Fe3+ release from FeNTf at pH 5.5 and 7.4.


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EXPERIMENTAL SECTION

Materials and methods. Bovine serum apo-transferrin (apo-sTf) (T0178), NaCl, NaHCO3, NaClO4, EDTA, FeCl3.6H2O, nitriloacetic acid, bathophenanthroline disulfonate (BPS), crowding agents (dextran 40, dextran 70), NaOH, sodium dithionite, and salts of buffers (glycine, 4-(2Hydroxyethyl)-1-piperazineethanesulfonate

(HEPES),

and

2-(N-mopholino)

ethanesulfonate

(MES)) were purchased from Sigma. Ultra pure urea was purchased from USB. Ficoll 70 was purchased from GE health care. Centricon filter of 10 kDa molecular mass cut-off was purchased from Millipore, Bedford, MA, USA. pH of samples were adjusted by using concentrated NaOH or HCl. The concentrations of the urea before and after measurements were determined by refractive index measurements on an Abbe refractometer. The final concentrations of urea are those taken after the measurements. The pH of protein samples were measured both before and after the experiments and the reported pH values are those after the data collection. Sigma Plot (v. 9) was used to analyze the kinetic and thermodynamic data.

Preparation of Fe2Tf and FeNTf. Fe2Tf was prepared by a well established method used earlier for Fe2Tfs.34,86-87 Iron nitrilotriacetate solution was prepared by dissolving nitriloacetic acid (~100 mol) and FeCl3.6H2O (~50 mol) in 2 mL solution of 6 M HCl. The pH was adjusted to 4.0 using NaOH and then water was added to 10 mL. Apo-transferrin (apo-Tf) (80 mg, 1 mol) was dissolved in 3 mL of buffer (50 mM HEPES, pH 7.4, 20 mM NaHCO3). Then the protein solution was diluted upto 5 mL by adding the freshly prepared iron nitrilotriacetate solution. Thus prepared protein solution was incubated overnight at ~37 C. To remove the unbound iron, protein solution was exchanged at least five times with original buffer (50 mM HEPES, pH 7.4)



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by using a Centricon filter of 10 kDa molecular mass cut-off. The iron saturation of Tf was confirmed by urea–polyacrylamide gel electrophoresis. The concentration of Fe2Tf was determined spectrophotometrically on the basis of ε465 nm = 5103 M─1 cm─1.88 The percentage of protein iron saturation was above 95%. N-lobe of Tf (FeNTf) was also prepared by a well established method used earlier for FeNTf and N-lobe of oTf (FeNoTF).34,87 The ClO4– accelerates the rate of iron removal from the C-lobe 260 times more than the N-lobe of Tf.43 Thus, the concept of selective removal of iron from the C-lobe of Fe2Tf is used to prepare the FeNTf. Fe2Tf (~250 M) was incubated for ~2 hrs in 0.1 M HEPES buffer that also contained 0.1 M EDTA and 2.7 M NaClO4 at 37 C. Then to remove the EDTA and NaClO4, the protein solution was exchanged at least five times with original buffer (50 mM HEPES, pH 7.4) by using a Centricon filter of 10 kDa. The iron saturation of FeNTf was confirmed by urea–polyacrylamide gel electrophoresis. The concentration of FeNTf was determined spectrophotometrically on the basis of ε465

nm

=

2150 M─1 cm─1.86

Measurement of the far-UV CD monitored denaturant-induced equilibrium unfolding transitions of proteins. To test the effect of crowding agents on thermodynamic stability of Fe2Tf at pH 7.4 and pH 5.5, the far-UV CD-monitored (222 nm) urea-induced unfolding curves of Fe2Tf (0.05 M HEPES (pH 7.4) or 0.05 M MES (pH 5.5)) were measured in the presence of different concentrations of crowding agents (dextran 40, dextran 70 or ficoll70) at 25 C. To evaluate the effect of crowding agents on salt-dependence of thermodynamic stability of Fe2Tf at pH 7.4 and pH 5.5, the far-UV CD-monitored urea-induced unfolding curves of Fe2Tf (0.05 M HEPES (pH 7.4) or 0.05 M MES (pH 5.5)) were measured in the presence of different


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concentrations of NaCl in the absence and presence of 200 mg ml-1 dextran 40. The final concentration of protein was ~5 M. Prior to data collection, the protein samples were incubated for ~5 hrs at 25 C. The fraction of unfolded protein, fD, was estimated from the far-UV (222 nm) CD data. Far-UV CD (250-200 nm, 1.0 mm cell) spectra were taken on JASCO J815 spectropolarimeter. The unfolding transitions were analyzed assuming a two state transition between the folded (N) and unfolded (U) conformations by using the procedure of Santoro and Bolen eq 1,89

Sobs 

GD  mg[ D] ) RT GD  mg[ D] 1  exp( ) RT

( cf  mf [ D])  (cu  mu[ D]) exp(

(1)

where Sobs is the CD signal at given denaturant concentration, D, cf and cu, and mf and mu represent intercepts and slopes of native and denatured states baselines, respectively, ∆GD is the denaturation free energy in the absence of denaturant, and mg is the surface area exposed by the solvent.

Measurement of the absorbance monitored iron release profiles of Fe2Tf as a function of pH and urea. Low pH and high concentration of denaturant promote iron release from Fe2Tfs.2324,30-47

To determine the effect of crowding agents on pH-induced iron release from Fe2Tf, the

solutions of Fe2Tf were prepared at different pH in dextran 40, dextran 70, ficoll 70. To further evaluate the effect of crowding agents on salt-dependence of pH-induced iron release from Fe2Tf, Fe2Tf samples were prepared at different pH containing NaCl (0.0 to 1.5 M) both in absence and presence of 200 mg ml-1dextran 40. The mixture of buffers (0.1 M HEPES + 0.05 M MES) was used to prepare the Fe2Tf samples of different pH ranging between pH 7.5-2.0. Before



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the data collections, the protein samples were incubated for ~6 hrs at 25 C. The decrease in absorbance maximum at 465 nm was used as reliable markers for iron release from Fe2Tfs.5-6,3134

So the pH equilibrium profiles for Fe2Tf were measured by monitoring changes in absorbance

at 465 nm and the data were fitted to eq 2,33-34  c f  cu 10 Cm* pH      y  (2)  1  10 Cm* pH     where cf and cu are the normalized absorbance signals for the iron-loaded (native state) and iron-

free (acid-denatured) state, respectively, and Cm* is the pH midpoint where the Fe2Tf lost its half of the iron. To determine the effect of crowding agent on the urea-induced iron release from Fe2Tf, the protein samples (~6 M) were prepared in a buffer (0.1 M HEPES, pH 7.4 or 0.1 M MES, pH 5.7) containing different concentrations of urea and a desired concentration of crowding agent (dextran 40, dextran 70, ficoll 70). To test the effect of crowding agents on saltdependence of urea-induced iron release from Fe2Tf, Fe2Tf (~6 M) solutions were prepared in different concentrations of urea that contained variable concentrations of NaCl in the absence and presence of 200 mg ml-1dextran 40. Before the data collection, the protein samples at 7.4 and pH 5.7 were incubated for ~20 and ~3 hrs (25 C), respectively. On the basis of absorbance at 465 nm, the fraction of iron released was estimated. The normalized urea-induced denaturation curves for iron release were fitted to eq 1. The visible absorbance spectra (390-710 nm) were collected on Shimadzu 2450 spectrophotometer.

