A High-throughput Method for the Quantification of Iron Saturation in Lactoferrin Preparations

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Anal Bioanal Chem DOI 10.1007/s00216-013-6943-9 RESEARCH PAPER A high-throughput method for the quantification of iron saturation in lactoferrin preparations Grzegorz Majka & Klaudyna Śpiewak & Katarzyna Kurpiewska & Piotr Heczko & Grażyna Stochel & Magdalena Strus & Małgorzata Brindell Received: 21 January 2013 / Revised: 14 March 2013 / Accepted: 25 March 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Lactoferrin is considered as a part o
  RESEARCH PAPER  A high-throughput method for the quantification of ironsaturation in lactoferrin preparations Grzegorz Majka & Klaudyna Ś piewak  & Katarzyna Kurpiewska & Piotr Heczko & Gra ż yna Stochel & Magdalena Strus & Ma ł gorzata Brindell Received: 21 January 2013 /Revised: 14 March 2013 /Accepted: 25 March 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Lactoferrin is considered as a part of the innateimmune system that plays a crucial role in preventing bac-terial growth, mostly via an iron sequestration mechanism.Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by servingas an iron chelator for some bacterial strains; thus, it is veryimportant to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin ironsaturation is essential not only because of its clinical appli-cations but also for a wide range of biochemical experi-ments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed toobtain a calibration curve enabling the determination of ironsaturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm (  A 280/466 ). To achieve thisgoal, selected techniques such as spectrophotometry, ELISA,and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simpleandfastwayhasbeenproventobeveryuseful.Furthermore,asimilar approach could easily be implemented to facilitate thedeterminationofironsaturationlevelforothermetalloproteinsin which metal binding results in the appearance of a distinct  band in the visible part of the spectrum. Keywords Lactoferrin.Ironsaturation.Absorptionratio.ICP-MS Introduction Lactoferrin (Lf), a protein being a member of the transferrinfamily, is highly abundant in colostrum and milk, but is also present in other extracellular excretions. This glycoprotein bears remarkable resemblance to transferrin  —   both proteinshave a molecular weight of about 80 kDa and their structurecomprises two lobes, each one capable of binding one ferricion. The most important difference is lactoferrin possessingthe ability to bind iron more tightly (binding constants of  ∼ 10 22  –  10 24 and 10 20  –  10 21 M − 1 for lactoferrin and transfer-rin, respectively, were reported) [1  –  3] and retain it at muchlower pH values [4] than serotransferrin. Another lactoferrin-specific feature is a highly basic N-terminal re-gion that allows interaction with negatively charged mole-cules such as lipopolysaccharide [5] or glycosaminoglycans[6]. Those two properties grant its ability to act as anantimicrobial agent and affecting immune response by bind-ing to receptors on target cells.Lactoferrin undergoes glycosylation in various species, but the number and location of glycosylation sites varydepending on the protein srcin. Human lactoferrin has three potential N  -glycosylation sites (Asn137, Asn478, and Grzegorz Majka and Klaudyna Ś  piewak contributed equally to thiswork. Electronic supplementary material The online version of this article(doi:10.1007/s00216-013-6943-9) contains supplementary material,which is available to authorized users.G. Majka : K. Ś  piewak  : G. Stochel : M. Brindell ( * )Department of Inorganic Chemistry, Faculty of Chemistry,Jagiellonian University, Ingardena 3,30-060 Krakow, Polande-mail: brindell@chemia.uj.edu.plG. Majka : K. Ś  piewak  : P. Heczko : M. Strus ( * )Chair of Microbiology, Jagiellonian University Medical College,Czysta 18,31-121 Krakow, Polande-mail: magdalena.strus@uj.edu.plK. KurpiewskaDepartment of Crystal Chemistry and Crystal Physics, Facultyof Chemistry, Jagiellonian University, Ingardena 3,30-060 Krakow, PolandAnal Bioanal ChemDOI 10.1007/s00216-013-6943-9  Asn623); cow, goat, and sheep lactoferrin has five (Asn233,Asn281, Asn368, Asn476, and Ans545) [7]. Folding of human lactoferrin does not depend on the number andlocation of bound glycan chains, and the role of glycosyla-tion has not been