Selective separation of ferric and non-ferric forms of humantransferrin by capillary micellar electrokinetic chromatography

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Journal of Chromatography A, 1341 (2014) 73–78

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

elective separation of ferric and non-ferric forms of humanransferrin by capillary micellar electrokinetic chromatography

aweł Nowaka, Klaudyna Spiewakb, Julia Nowaka, Małgorzata Brindellb,ichał Wozniakiewicza,∗, Grazyna Stochelb, Paweł Koscielniaka

Jagiellonian University in Kraków, Faculty of Chemistry, Department of Analytical Chemistry, Kraków, PolandJagiellonian University in Kraków, Faculty of Chemistry, Department of Inorganic Chemistry, Kraków, Poland

r t i c l e i n f o

rticle history:eceived 5 February 2014eceived in revised form 9 March 2014ccepted 11 March 2014vailable online 18 March 2014

eywords:lectrophoretically mediated microanalysisron saturation

icellar electrokinetic chromatographyn-line reaction

a b s t r a c t

The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed.Using a surface response methodology and a three-factorial Doehlert design we have established a newoptimized running buffer composition: 50 mM Tris–HCl, pH 8.5, 22.5% (v/v) methanol, 17.5 mM SDS.As a result, two previously unobserved monoferric forms of protein have been separated and identified,moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerablyreduced by methanol, and the method selectivity has been yet increased resulting in a total separationof proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowedus to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein withthe buffers with different concentrations of ferric ions. It revealed continuously changing contribution of

hort-end injectionransferrin

monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobesof protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside thecapillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion medi-ated and electrophoretically mediated ones. Finally, the total time of separation has been decreased downto 4 min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Human serum transferrin (Tf) is the iron-binding transport pro-ein essential for delivering of iron ions into the cells. It binds tworivalent iron (ferric) ions to the N- and C-binding sites, respec-ively, resulting in the entirely iron-saturated form of protein (holo,-Tf). Alternatively only one ferric ion can be bound to the N- or C-inding site, hence two possible monoferric forms of Tf may exist:eN–Tf and FeC–Tf [1,2]. The efficient separation of iron-free (apo,-Tf), monoferric, and holo forms was a problematic task, becausehey exhibit the structural and chemical similarity [3].

Both a capillary zone electrophoresis and a micellar electroki-

etic chromatography (MEKC) have been reported to be suitableechniques for the analysis of proteins and peptides [4–6]. Therere numerous parameters which may be adjusted to improve the

∗ Corresponding author at: Jagiellonian University in Kraków, Ingardena 3, 30-060raków, Poland. Tel.: +48 12 663 20 84; fax: +48 12 663 20 84.

E-mail addresses: [email protected],[email protected] (M. Wozniakiewicz).

ttp://dx.doi.org/10.1016/j.chroma.2014.03.037021-9673/© 2014 Elsevier B.V. All rights reserved.

separation, however, the fast method optimization is concurrentlyimpeded. Therefore, the use of experimental design approach isadvisable, especially in the case when the results seem to bedependent on several factors with no determined correlation.

MEKC, due to the surfactant presence, minimizes the adsorptionof proteins to the capillary inner surface, thus making the resultsmore precise and reproducible [7–10]. Tf was a subject of severalCE-based analysis conducted usually with the use of MEKC. Thesestudies were mainly focused on its determination in the biologi-cal material, including determination of a carbohydrate-deficienttransferrin, a common marker of alcohol abuse [11–15]. Recently,we have also published a method allowing the separation of a-Tfand h-Tf using MEKC technique [16]. Our protocol was based onthe addition of 7 mM SDS to running buffer consisting of 50 mMTris–HCl, pH 8.5. Unfortunately, the presence of the two monofer-ric forms of Tf (FeN–Tf and FeC–Tf) could not be confirmed by usingdescribed method.

Our present work was aimed at the further method develop-ment to enable the detection of all four forms of Tf includingthe differentiation between FeN–Tf and FeC–Tf, prevention fromiron release during electrophoretic separation, and reduction in

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eparation time. A particular effort has been made to providehe evidence that the peaks observed on the electropherogramsre the assumed Tf forms, and to investigate the contribution ofach ferric form at different saturation levels. Distinct analyticalurpose was to attempt two modes of on-line protein saturationonducted directly inside the capillary, and to use the experi-ental design and the surface response methodology for method

ptimization.