Measurement of urea denaturation-induced and sodium dithionite reduction-induced iron release kinetic profiles of FeNTf. When FeNTfs was incubated with 9.0 M urea at pH 7.4 or 6.0


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M urea at pH 5.5, the absorbance of 465 nm band is decreased significantly with time. 34,90 To examine the effect of crowding agents on urea-induced iron release from FeNTf, FeNTf solution (50 L, pH 7.4) was mixed rapidly to HEPES buffer (0.1 M, 950 L, pH 7.4) (for measurement at pH 7.4) or MES buffer (0.1 M, 950 L, pH 5.5) (for measurement at pH 5.5) that contained 10.0 M (pH 7.4) or 6.0 M (pH 5.5) urea and a desired concentration of crowding agent (dextran 40, dextran 70 and ficoll 70) at 37 C. To measure the effect of crowding agents on saltdependent urea-induced iron release from FeNTf, FeNTf solution (50 L, pH 7.4) was mixed rapidly to HEPES buffer (0.1 M, 950 L, pH 7.4) (for measurement at pH 7.4) or MES buffer (0.1 M, 950 L, pH 5.5) (for measurement at pH 5.5) that contained 10.0 M (pH 7.4) or 6.0 M (pH 5.5) urea and different concentrations of NaCl in the absence and presence of 200 mg ml-1 dextran 40 at 37 C. For kinetic experiments of urea denaturation-induced iron release, the final concentration of FeNTf was ~15.0 M. The kinetics of iron release from FeNTf was measured by monitoring the change in absorbance at 465 nm, 37 C. To test the effect of crowding agents on reduction induced Fe2+ release from FeNTf at pH 7.4, 20 L (pH 7.4) of FeNTf solution was mixed rapidly to a deaerated HEPES buffer (0.1 M, 980 L, pH 7.4) that contained 10 mM sodium dithionite, 0.25 mM BPS and a desired concentration of crowding agent (dextran 40, dextran 70 and ficoll 70) at 25 C. To measure the effect of crowding agents on salt-dependent reduction induced Fe2+ release from FeNTf at pH 7.4, 20 L (pH 7.4) of FeNTf solution was mixed rapidly to a deaerated HEPES buffer (0.1 M, 980 L, pH 7.4) that contained 10 mM sodium dithionite, 0.25 mM BPS and different concentrations of NaCl in the absence and presence of 200 mg ml-1 dextran 40 at 25 C. At pH 5.5, the reductive iron release reaction is relatively faster, therefore, the kinetics of reductive iron



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release at this pH was measured by a Shimadzu 2450 spectrophotometer coupled with applied photophysics RX 2000 rapid kinetics stopped-flow mixing accessory. FeNTf (pH 7.4) was placed in one syringe and deaerated MES buffer (0.1 M, pH 5.4) that contained sodium dithionite, BPS, and a desired concentration of crowding agent or/and salt was placed in other stopped flow syringe (25 C). About 8 M and 10 M concentration of FeNTf was used for Fe2+ release kinetic experiments at pH 7.4 and pH 5.5, respectively and the kinetics was measured by Fe2+BPS complex absorbance at 538 nm.28,34

Urea concentration correction in the presence of crowding agent. Inert crowding agent increases the activity coefficient of other small species through decreasing its available volume through pure steric repulsion.80,91 To calculate the effective concentrations of urea in the presence of crowding agent (dextran 40, dextran 70, ficoll 70), the partial specific volume of dextran 40, dextran 70 and ficoll 70 were determined in the absence and presence of urea. The partial specific volume is same in the absence and presence of crowding agent suggest that no interaction between crowding agent and urea. The value partial specific volume for all the crowding agents is 0.670 (±0.004) ml gm-1 and did not dependent on the concentration of crowding agent. The effective concentration of urea was calculated as described previously.80,91 Urea concentration denotes the corrected effective concentration.

RESULTS

Effects of crowding agents and salt on the pH- and urea dependence of the absorbance spectra of Fe2Tf. At pH 7.4, the absorption spectrum of Fe2Tf shows a maximum at ~465 nm (Figures 1a and 1b), indicative of iron is associated with the protein. At pH 7.4, the presence of 12 ACS Paragon Plus Environment

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200 mg ml-1 dextran 40 or 1.0 M NaCl, has no significant effect on the absorbance maximum at 465 nm (Figure 1a). When pH is lowered from 7.4 to 4.0, the absorbance at 465 nm is completely diminished, suggestive of a decrease in pH from physiological to mildly acidic results in the complete release of iron from Fe2Tf (Figure 1a). When urea concentration is increased from 0.0 to 10.0 M at pH 7.4 (Figure 1b) or 8.5 M at pH 5.7 (Figure 1c), the absorbance at 465 nm is completely diminished (Figures 1b and 1c), indicating that the high concentration of urea results in complete release of iron from the Fe2Tf at both physiological and endosomal pH conditions. At pH 5.0 (Figure 1a) or at pH 7.4 with 5 M urea (Figure 1b) or at pH 5.7 with 3.5 M urea (Figure 1c), the absorbance at 465 nm is diminished by around half (Figures 1a, 1b and 1c). Interestingly, when 200 mg ml-1 dextran 40 is included, the absorbance maximum at 465 nm does not decrease significantly at pH 5.0 (Figure 1a) or at pH 7.4 with 5 M urea (Figure 1b) or at pH 5.7 with 3.5 M urea (Figure 1c), indicating that the presence of crowding agent inhibits the pH- and urea-denaturation induced iron release from Fe2Tf at mild acidic pH (~5.0) or at moderate urea concentration (5.0 M at pH 7.4 and 3.5 M at pH 5.7). However, the inclusion of 0.3 M NaCl both in the absence and presence of 200 mg ml-1 dextran 40 at pH 5.0 (Figure 1a), at pH 7.4 with 5 M urea (Figure 1b) or at pH 5.7 with 3.5 M urea (Figure 1c), leads to significant decrease of the absorbance maximum at 465, indicating that salt exhibits additive effect on the mild acidic pH- and moderate urea-denaturation induced iron release from Fe2Tf. Based on these observations, the pH and urea-denaturation equilibrium titrations of Fe2Tf were carried out under: (i) different concentrations of dextran 40, dextran 70 and ficoll 70 and (ii) different concentrations of salt both in the absence and presence of 200 mg ml-1 dextran 40.



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Effects of crowding agents on the pH- and urea-denaturation induced iron release from Fe2Tf. The pH- and urea denaturation-induced iron release profiles of Fe2Tf measured in the absence and presence of 200 mg ml-1 crowding agent (ficoll 70, dextran 40 and dextran 70) show that the crowding presence shifts the pH-induced iron release profile toward the lower pH (Figure 2a) while the urea-denaturation induced iron release profile toward the higher concentration of urea (Figures 2b (pH 7.4) and 2c (pH 5.7)). Furthermore, the crowding agentinduced shifts in the pH profile (toward the lower pH) and urea profile (toward the higher urea concentrations) are more pronounced for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70). The urea and pH equilibrium titrations were fitted according to eqs 1 and 2, respectively. The estimated urea denaturation free energy (∆GD), surface area exposed by solvent (mg), and urea-denaturation midpoint for iron release (Cm = ∆GD/mg) are summarized in Table 1. The estimated pH-midpoint for iron release (Cm*) decreases in the presence of crowding agents, and typically follows the order, dextran 70 > dextran 40 > ficoll 70 (Figure 2d). At both pH 7.4 and pH 5.7, the estimated Cm (Figures 2e and 2f) and ∆GD (Inset of Figures 2e and 2f) for iron release increase in the presence of crowding agents and typically follows the order, dextran 70 > dextran 40 > ficoll 70. These findings indicate that the crowder environment counteracts the pHand urea-denaturation induced iron release form Fe2Tf and this effect is appreciably more pronounced for dextran 70 and less for ficoll 70 (dextran 70 > dextran 40 > ficoll 70).

Effects of crowding agent on the salt-dependence of pH- and urea-denaturation induced iron release from Fe2Tf. The pH- and urea denaturation-induced iron release profiles of Fe2Tf measured at different concentration of NaCl in the absence and presence of 200 mg ml -1 crowding agent (ficoll 70, dextran 40 and dextran 70) show that the salt presence in reaction medium shifts the pH- and urea-denaturation induced iron release profiles of Fe2Tf toward the 14 ACS Paragon Plus Environment

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higher pH (Figure 3a) and lower urea concentrations (Figures 3b (pH 7.4) and 3c (pH 5.7)), respectively, which reveals that the salt presence decreases the Tf-Fe3+ complex stability. However, these salt-induced shifts in the pH and urea profiles are less pronounced in the presence of crowder than in its absence (Figures 3a, 3b and 3c). The urea and pH titrations were fitted according to eqs 1 and 2, respectively. The estimated urea denaturation free energy (∆GD), and surface area exposed by solvent (mg) for iron release are summarized in Table 2. Both in the absence and presence of crowder, as [NaCl] is increased, the estimated pH-midpoint (Cm*) (Figures 3d) and urea-denaturation midpoint (Cm=∆GD/mg)) (Figures 3e (pH 7.4) and 3f (pH 5.7)) for iron release increase and decrease monoexponentially, respectively and plateaus at ~0.5 M. These data suggest that both in the absence and presence of crowding agent, the salt effect destabilizes the Tf-Fe3+ complex stability.