elucidated so far. Several studies revealedthat deglycosylation of human lactoferrin does not affect the binding of iron and other molecules, but decreases the proteolytic resistance of lactoferrin [8]. However, it has beenreported that the glycosylation of camel lactoferrin influ-ences the protein structure and iron binding mode within thecleft of the C-lobe. Supposedly, localization of the glyco-sylation site in the C-lobe inhibits the domain closer andweakens iron binding [9]. Lactoferrin is a basic protein withan isoelectric point of 8.7. To our knowledge, there are nodata showing the influence of glycosylation or iron satura-tion on protein p  I  value. Basing on results reported for transferrin glycoforms (pentasialotransferrin, p  I  =5.2;tetrasialotransferrin, p  I  =5.4; trisialotransferrin, p  I  =5.6; anddisialotransferrin, p  I  =5.7) [10], it should be expected that glycosylation should decrease the observed protein isoelec-tric point as a consequence of increasing the number of glycosylated arginine residues. Noticeable changes of the p  I  values should be anticipated upon saturation of latoferrin withiron (p  I  of 5.2 and 5.6 were reported for Fe 2 -Tf and Fe-Tf,respectively) [11]. The natural microheterogeneity of lactoferrin (different glycosylation status and iron satu-ration levels) makes lactoferrin isoforms difficult toseparate.Since nearly all living organisms need iron to live, ironscavengers  —  such as lactoferrin  —  might have a static effect on the growth of microorganisms in the gastrointestinaltract. The antimicrobial activity of lactoferrin has been fre-quently reported  —  its influence on bacterial, viral, protozo-an, and fungal pathogen growth has been numerouslyreviewed [12  –  14]. However, very little attention has been paid to the iron saturation level of lactoferrin used in thosestudies. The iron content of lactoferrin preparations is anaspect somewhat neglected in the literature  —  only a fewgroups investigated the more accurate determination of the percentage saturation of lactoferrin with ferric ions anddifferentiation between the effects of the apo and holo formson the studied pathogen species or target human cells. It has been shown that iron-free and iron-saturated lactoferrin af-fects human enterocytes in a different way [15,16]. Fur- thermore, apolactoferrin has been described as more potent in reducing the growth of numerous bacterial strains[17  –  20]. Neither accurate determination of Lf iron content nor the preparation of apo- and hololactoferrin is a trivial issue. Thesimplest way of calculating the iron content in the prepara-tion is based on spectrophotometric measurement. Total protein concentration can be determined using the absorp-tion coefficient at 280 nm, while the concentration of theholo form can be estimated by employing molar absorptioncoefficient at 466 nm. The band in the visible part of spectrum with maximum approx. 470 nm is characteristicof iron-saturated lactoferrin. In the literature, several differ-ent absorption coefficients were described for both apo- andhololactoferrin; some of them are presented in electronicsupplementary material (ESM) TableS1. However, theapplication of absorption coefficients to calculate the protein concentration is limited to the situation whenonly the apo or the holo form is present in solution.When the preparation is a mixture of both forms, theuse of this method can lead to substantial deviations. Toimprove the accuracy of protein content determination,some groups applied ELISA [21], the method of Lowry[22], or the method of Dumas using a nitrogen  –   proteinanalyzer [23,24]. Calculation of the iron content using the spectrophotometric method faces a problem related with thecoexistence of diferric and monoferric lactoferrin forms.About 47  –  58 % of human serum transferrin is monoferric,while 16  –  52 % is in the apo form and differric transferrinaccounts for 2  –  31 % depending on the srcin of the bloodsamples [25]. To our knowledge, there are no analogous datareported for lactoferrin. Human lactoferrin was found to besaturated in 20 % [26], while bovine lactoferrin is saturatedwithironin15  –  19%[27,28].Thesimilarityofproteinsofthe lactoferrin family suggests that a significant percentage of lactoferrin may be in the monoferric form. The iron content of lactoferrin preparations can be accurately determined usinginductively coupled plasma  –  mass spectrometry (ICP-MS)[29,30] or optical emission spectrometry [31,32] as well as atomic absorption spectrometry techniques. Recently, thecommercially available iron/TIBC kit was also appliedfor the determination of iron saturation levels in apo-and holotransferrin, with no data for lactoferrin avail-able yet [33].The most convenient and easily accessible way of determining the iron content in a lactoferrin preparation,which is valuable not only for biomedical research but also for drug development sector, would be the usage of the ratio of absorbances at 280 and 466 nm. Suchapproach has been described several times in the litera-ture. In the early 1980s, the value of 27  –  28 was pro- posed for the bovine lactoferrin fully saturated with iron[34], while quite recently, the value of 22 wassuggested for fully saturated non-glycosylated humanserum transferrin [35]. However, there is no informationabout using this parameter for lactoferrin only partiallysaturated with iron. To address the aforementioned prob-lems, we have combined several techniques such asspectrophotometry, ELISA, and ICP-MS in order to prepare the calibration curve enabling determination of the iron saturation level of bovine lactoferrin relyingsolely on the A 280/466 ratio. G. Majka et al.  Materials and methods MaterialsBovine lactoferrin was purchased from DMV International(estimated purity, 95 %; Veghel, the Netherlands) and thereference sample from Sigma-Aldrich Co. ( ≥ 85 % pure; St.Louis, MO, USA) and stored at 4 °C. Citric acid, acetic acid,and sodium chloride were analytical grade reagents supplied by POCH S.A. (Gliwice, Poland). Bovine β -lactoglobulin,lysozyme from chicken egg, ribonuclease A from bovine pancreas, bovine serum albumin, ferritin from equine spleentype I (saline solution), β -galactosidase from Escherichiacoli , and other chemicals were of analytical grade and pur-chased from Sigma-Aldrich Co. Buffers and reagents in allexperiments were prepared using Milli-Q water.Electrophoresis: SDS-PAGEThe abundance of different protein species was examinedusing 10 % polyacrylamide gel electrophoresis under dena-turing conditions [36]. Gels were stained with CoomassieBrilliant Blue and documented using GelDoc-It  ™ 310 Im-aging System (Upland, CA, USA).Preparation of bovine apo- and hololactoferrinsLactoferrin supplied by the manufacturers was used without further purification. For apolactoferrin, preparation containing50 mg/mL protein was dissolved in water and dialyzed exten-sively against 100 mM citrate buffer for 24 h, followed bydialysis against distilled water for 24 h [37]. Temperature(4 and 20 °C) and buffer pH (2.0  –  5.0) were modified in order to monitor their impact on iron desaturation. Iron saturationwas calculated based on the A 280 /   A 466 ratio according to thecalibration curve presented in “ Results and discussion. ” Wedefine the iron saturation level of lactoferrin as the percentageof iron-binding sites occupied by ferric ions assuming that 2 mol of iron(III) ions is bound per 1 mol of protein. Thus,the given values refer to the percentage of differic lactoferrin.For some of these samples, the ICP-MS and ELISA tests werecarried out and were used to prepare a calibration curve.Hololactoferrin was prepared by the reaction of 50 mg/mLlactoferrin solution in 50 mM Tris  –  HCl, 150 mM NaCl(pH 7.4) with ferric nitrate salt in the presence of nitrilotriacetic acid (NTA) as well as different concentrationsof sodium bicarbonate [24]. After incubation, excess iron wasremoved by dialysis against the same buffer solution without ferricsaltsfor24handagainstwaterforanother24h.Variousincubationtimes,temperatures,aswellasratiosofLf/Fe/NTAwere employed to examine their effect on iron saturationefficiency; detailed conditions are depicted in the captions of figures.Resaturation of apolactoferrinThe apolactoferrin prepared by dialyzing a sample of the src-inal lactoferrin to citrate buffer (100 mM, pH 2.0, 3.0, and 4.0)at room temperature as described above was resaturated withiron ions in order to evaluate its chelation. Lf was incubated(1h,roomtemperature)withferricnitratesaltinthepresenceof nitrilotriacetic acid at a Lf/Fe/NTA ratio of 1:4:4.FPLC proceduresThe chromatographic system ÄKTA Purifier 10 (GEHealthcare, Life Sciences), with an injection loop of 50 μ  L, fraction collector FRAC-901, and UV/VIS (Monitor UV-900, GE Pharmacia) as well as