. Materials and methods

Human a-Tf, human h-Tf (powder, BioReagent, suitable forell culture, ≥98%), and human serum albumin (HSA) (powder,atty acids free, globulin free, ≥99%), iron(III) nitrate nonahydrate,nd nitrilotriacetic acid (NTA) were obtained from Sigma–AldrichGermany). Chemicals of analytical reagent grade: sodium hydrox-de and ethanol were supplied by POCH S.A. (Gliwice, Poland), whileC–MS grade solvents: methanol, isopropanol an acetonitrile wereurchased from Sigma–Aldrich (Germany).

All solutions were prepared in MiliQ quality water and filteredhrough 45 �m regenerated cellulose membrane and degassed byentrifugation. All separation buffers were stored at +4 ◦C, whileaOH and HCl solutions at ambient temperature. The samples ofroteins were prepared in 50 mM Tris buffer, pH 8.5 with neitherurfactant nor organic solvent addition, at the final concentrationanging from 0.10 to 0.50 mg/mL. The accurate value of buffer pHas obtained by using concentrated HCl (Tris–HCl). Before each

nalysis, samples were centrifuged for 5 min, 5000 × g. The minimalolume of the sample was 50 �L.

The measurements were performed using a P/ACE Capillarylectrophoresis System (Beckman-Coulter, USA) equipped with

diode array detector. During all experiments the whole spec-ra within the range of 200–600 nm were collected, however, theesults obtained at 200 nm were used in further processing. Thencoated fused-silica capillaries (laser burned detection window)ere of 60 cm × 50 �m i.d. or 30 cm × 50 �m (Beckman-Coulter),ith a 50 or 20 cm distance to the detector, respectively. Temper-

ture of the sample tray and capillary were set up to 22 ◦C. Theapillary was being rinsed between runs as follows: 0.138 MPa20 psi) of MiliQ water for 2 min, 0.138 MPa of 0.1 M NaOH for

min; and 0.138 MPa of running buffer for 3 min. Before the firstun at a working day the rinsing protocol involved 0.138 MPa ofethanol for 6 min; 0.138 MPa of 0.1 M HCl for 4 min; 0.138 MPa

f MiliQ water for 3 min; 0.138 MPa of 0.1 M NaOH for 10 min; and.138 MPa of running buffer for 10 min, whereas in case of the firstse of the capillary after mounting in cartridge the same procedureas been used, but the duration of all steps was doubled. Sample

njection was performed using a forward pressure at anodic side,pplying: 3.4 kPa (0.5 psi) for 5 s, unless stated otherwise. Forwardoltage ranging from 15 kV to 30 kV was being applied. Each anal-sis has been repeated at least three times. The instrumental noiseroduced during detection has been smoothed out using Origin 8.0oftware (OriginLab Corporation, USA). During chemometric cal-ulations Statistica 10 (StatSoft Inc., Tulsa, OK, USA) software wassed.

Conductivity measurements were performed using a microcom-uter conductivity meter (Elmetron CC-551) with a conductivityensor (CD-2 type, 0.51 cm−1 sensor constant).

. Results and discussion

.1. Addition of organic solvent

In the beginning, the effect of four different organic sol-ents as the buffer additives has been tested: methanol, ethanol,

r. A 1341 (2014) 73–78

propan-2-ol, and acetonitrile, each one in final 20% (v/v) concen-tration. The solvents were added to the 50 mM Tris buffer, pH 8.5,containing 10 mM SDS. According to the previously reported data,in this buffer SDS is able to form the micelles, probably a crucial fac-tor enabling the separation of holo and apo forms of Tf. A referencebuffer was the buffer without the addition of any organic modi-fier. The results have been generally compared by separating thesample containing mixed h-Tf, a-Tf and HSA. HSA was used as aninternal standard to compare the obtained relative migration timesstanding for the particular transferrin forms and for calculation ofrelative peak areas.

Among the four tested organic solvents, each one resulted inaltered electropherogram, however only in the case of methanolthe appearance of the two minor, totally separated peaks has beenreported, localized in a gap between the peaks corresponding to h-Tf and a-Tf. Another effect which has been observed for methanolwas the change in peak intensity, i.e. the peak standing for h-Tfhad similar intensity as that for a-Tf, contrary to the buffer with-out methanol where the peak of h-Tf was considerably diminished.It has supported us to conclude, that the loss of iron by h-Tf canbe minimized in presence of the methanol (see SupplementaryMaterial for more detailed investigations of the role played bymethanol).

Apart from the organic solvents, also the addition of urea infinal concentration of 6 M, 1 M, and 1 M with combination with20% methanol, has been investigated. Urea was reported to improveseparation of Tf forms according to the iron saturation in gel elec-trophoresis [17]. In our case it has turn out, that the changes inelectropherograms have been more extensive than those causedby the solvents, however the peaks were incompletely separatedand vastly diminished, i.e. the sensitivity of the method was con-siderably weaker after addition of urea. In the end, we haveconcluded that methanol without urea is the most promising bufferadditive.