Effects of crowding agents and salt on the far-UV CD spectra of native and urea-denatured Fe2Tf. Far-UV CD spectrum of native Fe2Tf shows negative extrema at ~208 nm and shoulder at around 215-225 nm, which reflects the secondary structure of Fe2Tf. At pH 7.4 and pH 5.5, in the presence of varying concentrations of crowding agent (ficoll 70, dextran 40 and dextran 70) or 1.0 M NaCl, the far-UV CD spectrum of Fe2Tf is not greatly altered (Figures S1a, S1b, S1c, S1d, and S1f). However, the negative extrema at 208 nm and shoulder around 215-225 nm in the presence of 10.8 M urea at pH 7.4 and 8.5 M urea at pH 5.5 are substantially disrupted (Figures S1g and S1h), indicating that the high concentration of urea significantly denatured the secondary structure of the protein. In the presence of 8.0 M urea at pH 7.4 and 4.5 M urea at pH 5.5, the negative extrema at 208 nm and shoulder around 215-225 nm are partially disrupted but with 1.0 M NaCl, these features are significantly disrupted (Figures S1g and S1h), indicating that



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the salt exhibits the additive effect on the urea-induced destabilization of secondary structure of Fe2Tf. However, with inclusion of 200 mg ml-1 dextran 40 both in the absence and presence of 1.0 M NaCl, these features are not greatly altered (Figures S1g and S1h). This finding reveals that at moderate urea concentrations (8.0 M at pH 7.4 and 4.5 M at pH 5.5), both in the absence and presence of 1.0 M NaCl, the presence of crowding agent prevents the destabilization of secondary structure of Fe2Tf. Based on these observations, the urea-induced unfolding of Fe2Tf were carried out under (i) different concentrations of dextran 40, dextran 70 and ficoll 70 at pH 7.4 and 5.5, and (ii) different concentrations of salt both in the absence and presence of 200 mg ml-1 dextran 40 at pH 7.4 and 5.5.

Effects of crowding agents on the urea-induced unfolding of Fe2Tf. The urea-denaturation induced unfolding curves of Fe2Tf (CD 222 nm) measured at different concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) at pH 7.4 and pH 5.5 show that the crowding presence shifts the urea-denaturation induced unfolding curve (based on ellipticity at 222 nm) toward the higher concentrations of urea (Figures 4a and 4b). In addition, the crowdinginduced shift in the urea-denaturation induced unfolding curve at pH 7.4 and 5.5 is more pronounced for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70). The ureadenaturation induced unfolding curves were analyzed using eq 1. The resulting urea unfolding free energy (∆GD), surface area exposed by solvent (mg), and urea unfolding midpoint (Cm=∆GD/mg) are summarized in Table 3. At pH 7.4 and 5.5, as [Crowding agent] is increased, the Cm and ∆GD for unfolding of Fe2Tf increase, and typically follows the order, dextran 70 > dextran 40 > ficoll 70 (Figures 4c and 4d). This finding suggests that the crowder environment


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increases the thermodynamic stability of Fe2Tf and this effect is more pronounced for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70).

Effects of crowding agent on the salt dependence of the urea-induced unfolding of Fe2Tf. The urea-denaturation induced unfolding curves of Fe2Tf (CD 222 nm) measured at different concentration of NaCl in the absence and presence of 200 mg ml-1 crowding agent (ficoll 70, dextran 40 and dextran 70) at pH 7.4 and pH 5.5 show that the salt presence shifts the ureadenaturation induced unfolding curve towards the lower urea concentrations, which suggests that the salt decreases the structural stability of Fe2Tf (Figures 5a and 5b). However, the salt-induced shift in urea-induced unfolding curve is less pronounced in the presence of crowder than in its absence (Figures 5a and 5b). The urea-denaturation induced unfolding curves were analyzed using eq 1. The resulting urea unfolding free energy (∆GD), surface area exposed by solvent (mg), and urea unfolding midpoint (Cm=∆GD/mg) are summarized in Table 4. At pH 7.4 and pH 5.5, both in the absence and presence of crowder, as [NaCl] is increased, the Cm and ∆GD for unfolding decrease mono-exponentially and plateaus at ~0.5 M (Figures 5c, 5d, 5e and 5f). This finding suggests that both in the absence and presence of crowding agent, the salt presence decreases the structural stability of Fe2Tf.

Effects of crowding agents on the reduction and urea-denaturation induced iron release from FeNTf. Figures 6a and 6b show the representative reduction induced Fe2+ release kinetic traces of FeNTf measured in the absence and presence of 200 mg ml-1 dextran 40 at pH 7.4 and pH 5.5, respectively. The representative urea denaturation-induced Fe3+ release kinetic traces of FeNTf measured in the absence and presence of 200 mg ml-1 dextran 40 at pH 7.4 and pH 5.5 are shown in Figures 6c and 6d, respectively. The observed rate constant, kobs for Fe2+ and Fe3+ 17 ACS Paragon Plus Environment


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release at different concentrations of crowding agents are summarized in Tables S1 and S2, respectively. The variations of kobs for Fe2+ release with [Crowding agent] at pH 7.4 and 5.5 are shown in Figures 6e and 6f, respectively. Figures 6g and 6h show the variations of kobs for Fe3+ release with [Crowding agent] at pH 7.4 and 5.5, respectively. As [Crowding agent] is increased, the rate constants for Fe2+ and Fe3+ release decrease at pH 7.4 (Figures 6e and 6g) and pH 5.5 (Figures 6f and 6h), which reveals that the crowder environment retards the iron release from FeNTf. Furthermore, the crowder-mediated retardation in iron release typically follows the order, dextran 70 > dextran 40 > ficoll 70, which suggest that dextran 70 is most and ficoll 70 is least effective in retardation of iron release from FeNTf under physiological and endosomal pH conditions.

Effects of crowding agent on salt-dependence of reduction and urea-denaturation induced iron release from FeNTf. The representative kinetic traces of Fe2+ release from FeNTf in the absence and presence of 0.5 M NaCl, 200 mg ml-1 dextran 40, 0.5 M NaCl with 200 mg ml-1 dextran 40 at pH 7.4 and pH 5.5 are shown in Figures 7a and 7b, respectively. Figures 7c and 7d present the representative urea denaturation-induced Fe3+ release kinetic traces of FeNTf at pH 7.4 and pH 5.5, respectively, measured in the absence and presence of 0.6 M NaCl, 200 mg ml-1 dextran 40, 0.6 M NaCl with 200 mg ml-1 dextran 40. The observed rate constants, for Fe2+ and Fe3+ release measured at different [NaCl] in the absence and presence of 200 mg ml-1 dextran 40 at pH 7.4 and 5.5, are summarized in Tables S3 and S4. Figures 7e,f and 7g,h show the variation of kobs for Fe2+ and Fe3+ release, respectively, as a function of [NaCl] in the absence and presence of 200 mg ml-1 dextran 40 at pH 7.4 (Figures 7e and 7g) and pH 5.5 (Figures 7f and 7h). Both in the absence and presence of crowding as [NaCl] is increased, the kobs for Fe2+ and Fe3+ release increase monoexponentially at pH 7.4 (Figures 7e 18 ACS Paragon Plus Environment

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and 7g) and pH 5.5 (Figures 7f and 7h), and plateaus at ~0.5 M. Earlier reports also showed that in the absence of crowder, the rate constant for Fe2+ and Fe3+ release from FeNTf and FeNoTf at pH 5.6 increases mono-exponentially with salt concentration.34,47 Both in the absence and presence of crowding agent, the exponential saturation of anion effect on kobs for Fe2+ and Fe3+ release at pH 7.4 and 5.5 provides two important information, (i) anion-binding-induced conformational change promotes the iron release from FeNTfs,5-6,34,42,45,47,92-94 and (ii) under physiological and endosomal pH conditions, the crowder environment does not alter the anion effect on Fe2+ and Fe3+ release from FeNTf. Previously we have reported that at low to intermediate concentrations of salt, an extended Debye-Hückel model captures the role of ionic strength (I) on the rate of iron release from Fe2Tf,33 and FeNoTf.34 Both in the absence and presence of crowding agent, the log kobs for Fe2+ and Fe3+ release increases linearly with [I1/2/(1+I1/2] at pH 7.4 (Figures 7e and 7g) and 5.5 (Figures 7f and 7h).

Effects of crowding agent on the activation thermodynamic parameters of reduction induced iron release from FeNTf. To test whether the crowding presence affects the thermodynamic activation parameter for reductive iron release, the Eyring plots for Fe2+ release reaction of FeNTf obtained in the absence and presence of 200 mg ml-1 crowding agent (dextran 40, dextran 70 and ficoll 70) at pH 7.4 (Figure 8a) and pH 5.5 (Figure 8b) were analyzed using eq 3,95

ln(kobs h/kB T ) =(ΔS ‡ /R)- (ΔH ‡ /RT)

(3)

The estimated values of ΔH‡ and ΔS‡ are summarized in Table 5. By using the ΔH‡ and ΔS‡ values, the corresponding (ΔG‡) is also calculated at 25 C using Gibbs free energy eq (ΔG‡= ΔH‡−TΔS‡) (Table 5). The data in Table 5 clearly show that the crowding presence increases the



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enthalpic barrier (ΔH‡) for Fe2+ release reaction at pH 7.4 and 5.5, and which is more increased for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70. To find out whether the crowding presence affects the salt-dependence of ΔH ‡ and ΔS ‡ for reductive iron release, the Eyring plots for Fe2+ release reaction of FeNTf obtained in the absence and presence of 0.15 M NaCl, 200 mg ml-1 dextran 40, and 0.5 M NaCl with 200 mg ml1

dextran 40 at pH 7.4 (Figure 8c) and pH 5.5 (Figure 8d) were analyzed using eq 3.95 The

activation thermodynamic parameters are summarized in Table 6, which shows that the salt presence decreases the enthalpic barrier (ΔH‡) for Fe2+ reaction at pH 7.4 and 5.5, and which is less decreased in the presence of crowding agent than in its absence. As described earlier,95-96 the enthalpy-entropy plot could be used to determine enthalpic and entropic contributions toward the stability, folding and dynamics of proteins. The enthalpyentropy plot has four sectors (Figures 8e and 8f). Sectors 1 and 2 correspond to stabilizing cosolutes while sectors 3 and 4 correspond to destabilizing cosolutes. Furthermore, sectors 1 and 3 represent the enthalpically dominated effect while sectors 2 and 4 represent entropically dominated effect.95-96 The enthalpy-entropy plots for reductive iron release from FeNTf, collected in the absence and presence of 200 mg ml−1 of dextran 40, dextran 70 and ficoll 70 at pH 7.4 and 5.5 lie in sector 1 (Figures 8e and 8f), which is in general agreement with the models that describe enthalpically dominated stabilization.95-96

DISCUSSION This is the first assessment of the effect of non-charged synthetic crowding agents on the stability and iron release kinetics of Tf at physiological and endosomal pH. Besides non-charged synthetic crowders such as dextrans and ficolls, the use of physiologically relevant charged


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crowders also appear to be advanced models for explaining the effects of macromolecular crowding.60,82-84 In general, the crowding effect by synthetic polymers on proteins is stabilizing, however, the crowding effect by individual proteins could be destabilizing on globular proteins (see refs 60, 82-84 and references therein for examples). The net charge of protein crowder against the net charge of test protein determines the effect of protein crowder on protein stability.82 Using the current techniques, the use of in vivo crowders of varying sizes is avoided because (i) the Tf is studied under different conditions of pH (ranging between pH 2.5 to 7.4) and which may change the charge (also size and shape) of protein crowder and alter the chargecharge interaction between protein crowder and target protein or between protein crowder itself83 and ultimately gives a complex picture of the crowding effect, and (ii) under different conditions of urea, the denaturation of protein crowder may interfare the CD signal of protein of interest which may makes the analysis difficult. Analysis of the pH and urea-denaturation (pH 7.4 and 5.7) profiles for iron release of Fe2Tf, collected in the absence and presence of different concentration of crowder (dextran 40, dextran 70, ficoll 70) revealed that crowder’s environment counteracts the pH- and urea-denaturation induced iron release (Figures 2d, 2e and 2f; Table 1) by stabilizing the protein against loss of iron under physiological and endosomal pH conditions (Figures 2d, 2e and 2f; Table 1), and this stabilizing effect is more for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70) (Figures 2d, 2e and 2f; Table 1). As dextran 70 has larger size than dextran 4081 and different shape than ficoll 7080, the results indicate that size and shape of crowding agents plays a vital role in the stability of iron centers of Fe2Tf. On the basis of current observations, it is expected that if twice as large or half as small synthetic crowders are used in the current experiments, the effect of larger sized crowder will be more than the smaller sized one if the shape of crowders is same. The binding constants for the two iron binding sites



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in Tf differ by about a log unit.12 Despite this difference, our previous thermal denaturationinduced iron release profiles for Fe2Tf33 and urea denaturation induced iron release profile for Fe2oTf showed single step behavior for iron release.34 It may be possible that the binding constants difference for the two binding sites is too small to be detected in the equilibrium data for urea-denaturation induced iron release from Fe2Tf. In addition, it is also possible that the crowding effect may further decrease the difference in iron binding affinity between the two sites. The secondary structural content in natively folded Fe2Tf is not much altered in the presence of crowder (Figures S1a, S1b, S1c, S1d, S1e and S1f). Similar results were found for cytochrome c, apoazurin and cellular retinoic acid-binding protein I in which the secondary structural content was not much affected by the crowders.80,97-98 However, crowding agents induce the secondary structure in the natively folded apo and holo-flavodoxin, and VlsE.79,99-100 Analysis of the urea-denaturation induced unfolding curves of Fe2Tf at pH 7.4 and 5.5, collected in the absence and presence of different concentration of crowders (dextran 40, dextran 70, ficoll 70) showed that the crowder environment increases the Fe2Tf stability against overall denaturation, and which is found to be increased more for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70) (Figures 4c and 4d; Table 3). These findings reveal that the size, shape and concentration of crowding agents also control the structural stability of Fe2Tf under physiological and endosomal pH conditions. Previous reports concluded that crowder environment increases the thermal and structural stability of proteins.79-80,97-100 The guanidine hydrochloride (GdnHCl)-induced equilibrium unfolding of native apoazurin revealed that the stabilizing effect of ficoll 70 is less than any of the dextrans (dextran 20, dextran 40 and dextran 70) and among the dextrans no size dependent effect was observed.97 However, GdnHCl-induced


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equilibrium unfolding transitions of ferrocytochrome c at pH 7 showed that the smaller sized crowder (dextran 40) has a greater impact on the thermodynamic stability of protein and typically follows the order, dextran 40 > dextran 70 > ficoll 70.95 The thermal stability of flavodoxin is increased more in the presence of dextran 70 than ficoll 70.99 These studies thus suggest that crowding agents of different sizes and shapes have different stabilizing effects on the different proteins. The increase in ∆GD values measured for urea-denaturation induced iron release (Table 1) and unfolding (Table 3) typically follows the order, dextran 70 > dextran 40 > ficoll 70. This trend suggests that the efficiency of macromolecular crowding is dependent on a ratio between the hydrodynamic dimensions of the crowder and the test protein, with the most effective conditions being those where the volumes occupied by the crowder and the test protein are of similar size. Thus, the information on the hydrodynamic radii of the crowder and the test protein is significant for the rational selection of suitable crowding environment. The more complicated trends of the effect of crowding agents on the enzyme kinetics were observed because either increase, ,101-102 decrease,103-104 or have no effect on enzymatic activity.105 Moreover, concentration dependent effect of macromolecular crowding was also observed.106 In the present study, the kinetic and thermodynamic analysis of the reduction and urea-denaturation induced iron release at pH 7.4 and 5.5 under different concentration of crowder (dextran 40, dextran 70 and ficoll 70) revealed that: (i) the presence of crowding agent retards the iron release (Figures 6e, 6f, 6g and 6h; Tables S1 and S2) by increasing enthalpic barrier (ΔH‡), (ii) the crowding agent-mediated retardation in iron release is more pronounced for dextran 70 and least for ficoll 70 (dextran 70 > dextran 40 > ficoll 70) (Figures 6e, 6f, 6g and 6h; Tables S1 and S2), which indicates that size, shape and viscosity of crowding agents control iron release process of FeNsTf, and (iii) the increase in ΔH‡ in presence of crowding agent is