Then, the effect of increasing concentration of methanol inthe running buffer has been investigated, as it has been shownin Fig. 1. Final concentration of Tris 50 mM, SDS 10 mM andpH 8.5 were kept for each buffer. The methanol-free buffer andthe buffers containing: 10.0%, 17.5% and 25.0% (v/v) methanolwere compared. It has revealed the strong and increasing withconcentration effect of this additive on the peak area ratiobetween holo and apo forms, and the appearance of two novelpeaks. We have hypothesized, that these peaks may originatefrom monoferric forms of protein, previously unobserved. Theanalysis performed for the pure h-Tf and a-Tf resulted also inthe appearance of these peaks, but their intensity was verylow.

3.2. Experimental design

Taking into account that finding of the optimal conditions fora method involving addition of the two different but crucial buffercomponents is not a trivial task, we have decided to use experimen-tal design approach. For that purpose, set of experiments based on athree-factorial Doehlert uniform shell design and surface responsemethodology was conducted [18]. The optimized factors were: con-centration of methanol cMeOH, concentration of SDS cSDS and pH.Preliminary experiments performed initially helped us to pointout the range of optimized factors (see Supplementary Materialfor more details) [19]. Three system responses have been chosen:time of analysis defined as time of the end of HSA peak, sum ofFWHM (Full Width at Half Maximum) for all five peaks of inter-

est, and inverse resolution between h-Tf and HSA peaks. Suchdefined responses reflect the improvement in separation if theirvalues are decreasing. According to the surface response method-ology, a quadratic polynomial response model for the sample was

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P. Nowak et al. / J. Chromatogr. A 1341 (2014) 73–78 75

Fig. 1. Electropherograms presenting the separation of the mixed sample containing h-Tf, a-Tf, and HSA, with reference to the increasing concentration of methanol, as wellas the samples containing only h-Tf or a-Tf with HSA in the highest methanol concentration. Each Tf form was prepared at final concentration 0.33 mg/mL, while HSA at0 voltam ties. E

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.33 mg/mL for mixed sample and 0.16 mg/mL for Tf standards sample. The value ofethanol, and 30 kV for 25% methanol, to ensure the similar electrophoretic mobili

stimated, according to formula (1).

k = ˇ0 + ˇ1cMeoH + ˇ2cSDS + ˇ3pH + ˇ11c2MeoH + ˇ33c2

SDS

+ ˇ33pH2 + ˇ12cMeOHcSDS + ˇ13cMeOHpH + ˇ23cSDSpH (1)

here yk is the given response of the system (time of analysis, sumf FWHM or inverse resolution). The coefficients ˇi and ˇij werealculated by constructing the model by means of general linearegression, with the application of effective hypothesis test [20].tatistical significance of coefficients ˇi and ˇij was evaluated withhe use of the ANOVA test at the level = 0.05.

According to the developed models, all three factors (cMeOH, cSDS,nd pH) have significant influence on analysis time, both methanolnd SDS concentration and SDS are significant with regard to theWHM values, inverse resolution of chosen peaks is influenced byone of these factors. This may be caused by relatively good resolu-ion in most of the experiments. The correlations between factorsere found insignificant and the calculated model reveals mostly

inear relations between factors and analyzed responses.In order to estimate conditions optimal for all of the responses of

he system, a Derringer and Suich desirability function (D function)21], has been applied to the calculated model. The desirability val-es for the analyzed factors were between 0 for the lowest responseyk) values and 1 for the highest. The overall function D has been cal-ulated as the geometric mean of the individual desirability values.unction D surfaces diagrams are presented in Fig. 2. The optimum

onditions correspond to a local maximum value of function D, andhey are as follows: cMeOH = 22.5% (v/v), cSDS = 17.5 mM (w/v), pH.5. Since that time, these values have been kept for running bufferuring the all next experiments.

ge was: 15 kV for buffer without methanol, 20 kV for 10% methanol, 25 kV for 17.5%ffective capillary length was 50 cm.

3.3. Gradual Tf saturation

In the next step, an experiment assuming the preparation ofsamples characterized by various iron saturation of a-Tf by ferricions has been carried out. To this end, 6 mixtures with different a-Tfand ferric ions ratios (added as the nitrilotriacetic-Fe(III) complex,Fe-NTA) have been prepared [22], and incubated 20 min at ambienttemperature in vials placed directly on a sample tray in CE instru-ment. Hence, it was an example of an off-line process conductedin an at-inlet format. After incubation, the samples were subjectedto the CE analysis in optimized conditions and using a truncatedcapillary to 20 cm of effective length and 15 kV voltage. Electro-pherograms obtained for all of the 6 samples have been shown inFig. 3.