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accompanied by the decrease in entropy change, −TΔS‡ (Table 5). The increase in ΔH‡ by crowding agents suggests that some crowding agents form attractive interactions with FeNTf and thus block the iron release from FeNTf under physiological and endosomal pH conditions. Some earlier reports revealed that the crowding agent forms the attractive interaction with cytochrome c and ubiquitin.107-109 The direction and magnitude of crowding effects on macromolecular reactions mainly depend on nature and strength of both hard and soft interactions between crowder and reactant/product molecules107-110 as well as between crowder molecules.110 The nonspecific chemical interactions can be attractive or repulsive. The repulsive chemical interactions contribute entropically and reinforce the hard-core repulsions and lead to protein stabilizing effect.84 On the other hand, the attractive chemical interactions contribute enthalpicaly and counteract the effect of hard-core repulsions and lead to protein destabilizing effect.84 The binding of non-inert crowders to target proteins may also take place. The enthalpy-entropy plots for reductive iron release from FeNTf, collected in the absence and presence of crowder at pH 7.4 and 5.5 reveal that in controlling iron release from FeNTf in presence of crowding agent under physiological and endosomal pH conditions, the enthalpic effect is more dominated than the entropic one (Figures 8e and 8f). Previously it was established that crowder-induced protein stabilization through purely steric excluded volume effect is entropic.73,84,111 However, recent studies showed that the observed effect of crowders on protein stability and folding have a significant enthalpic contributions.95,112-113 Moreover enthalpic effect even dominates over entropic contributions.95,112-113 To account for enthalpic as well as entropic effects of crowders, some recent reports suggest that it is necessary to augment the steric excluded volume effects with other nonspecific interactions.84,95,110,112-113 Therefore the present study highlights that both


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hard-core repulsions and nonspecific soft chemical interactions must be considered to understand the effects of crowding agent on the iron release from FeNTf under physiological and endosomal pH conditions. Current result suggests that hard and soft interactions are always present, but their relative influences on protein depend on the size, shape, and chemical nature of the crowders. Within the biological cell, the distribution of crowders is heterogeneous and the size of crowding molecules can also be distributed.52 Such properties of the cell cytoplasm may control the speeds of biochemical reactions including polymer dynamics and particle diffusion.114-116 Molecular dynamics simulations on model system can be used to investigate the tracer diffusion in crowded environment.114-116 Recently, pioneering computer simulations works by Ralf Metzler group have provided implications for the diffusion, transport, and spreading of chemical components in highly crowded environments inside living cells and other structured liquids.115116

They studied the tracer diffusion in a heterogeneously crowded environment115 and

subsequently using a continuum lattice made of static obstacles.116 Moreover, different physical origins dominate for different tracer sizes and shapes, as well as length and time scales of the diffusion.116 Here we created the homogeneous crowding of different viscosities by varying concentrations of crowders of different sizes and shapes, which also suggests that the size, shape and viscosity of crowding alter the stability and iron release from Tf. Therefore, besides above discussed interactions, the alternation in diffusion behavior of Tf under crowding cannot be ignored and warrants further analysis. The effect of salt ions on protein stability is typically attributed to electrostatic (DebyeHückel) screening of Coulombic interactions (i.e., at low to intermediate salt concentration),3334,117

to specific ion binding118 or to increased surface tension of water that modulate hydrophobic

interactions (the Hofmeister effect).33-34,119 The effect of charged crowders on target protein



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stability could be altered by presence of salt ions because salt ions may also screen the surface charge of protein crowder.82 However, this salt mediated charge screening of protein crowder is not expected for non-charged synthetic crowders. Thus, our experimental setup using noncharged synthetic crowders provides fundamentally improved system to study the anion effect on stability and iron release kinetics of Tf. Analysis of the pH-induced iron release profiles and urea-denaturation-induced unfolding and iron release profiles of Fe2Tf, collected under varying concentration of NaCl both in the absence and presence of dextran 40 revealed that: (i) the salt presence in reaction medium decreases the Fe2Tf stability against the loss of iron (Figures 3d, 3e and 3f; Table 2) and overall denaturation (Figures 5c, 5d, 5f and 5f; Table 4) due to ionic screening of electrostatic interactions,33-34 while the crowding agent increases the Fe2Tf stability against the loss of iron (Figures 3d, 3e and 3f; Table 2) and overall denaturation (Figures 5c, 5d, 5e and 5f; Table 4) due to macromolecular crowding effect, and (ii) salt also decreases the Fe2Tf stability against the loss of iron (Figures 3d, 3e and 3f; Table 2) and overall denaturation at pH 7.4 and 5.6 (Figures 5c, 5d, 5e and 5f; Table 4) in the presence of crowding agent, which demonstrates that under physiological and endosomal pH conditions, the crowders environment is unable to diminish the salt-induced electrostatic destabilization of Fe2Tf against the loss of iron and overall denaturation. These observations suggest that in crowder-salt mixture, both crowder and salt act independently and act through different mode of interactions. Kinetics and thermodynamic analysis of reduction and urea-denaturation induced iron release from FeNTf at different concentration of NaCl in the absence and presence of 200 mg ml1

dextran 40 at pH 7.4 and 5.5 revealed that: (i) under physiological and endosomal pH

conditions, both in the absence and presence crowding agent, the ionic screening of diffusive counterions promotes the release of iron from FeNTf34 (Insets of Figures 7e, 7f, 7g and 7h), (ii)


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the crowders environment is unable to diminish the ionic screening of diffusive counterions (Insets of Figures 7e, 7f, 7g and 7h) responsible to promote iron release from FeNTf, (iii) the presence of salt in reaction medium decreases enthalpic barrier (ΔH‡) for Fe2+ release at pH 7.4 and 5.5 (Figures 8c and 8d; Table 6), and which is less decreased in the presence of crowding agent than in its absence (Figures 8c and 8d; Table 6), and (iv) both in the absence and presence of crowding agent, the salt mediated decrease in ΔH‡ is accompanied by the increase in entropy change, −TΔS‡ (Figures 8c and 8d; Table 6). In order to emphasize the anions mediated allosteric effect on iron release from Tf, Egan et al.30, 44,120,121 proposed sites to which anions bind as ‘‘kinetically significant anion binding’’ or KISAB sites. It is well established that the anions binding to KISAB sites alter the kinetics of iron release from Tf (see ref 94 and references therein). However, the KISAB sites have remained less characterized94 and many KISAB sites exist for each lobe of Tf.24,41,44-47,94,122 Furthermore, the kinetic effects of anions can vary considerably between the two lobes43 We have recently reported that both ionic strength effect and anions binding to the KISAB sites alter the rates of iron release from monoferric N-lobe of Tfs, the extent of these two effects on rates of iron release should depend on the anions concentration and pH.34 At pH 7.4, under low concentration of NaCl, the ionic strength effect may majorly assists the iron release from FeNTf and anions binding to KISAB sites may have small effect on iron release because at neutral pH only weak interactions exist between the anions and KISAB sites.47 However, at pH 5.5, the anions binding strength to KISAB sites increases so it is expected that both anions binding to the KISAB sites and ionic strength effect together facilitate the iron release from FeNTf. The current results suggest that both ionic strength effect and anion binding to KISAB sites may play a vital role in controlling the kinetics of iron release from FeNTf, however, overall picture is still much



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more complex because ionic strength effect and anion binding to KISAB sites depends mainly on the pH and concentration of anion.

CONCLUSIONS Analysis of pH profiles for iron release and urea-denaturation profiles for iron release and unfolding (pH 7.4 and 5.7) of Fe2Tf, measured at different concentration of crowding agents (dextran 40, dextran 70, ficoll 70) and at fixed concentration of dextran 40 under variable concentration of NaCl concluded that (i) the size, shape and concentration of synthetic inert non-charged crowders play an important role in the stabilization of Tf against the loss of iron and overall denaturation, (ii) the salt presence decreases the Fe2Tf stability against the loss of iron and overall denaturation due to ionic screening of electrostatic interactions and anion binding to KISAB sites, and (iii) the crowders presence is unable to diminish the salt effect on iron release and protein stability. Kinetic and thermodynamic analysis of Fe2+ and Fe3+ release reaction of FeNTf, measured at different concentration of crowding agents (dextran 40, dextran 70, ficoll 70) and at fixed concentration of dextran 40 under variable concentration of NaCl at pH 7.4 and 5.5 concluded that (i) the size, shape, concentration, and viscosity of crowder play vital role in controlling the iron release from FeNTf, (ii) the salt presence promotes the iron release from FeNTf due to ionic strength effect and anion binding to KISAB sites but their relative influences on iron release depend on the concentration and pH of reaction medium, (iii) the crowder environment increases the enthalpic barrier (ΔH‡) for iron release while salt presence decreases it and this salt-mediated decrease in ΔH‡ is accompanied by the increase in entropy change, −TΔS‡, and (iv) in controlling the iron release from FeNTf, the enthalpic effect is more dominated than the entropic one. The current results thus concluded that besides the hard-core repulsions,


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the nonspecific soft interactions also play an important role in the biochemical processes. Still, more experimental and theoretical studies needed to explain the nature and origin of these soft interactions. We hope that our work will also initiate the development of more realistic models for explaining the effects of macromolecular crowding on the iron release from Tf using the more physiologically relevant crowders of different physicochemical properties. It will also be of interest to extend our results, in particular to the superdense cytoplasm of biological cells. The biological significance of the present study is evidenced by the salt induced modulation of iron release from Tf in the presence of crowding agent. On the basis of the current findings the effect of macromolecular crowding can be taken into account in future studies to describe the mechanism of iron release from Tfs.

ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Tables S1 to S2 show effects of crowding agents on the kobs for Fe2+ and Fe3+ release kinetics of FeNTf complex at pH 7.4 and pH 5.5, respectively. Tables S3 to S4 show effects of crowding agent on the salt dependence of kobs for Fe2+ and Fe3+ release kinetics of FeNTf complex at pH 7.4 and pH 5.5, respectively. Figure S1 shows effects of crowding agents and salt dependence of the far-UV CD spectra of native and urea-denatured Fe2Tf at 25 C.

ACKNOWLEDGEMENTS



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This work was supported by DBT grant (BT/PR11684/BRB/10/1300/2014), DST-SERB grant (EMR/2014/000242) and ICMR grant (F.No. 52/6/2013-BMS), Government of India.

Author Contributions: R.K. conceived the ideas. S.K., D.S., and R.K. designed the research. S.K. performed the research. S.K., D.S., and R.K analyzed the data. R.K., S.K. and D.S. wrote the paper.


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102. Dhar, A.; Samiotakis, A.; Ebbinghaus, S.; Nienhaus, L.; Homouz, D.; Gruebele, M.; Cheung, M. S. Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 17586–17591. 103. Homchaudhuri, L.; Sarma, N.; Swaminathan, R. Effect of crowding by dextrans and Ficolls on the rate of alkaline phosphatase–catalyzed hydrolysis: A size‐dependent investigation. Biopolymers 2006, 83, 477–486. 104. Pastor, I.; Vilaseca, E.; Madurga, S.; Garces, J. L.; Cascante, M.; Mas, F. Effect of crowding by dextrans on the hydrolysis of N-succinyl-l-phenyl-ala-p-nitroanilide catalyzed by α-chymotrypsin. J. Phys. Chem. B 2011, 115, 1115–1121. 105. Vopel, T.; Makhatadze, G. I. Enzyme activity in the crowded milieu. PLoS One 2012, 7, e39418. 106. Pozdnyakova, I.; Wittung-Stafshede, P. Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase. Biochim. Biophys. Acta. Proteins Proteomics 2010, 1804, 740–744. 107. Crowley, P. B.; Brett, K.; Muldoon, J. NMR Spectroscopy reveals cytochrome c–poly (ethylene glycol) interactions. ChemBioChem 2008, 9, 685–688. 108. Kim, Y. C.; Mittal, J. Crowding induced entropy–enthalpy compensation in protein association equilibria. Phys. Rev. Lett. 2013, 110, 208102–208105. 109. Mukherjee, S. K.; Gautam, S.; Biswas, S.; Kundu, J.; Chowdhury, P. K. Do macromolecular crowding agents exert only an excluded volume effect? A protein salvation study. J. Phys. Chem. B 2015, 119, 14145-14156.



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110. Minton, A. P. Explicit incorporation of hard and soft protein-protein interactions into models for crowding effects in protein mixtures. 2. Effects of varying hard and soft interactions upon prototypical chemical equilibra. J. Phys. Chem. B 2017, 121, 5515– 5522 111. Zhou, H. X. Polymer crowders and protein crowders act similarly on protein folding stability. FEBS Lett. 2013, 587, 394−397. 112. Senske, M.; Törk, L.; Born, B.; Havenith, M.; Herrmann, C.; Ebbinghaus, S. Protein stabilization by macromolecular crowding through enthalpy rather than entropy. J. Am. Chem. Soc. 2014, 136, 9036−9041. 113. Benton, L. A.; Smith, A. E.; Young, G. B.; Pielak, G. J. Unexpected effects of macromolecular crowding on protein stability. Biochemistry 2012, 51, 9773−9775. 114. Ando, T.; Yu, I.; Feig, M.; Sugita, Y. Thermodynamics of macromolecular association in heterogeneous crowding environments: theoretical and simulation studies with a simplified model. J. Phys. Chem. B 2016, 120, 11856−11865. 115. Ghosh, S. K.; Cherstvy, A. G.; Grebenkov, D. S.; Metzler, R. Anomalous, non-Gaussian tracer diffusion in crowded two-dimensional environments. New J. Phys. 2016, 18, 013027. 116. Ghosh, S. K.; Cherstvy, A. G.; Metzler, R. Non-universal tracer diffusion in crowded media of non-inert obstacles. Phys. Chem. Chem. Phys. 2015, 17, 1847−1858. 117. Elcock, A. H.; McCammon, J. A. Electrostatic contributions to the stability of halophilic proteins. J. Mol. Biol. 1998, 280, 731–748.


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118. Perez-Jimenez, R.; Godoy-Ruiz, R.; Ibarra-Molero, B.; SanchezRuiz, J. M. The efficiency of different salts to screen charge interactions in proteins: a Hofmeister effect? Biophys. J. 2004, 86, 2414–2429. 119. Pegram, L. M.; Record, M. T. Jr. Thermodynamic origin of Hofmeister ion effects. J. Phys. Chem. B 2008, 112, 9428–9436. 120. Marques HM, Egan TJ, Pattrick G. The non-reductive removal of iron from human serum N-terminal monoferric transferrin by pyrophosphate. S. Afr. J .Sci. 1990, 86, 21– 24. 121. Egan, T. J.; Ross, D. C.; Purves, L. R.; Adams, P.A. Mechanism of iron release from human

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Table 1. Crowding agent dependence of Cm, ∆GD, and mg derived for urea-denaturation induced iron release from Fe2Tf at pH 7.4 and pH 5.7 as monitored by absorbance at 465 nm.* pH 7.4 pH 5.7 ∆GD mg Cm ∆GD mg Cm Control 6.2 1.20 5.2 4.1 1.13 3.6 dextran 40 100 mg ml-1 6.6 1.10 6.0 4.5 1.08 4.2 dextran 40 200 mg ml-1 7.8 1.22 6.4 5.6 1.24 4.5 7.4 1.19 6.2 4.8 1.11 4.3 dextran 70 100 mg ml-1 -1 dextran 70 200 mg ml 8.1 1.18 6.9 5.9 1.14 5.2 ficoll 70 100 mg ml-1 6.7 1.22 5.5 4.2 1.10 3.8 -1 ficoll 70 200 mg ml 7.2 1.23 5.9 4.6 1.06 4.3 *Cm, ∆GD, and mg are reported as M, kcal mol-1, and kcal mol-1 M-1. The uncertainty of Cm, ∆GD, and mg values reported here is 0.1 M, 0.2 kcal mol-1, and 0.05 kcal mol-1 M-1. Table 2. Effect of crowding agent on the salt dependence of Cm, ∆GD, and mg derived for ureainduced iron release from Fe2Tf at pH 7.4 and pH 5.7 as monitored by absorbance at 465 nm.* pH 7.4 pH 5.7 ∆GD mg Cm ∆GD mg Cm Control 6.2 1.20 5.2 4.1 1.13 3.6 dextran 40 200 mg ml-1 7.8 1.22 6.4 5.6 1.24 4.5 0.3 M NaCl 5.8 1.25 4.6 3.4 1.06 3.2 0.6 M NaCl 5.1 1.15 4.4 3.3 1.23 2.7 1.2 M NaCl 4.9 1.11 4.4 3.2 1.23 2.6 -1 0.3 M NaCl + dextran 40 200 mg ml 7.6 1.28 5.9 5.4 1.24 4.4 0.6 M NaCl + dextran 40 200 mg ml-1 7.3 1.28 5.7 4.9 1.16 4.2 -1 1.2 M NaCl + dextran 40 200 mg ml 6.6 1.20 5.5 4.5 1.09 4.1 *Cm, ∆GD, and mg are reported as M, kcal mol-1, and kcal mol-1 M-1. The uncertainty of Cm, ∆GD, and mg values reported here is 0.1 M, 0.2 kcal mol-1, and 0.05 kcal mol-1 M-1.