As it can be seen, this experiment allowed us to monitor thegradually increasing saturation of a-Tf by ferric ions, revealing thatintensities of the two novel peaks are considerably changing withincreasing concentration of iron in sample. Moreover, differentbehavior of these peaks has been reported. One of them is rapidlydeclining, while the second one is gradually growing up, reachingthe intensity similar as that of h-Tf in a fourth sample. For the lastsample, a complete saturation of a-Tf to h-Tf was observed. Theseresults prove, that the peaks localized between h-Tf and a-Tf arethe two predicted monoferric forms of protein, completely sepa-rated by this method and sensitive on the concentration of iron insample. After calculation of the relative peak areas (Fig. S-3) wehave concluded, that obtained outcomes are consistent with the

data reporting on different binding affinities of the two iron bidingsites and an existing cooperativity between them (see Supplemen-tary Material for more details). As a consequence, monoferric formshave been identified, i.e. FeC–Tf is a faster migrating form than

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Fig. 2. Diagrams of function D surfaces: cSDS–cMeOH, cSDS–pH, cMeOH–pH, respectively.It is worth highlighting, that searching for a maximum value of function D beyondthe considered ranges of parameters has been excluded on the basis of the firstpreliminary set of experiments. In particular, cMeOH lower than 22.5% (v/v) initiatedus

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Fig. 3. Electropherograms obtained for the samples containing equal amount of a-Tf (0.16 mg/mL) and HSA (0.10 mg/mL) in buffer composed of 50 mM Tris, 100 mM

3.4. Short-end injection

ndesirable iron release, and pH higher than 8.5 strongly deteriorated the peakhape of h-Tf (see Supplementary Material).

eN–Tf. To our knowledge, this is the first demonstration of a grad-al saturation of a-Tf by iron using the chromatography-relatedechnique able to distinguish between all possible forms of protein.

Subsequently, the potential of the CE instrument for perform-ng on-line reactions has been attempted. In this case, the bindingetween protein and metal ions were assumed to be possible

NaCl, 25 mM NaHCO3, pH 7.4, and different concentration of iron added as Fe-NTA,incubated at-inlet for 20 min. Molar ratio of a-Tf to iron amounting to 1:2 theoreti-cally enables the complete saturation of protein.

directly inside the capillary, as the effect of interactions betweenthe distinct zones injected consecutively into capillary. For thatpurpose two different methodologies are possible. Firstly, the reac-tion may be triggered by diffusion-mediated mixing, i.e. an idleprocess occurring when the zones get in a direct contact insidecapillary. Secondly, the efficiency of mixing may be improvedvia electrophoretically-mediated mixing, by applying the voltagedirectly after injections to mix properly the reactants. This method-ology was broadly used to investigate the activity of differenttypes of enzymes, and is known as an electrophoretically mediatedmicroanalysis (EMMA) [23–27]. In both cases, an additional incuba-tion step may be introduced without the voltage application, whenthe reaction might take place. Thus, during incubation period thecapillary played a role of a specific microreactor. The largest advan-tage of on-line approach is that the volumes of reactants needed tobe injected into capillary are even three orders of magnitude lowerthan in off-line approach.

Both mixing modes have been applied to study iron binding toa-Tf. The obtained electropherograms showing the similar level ofprotein saturation by iron are depicted in Fig. 4(A). In addition,the schematic illustration of these methodologies is depicted thereas well. The molar ratio between iron and protein at the point ofcontact between zones was 10:1, which reflects the concentrationof iron and protein in given zones. Electrophoretically-mediatedmixing combined with a step of an idle incubation of the sam-ple in capillary without the voltage application was more efficientthan the diffusion-mediated mode combined with the same step ofincubation. To reach similar iron saturation of Tf, the step of incu-bation required in the case of electrophoretically-mediated mixingwas five times shorter than that in the case of diffusion-mediatedapproach. More details concerning these experiments and addi-tional electropherograms obtained for different times of incubationare available in Supplementary Material.

At the end, we have decided to apply another variant ofCE allowing for the large reduction in time of analysis, i.e. a

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P. Nowak et al. / J. Chromatogr. A 1341 (2014) 73–78 77

Fig. 4. The outcomes obtained after the application of on-line reaction and short-end injection approaches. (A) – iron saturation of a-Tf performed inside capillary applyingdiffusion-mediated (top) and electrophoretically mediated (bottom) modes. In each case the step of incubation has been introduced directly after the injections: 5 min fordiffusion-mediated mode and 1 min for electrophoretically-mediated mode. The protein zones contained: a-Tf (0.5 mg/mL), HSA (0.3 mg/mL), 50 mM Tris, 100 mM NaCl,25 mM NaHCO3, pH 7.4, while iron was injected as Fe-NTA solution in the same buffer (10 times higher molar concentration than a-Tf). Effective capillary length was 20 cm,while voltage value 15 kV. (B) – separation of the mixture of h-Tf, a-Tf and HSA (each one 0.33 mg/mL) performed by long-end (top) and short-end (bottom) injections,a e, respd

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pplying 20 cm effective length – 30 kV and 10 cm effective length – 15 kV voltagepicted.

hort-end injection strategy [28]. Contrary to the conventionalong-end injection, it uses the shorter outlet part of capillary, whichormally is about 10 cm long. The sample vial is placed at the out-

et, and then negative pressure is applied for injection and theeverse polarity for separation. Therefore, this is one of the sim-lest way for reduction of separation time, unless the efficienteparation in 10 cm section of capillary cannot be obtained. In ourase, we have compared short-end injection performed using 10 cmapillary effective length and applying 15 kV voltage with long-nd injection using 20 cm capillary length and 30 kV (the sameapillary). Theoretically, both methods should give similar timesf migration. Indeed, the results shown in Fig. 4(B) confirm ourssumption. However, in the case of long-end injection the peaktanding for h-Tf was decreased, while one of the monoferric Tfnd a-Tf peaks increased. After performing additional experimentse have concluded, that the high electric field (1000 V/cm) gener-

ted in the relatively short capillary could be a direct or indirecteason for partial iron release (see Supplementary Material to seeore results).Interaction with SDS micelles depends on proteins structure,

harge and hydrophobic amino acids residues exposure [29].elease of iron from h-Tf is accompanied by conformationalhanges resulting in an “open” structure formation, characterizedy an increased exposure of aromatic residues [30,31]. Due to thatact, a-Tf can be more amenable to interactions with amphiphilicDS micelles. Two monoferric forms in turn, might be able to exert

n intermediate behavior, but slightly differing between each other.ethanol was possibly an additional enhancement of this phe-

omenon, and caused the preservation of ferric ions bounded tohe particular lobes of protein.

ectively. On the electropherograms, the schematic illustration of these modes is

4. Conclusions

To conclude, short end injection mode enabled the fully effi-cient separation of all protein forms within the time less than 4 min.10 cm was a sufficient effective length to separate all forms of pro-teins with the migration times associated rather with separation oflargely smaller molecules than of proteins. It is a clear-cut demon-stration of the method selectivity. It is worth highlighting, thatthe relatively large protein molecules exerting only a subtle differ-ence in mass caused by the binding of only one or two additionalatoms could be fully separated by this method. Moreover ferric ionsdo not alter the charge of molecule, since they replace H+ ions.Consequently, two monoferric forms were of the same mass-to-charge ratio, and they have been also separated to the baseline. Itproves that our method could be denoted as an ultraselective one.Such a good outcome can derive from the interactions betweenthe micelles formed by SDS and protein molecules. The faster h-Tf spends probably most of time being dissolved in liquid phase,while slower a-Tf is localized in micellar phase characterized bya reverse vector of electrophoretic mobility due to the negativelycharged SDS molecules.

The results concerning gradual Tf saturation turn out to be con-sistent with the theoretical assumptions. On-line methodology hasproven to be good alternative for conventional off-line strategy,using significantly lower amounts of reactants.

The method seems to be easily transferrable to a on-chip format,

where the separation time could be further shortened. Owing to thesignificant automation, sensitivity, and speed of analysis, we areconvinced that MEKC-based method is the most effective from theall reported to date Tf iron saturation assays. Such a good outcome

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ay encourage to the searching for the novel protein–ligand assaysmong CE-related techniques. Additionally it proves, that MEKCay give the great separation efficiency irrespective of difference

n mass-to-charge ratio of analytes.

cknowledgements

Author Paweł Nowak has received the financial support fromrakowskie Konsorcjum “Materia-Energia-Przyszłosc” within theubsidy KNOW.

The research was carried out with equipment purchased withnancial support from the European Regional Development Fundithin the framework of the Polish Innovation Economy Opera-

ional Program (contract no. POIG.0 2.01.00-12-0 23/08).

ppendix A. Supplementary data

Supplementary material related to this article can be found,n the online version, at http://dx.doi.org/10.1016/j.chroma.2014.3.037.

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