Table 3. Effect of crowding agent on the Cm, ∆GD, and mg derived for urea-induced unfolding of Fe2Tf at pH 7.4 and pH 5.5 as monitored by monitored by CD at 222.* pH 7.4 pH 5.5 ∆GD mg Cm ∆GD mg Cm Control 7.8 0.98 8.0 4.6 1.10 4.2 dextran 40 100 mg ml-1 8.4 1.03 8.2 5.1 1.06 4.8 dextran 40 200 mg ml-1 9.0 1.06 8.5 5.5 1.05 5.2 -1 9.7 1.10 8.8 6.0 0.99 6.1 dextran 40 300 mg ml 8.7 1.06 8.2 5.3 1.01 5.2 dextran 70 100 mg ml-1 -1 dextran 70 200 mg ml 9.7 1.10 8.8 5.7 0.98 5.8 dextran 70 300 mg ml-1 10.3 1.11 9.3 6.3 0.95 6.6 -1 ficoll 70 100 mg ml 8.1 1.00 8.1 5.1 1.11 4.6 ficoll 70 200 mg ml-1 8.5 1.03 8.3 5.5 1.07 5.1 46 ACS Paragon Plus Environment

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9.1 1.06 8.6 5.7 1.05 5.4 ficoll 70 300 mg ml-1 -1 -1 -1 *Cm, ∆GD, and mg are reported as M, kcal mol , and kcal mol M . The uncertainty of Cm, ∆GD, and mg values reported here is 0.1 M, 0.2 kcal mol-1, and 0.05 kcal mol-1 M-1.

Table 4. Effect of crowding agents on salt dependence of Cm, ∆GD, and mg derived for ureainduced unfolding of Fe2Tf at pH 7.4 and pH 5.5 as monitored by monitored by CD at 222.* pH 7.4 pH 5.5 ∆GD mg Cm ∆GD mg Cm Control 7.8 0.98 8.0 4.6 1.10 4.2 dextran 40 200 mg ml-1 9.0 1.06 8.5 5.5 1.05 5.2 0.3 M NaCl 7.2 0.97 7.4 4.5 1.21 3.7 0.6 M NaCl 6.7 0.98 6.8 3.7 1.20 3.1 1.2 M NaCl 6.7 0.97 6.9 3.6 1.35 2.7 0.3 M NaCl + dextran 40 200 mg ml-1 8.6 1.05 8.2 5.3 1.10 4.8 -1 0.6 M NaCl + dextran 40 200 mg ml 8.2 1.03 8.0 5.1 1.14 4.5 1.2 M NaCl + dextran 40 200 mg ml-1 8.1 1.00 8.1 5.0 1.17 4.3 -1 -1 -1 *Cm, ∆GD, and mg are reported as M, kcal mol , and kcal mol M . The uncertainty of Cm, ∆GD, and mg values reported here is 0.1 M, 0.2 kcal mol-1, and 0.05 kcal mol-1 M-1.

Table 5. Effects of crowding agents on the activation parameters for Fe2+ release reaction of FeNTf.* ∆G‡a ∆H‡ ∆S‡ −T∆S‡a -1 -1 -1 -1 (kcal mol ) (kcal mol ) (cal mol K ) (kcal mol-1) pH 7.4 Control 20.9(0.1) 8.9(0.3) -40.2(1.2) 12.0(0.3) 200 mg ml-1 dextran 40 21.4(0.1) 11.5(0.5) -33.2(1.8) 9.9(0.5) -1 200 mg ml dextran 70 21.2(0.1) 12.2(0.2) -31.2(0.7) 9.0(0.2) 200 mg ml-1 ficoll 70 21.3(0.1) 11.0(0.4) -34.7(1.3) 10.3(0.4) pH 5.5 Control 19.0(0.1) 3.2(0.2) -52.9(0.6) 15.8(0.2) -1 200 mg ml dextran 40 19.6(0.1) 5.5(0.2) -47.2(0.7) 14.1(0.2) 200 mg ml-1 dextran 70 19.7(0.1) 6.6(0.3) -43.8(0.9) 13.1(0.3) -1 200 mg ml ficoll 70 19.3(0.1) 4.8(0.2) -48.7(0.7) 14.5(0.2) a Activation free energy (∆G‡) and entropy changes (−T∆S‡) are given at 25 ºC. *The uncertainties (standard error) in H‡, −T∆S‡ and S‡ are indicated in parenthesis.



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Table 6. Effect of crowding agent on the salt dependence of the activation thermodynamic parameters for Fe2+ release reaction of FeNTf.* ∆G‡a ∆H‡ ∆S‡ −T∆S‡a (kcal mol-1) (kcal mol-1) (cal mol-1 K-1) (kcal mol-1) pH 7.4 Control 20.9(0.1) 8.9(0.3) -40.2(1.2) 12.0(0.3) 200 mg ml-1 dextran 40 21.4(0.1) 11.5(0.5) -33.2(1.8) 9.9(0.5) 0.5 M NaCl with 200 21.1(0.1) 10.1 (0.2) -37.1(0.6) 11.0(0.2) mg ml-1 dextran 40 0.15 M NaCl 20.7(0.1) 6.6 (0.3) -47.3(1.0) 14.1(0.3) pH 5.5 Control 19.0(0.1) 3.2(0.2) -52.9(0.6) 15.8(0.2) 200 mg ml-1 dextran 40 19.6(0.1) 5.5(0.2) -47.2(0.7) 14.1(0.2) 0.15 M NaCl 18.7(0.1) 1.9(0.1) -56.6(0.2) 16.8(0.1) 0.5 M NaCl with 200 19.3(0.1) 5.1(0.2) -47.7(0.7) 14.2(0.2) mg ml-1 dextran 40 a Activation free energy (∆G‡) and entropy changes (−T∆S‡) are given at 25 ºC. *The uncertainties (standard error) in H‡, −T∆S‡ and S‡ are indicated in parenthesis.


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Figure 1. Effect of crowding agent and salt on pH- and urea dependence of the absorbance spectra of Fe2Tf at 25 C. (a) Absorbance spectra of Fe2Tf: (1) pH 7.4, no additive; (2) pH 5.0, 200 mg ml-1 dextran 40; (3) pH 5.0, 200 mg ml-1 dextran 40 with 0.3 M NaCl; (4) pH 5.0, no additive; (5) pH 5.0, 0.3 M NaCl; (6) pH 4.0, no additive. Red and blue short dash line spectra correspond to Fe2Tf (pH 7.4) in the presence of 1.0 M NaCl and 200 mg ml -1 dextran 40, respectively. Panel (b) shows the absorbance spectra of Fe2Tf at pH 7.4. Curve labels 1 to 6 correspond to 0.0 M urea; 200 mg ml-1 dextran 40 with 5.0 M urea; 5.0 M urea with 0.3 M NaCl and 200 mg ml-1 dextran 40; 5.0 M urea; 5.0 M urea with 0.3 M NaCl; and 10.0 M urea, respectively. Panel (c) shows the absorbance spectra of Fe2Tf at pH 5.7. Curve labels 1 to 6 correspond to 0.0 M urea; 200 mg ml-1 dextran 40 with 3.5 M urea; 3.5 M urea with 0.3 M NaCl and 200 mg ml-1 dextran 40; 3.5 M urea; 3.5 M urea with 0.3 M NaCl; and 8.5 M urea, respectively.

Figure 2. Effect of crowding agents on the pH- and urea-denaturation induced iron release from Fe2Tf at 25 C. Panel (a) shows the pH-induced iron release profiles (monitored by absorbance at 465 nm) of Fe2Tf in the absence (black circles) and presence of 200 mg ml-1 crowding agent (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)). These pH-equilibrium titrations were fitted according to eq 2. Panels (b) and (c) show the ureadenaturation induced iron release profiles (monitored by absorbance at 465 nm) of Fe2Tf in the absence (black circles) and presence of 200 mg ml-1 crowding agent (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)) at pH 7.4 and pH 5.7, respectively. The solid lines in panels (b) and (c) represent a non-linear least squares fit of the data to eq 1. Panel (d) shows the dependence of pH-midpoint (Cm*) for iron release from Fe2Tf on [Crowding



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agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)). Panels (e) and (f) show the dependence of urea-midpoint Cm (=∆GD/mg) for iron release from Fe2Tf on [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)) at pH 7.4 and pH 5.7, respectively. Insets of panels (e) and (f) show the dependence of ∆GD on [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)) at pH 7.4 and pH 5.7, respectively.

Figure 3. Effect of crowding agent on the salt dependence of pH- and urea-denaturation induced iron release from Fe2Tf at 25 C. Panel (a) shows the pH-induced iron release profiles (monitored by absorbance at 465 nm) of Fe2Tf in the absence (black circles) and presence of 200 mg ml-1 dextran 40 (blue triangles), 0.15 M NaCl (green circles), 0.15 M NaCl with 200 mg ml -1 dextran (pink triangles). These pH-equilibrium titrations were fitted according to eq 2. Panels (b) and (c) present the urea-denaturation induced iron release profiles (monitored by absorbance at 465 nm) of Fe2Tf in the absence (black circles) and presence of 0.3 M NaCl (green circles), 200 mg ml-1 dextran 40 (blue triangles), and 0.3 M NaCl with 200 mg ml-1 dextran 40 (pink triangles) at pH 7.4 and pH 5.7, respectively. The solid lines in panels (b) and (c) represent a non-linear least squares fit of the data to eq 1. (d) The dependence of the pH-midpoint (Cm*) for the iron release from Fe2Tf on [NaCl] (in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles)). The solid lines in panel (d) represent non-linear least squares fit of the data to a single-exponential function. Panels (e) and (f) show the dependence of urea-midpoint Cm (=∆GD/mg) for the iron release from Fe2Tf on [NaCl] (in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles) at pH 7.4 and pH 5.7, respectively. Insets of panels (e) and (f) show the dependence of ∆GD on [NaCl] (in the absence (green circles) and presence of


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200 mg ml-1 dextran 40 (pink circles)) at pH 7.4 and pH 5.7, respectively. The solid lines in panels (e) and (f) represent non-linear least squares fit of the data to a single-exponential function.

Figure 4. Effect of crowding agents on the urea-induced unfolding of Fe2Tf at 25 C. Panels (a) and (b) show the far-UV CD (222 nm) monitored unfolding curves of Fe2Tf at pH 7.4 and pH 5.5, respectively, in the absence (black circles) and presence of 200 mg ml-1 crowding agent (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)). The solid lines in panels (a) and (b) represent a non-linear least squares fit of the data to eq 1. Panels (c) and (d) show the dependence of urea-midpoint Cm (=∆GD/mg) for the unfolding of Fe2Tf on [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)) at pH 7.4 and pH 5.5, respectively. Insets of panels (c) and (d) show the dependence of ∆GD on [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) or ficoll 70 (red squares)) at pH 7.4 and pH 5.5, respectively.

Figure 5. Effect of crowding agents on the salt dependence of the urea-induced unfolding of Fe2Tf at 25 C. Panels (a) and (b) show the far-UV CD (222 nm) monitored unfolding curves of Fe2Tf at pH 7.4 and pH 5.5, respectively, in the absence (black circles) and presence of 0.3 M NaCl (green circles), 200 mg ml-1 dextran 40 (blue triangles), and 0.3 M NaCl with 200 mg ml-1 dextran 40 (pink triangles). The solid lines in panels (a) and (b) represent a non-linear least squares fit of the data to eq 1. Panels (c) and (d) show the dependence of urea-midpoint Cm (=∆GD/mg) for the unfolding of Fe2Tf on [NaCl] (in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles)) at pH 7.4 and pH 5.7, respectively. Panels (e) and (f) show



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the dependence of ∆GD on [NaCl] (in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles)) at pH 7.4 and pH 5.5, respectively. The solid lines in panels (c) to (d) represent non-linear least squares fit of the data to a single-exponential function.

Figure 6. Effects of crowding agents on the reduction and urea-denaturation induced iron release from FeNTf. Panel (a) and (b) present the single-phase reductive iron release kinetic traces of FeNTf in the absence (trace 1) and in the presence of 200 mg ml-1 dextran 40 (trace 2) at pH 7.4 and pH 5.5 (25 C), respectively. Panels (c) and (d) present the single-phase urea denaturation induced iron release kinetic traces of FeNsTf in the absence (trace 1) and in the presence of 200 mg ml-1 dextran 40 (trace 2) at pH 7.4 and pH 5.5 (37 C), respectively. The solid line in panels (a) to (d) show least-squares fits of the data to a single exponential rate expression. Panels (e) and (f) show the variation of rate constants for reductive iron release, kobs, with [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) and ficoll 70 (red squares)) at pH 7.4 and pH 5.5 (25 C), respectively. Panels (g) and (h) show the variation of rate constants for urea denaturation induced iron release, kobs with [Crowding agent] (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) and ficoll 70 (red squares)) at pH 7.4 and pH 5.5 (37 C), respectively.

Figure 7. Effects of crowding agent on salt-dependence of the reduction and urea-denaturation induced iron iron release from FeNTf. Panels (a) and (b) present the single-phase reductive iron release kinetic traces of FeNTf in the absence (trace 1) and in the presence of different additives (0.5 M NaCl (trace 2), 200 mg ml-1 dextran 40 (trace 3) and 200 mg ml-1 dextran 40 with 0.5 M NaCl (trace 4)) at pH 7.4 and pH 5.5 (25 C) , respectively. Panels (c) and (d) present the single-


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phase urea denaturation induced iron release kinetic traces of FeNTf in the absence (trace 1) and in the presence of different additives (0.6 M NaCl (trace 2), 200 mg ml -1 dextran 40 (trace 3) and 200 mg ml-1 dextran 40 with 0.6 M NaCl (trace 4)) at pH 7.4 and pH 5.5 (37 C), respectively. The solid lines in panels (a) to (d) show least-squares fits of the data to a single exponential rate expression. Panels (e) and (f) show variation of rate constants for reductive iron release, kobs with [NaCl] in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles) at pH 7.4 and pH 5.5 (25 C), respectively. The insets of panels (e) and (f) show the dependence of log kobs on [I1/2/(1+I1/2] for Fe2+ release at pH 7.4 and 5.5, respectively in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles). Panels (g) and (h) show variation of rate constants for urea denaturation induced iron release, kobs [NaCl] in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles) at 7.4 and pH 5.5 (37 C), respectively. The Insets of panels (g) and (h) show the dependence of log kobs on [I1/2/(1+I1/2] for Fe3+ release at pH 7.4 and 5.5, respectively in the absence (green circles) and presence of 200 mg ml-1 dextran 40 (pink circles). The solid lines in panels (e) to (h) show least-squares fits of the data to a single exponential function. The solid lines in insets of panels (e) to (h) show linear least squares fits to the data.

Figure 8. Effects of crowding agent on thermodynamic activation parameter for reductive iron release at pH 7.4 and 5.5. The Panels (a) and (b) show Erying plots for the reduction-induced iron release from FeNTf at pH 7.4 and pH 5.5, respectively (no addition (black circles), 200 mg ml-1 crowding agent (dextran 40 (blue triangles), dextran 70 (cyan inverse triangles) and ficoll 70 (red squares)). Panels (c) and (d) show Erying plots for the reduction-induced iron release from FeNTf at pH 7.4 and pH 5.5, respectively (no addition (black circle), 200 mg ml -1 dextran 40



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(blue triangle), 0.15 M NaCl (green circle) and 0.5 M NaCl with 200 mg ml-1 dextran 40 (pink triangle)). Panels (e) and (f) show the TΔΔS and ΔΔH plot at pH 7.4 and pH 5.5, respectively. Data points correspond to in the absence (black circle) and presence of 200 mg ml −1 of crowding agent (dextran 40 (blue triangle), dextran 70 (cyan inverse triangle) and ficoll 70 (red square)).


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Fig. 1



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Fig. 2


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Fig. 3



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Fig. 4


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Fig. 5

]



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Fig. 6


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Fig. 7



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Fig. 8


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TOC Graphic 100 % Iron Remaining

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 no additive () dextran 40 ( ) dextran 70 ( ) ficoll 70 ( )

0 3

4

5 pH

6

7



Table of Contents (TOC) Image

100

% Iron Remaining

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

No additive (■) 200 mg ml-1 ficoll 70 (■) 200 mg ml-1 dextran 40 (■) 200 mg ml-1 dextran 70 (■)

50

0 3

4

5 pH

6

7

ACS Paragon Plus Environment

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Role of Macromolecular Crowding on Stability and Iron Release

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