conductivity (Monitor  pH/C-900, GE Pharmacia) detectors were applied for pro-tein separation. Size exclusion chromatography A Superdex 200 10/300 GL column (10×300-mm ID,13- μ  m particle diameter; Tricorn, GE Healthcare Life Sci-ence) was equilibrated with phosphate buffer (50 mM,150 mM NaCl, pH 8.4) at a flow rate of 0.5 mL/min andwas kept at 20 °C. All buffers before use in fast proteinliquid chromatography (FPLC) system were degassed andfiltered through a 0.45- μ  m filter. Calibration of the columnwas achievedbyusing the following molecular massmarkers:ribonuclease A (13.7 kDa), lysozyme (14.3 kDa), β -lactoglobulin (35.0 kDa), bovine serum albumin (66 kDa),ferritin(440kDa),and β -galactosidase(465kDa).Thesampleof lactoferrin (DMV International or Sigma-Aldrich) wasdiluted in the same buffer, filtered through a 0.22- μ  m mem- brane filter, and subsequently loaded into a 50- μ  l loop andinjected into the column. After sample introduction, the pro-tein was eluted with the aforementioned buffer. Detectionwavelength was set at 280 nm.  Ion exchange chromatography A MonoS 5/50 GL column (5×50-mm ID, 10- μ  m particlediameter; Tricorn, GE Healthcare, Life Science) was usedfor CIEX separation. Various conditions of the separation(different eluent compositions, pH, gradients, and flowrates) were applied to achieve the separation of three formsof lactoferrin: apo-, holo-, and monoferric. Finally, to obtaina homogenous Lf preparation, solutions of 50 mM potassi-um phosphate (pH 7.5), A, and 50 mM potassium phos- phate, 1.5 M potassium chloride (pH 7.5), B, were used aseluents. The column was equilibrated at 1 mL/min with buffer A. Separation was monitored spectrophotometricallyat 280 nm as well as by conductivity measurement, whilethe temperature of the column was kept at 21 °C. Lactoferrin Quantification of iron saturation in lactoferrin preparations  solution (10 mg/mL; DMV International) was diluted in buffer A, filtered through a 0.22- μ  m membrane filter, andsubsequently loaded into a 1-mL loop and injected into thecolumn. After sample application, the column was washedwith 5 column volumes at a flow rate of 2 mL/min with A.Lactoferrin adsorbed on the column was eluted with a stepgradient   —  from 0 to 25 % B and from 25 to 75 % B at a flowrate of 1 mL/min  —  after which the column was washed with5 column volumes with B. Fractions of 1 mL were collectedduring the chromatographic run. As a result of several purifications, no separation of peaks corresponding to thedifferent iron-saturated lactoferrins was achieved. In order to discriminate the effect of glycosylation from those of ironsaturation on protein separation, the fraction that was pooledfrom the central part of the major peak identified as a non-glycosylated form of Lf (Fig.2) was lyophilized and mod-ified to obtain both apo- and hololactoferrins, as describedin “ Preparation of bovine apo- and hololactoferrins. ” The prepared non-glycosylated apo- and hololactoferrin sampleswere loaded separately into a 50- μ  l loop, injected into theMonoS column, and chromatographic separation was performed by utilizing the procedure described above.A MonoQ 5/50 GL separation column was also used for AEX separation. Our attempts  —  involving usage of variousconditions for the ion exchange chromatographic method  —  toisolate peaks corresponding to different iron-saturatedlactoferrins were unsuccessful (results not shown).Spectroscopic measurementsUV/VIS spectra were recorded using a Perkin Elmer Lambda35 spectrophotometer, with photometric accuracy ±0.010 A(measured for potassium dichromate) and photometric repro-ducibility <0.001 A. Samples were appropriately diluted whennecessary to obtain absorbance below 1. The obtained  A 280 /   A 466 ratios were subsequently used to determine the ironsaturation of lactoferrin preparations applying the calibrationcurve presented in “ Results and discussion. ” Measurements of circulardichroism(CD)spectrawereconductedwithaJASCOJ-710 spectropolarimeter (JASCO, Tokyo, Japan). The rangeof measurement (250  –  187 nm) was selected after sample test scan. Measurements were carried out for lactoferrin purchasedfrom DMV International and Sigma-Aldrich as well as theapo-andhololactoferrinsobtainedbypurification(usingcationexchange chromatography) and iron saturation/desaturation of commerciallyavailableproducts.Allsampleswerepreparedin50 mM Tris  –  HCl buffer (pH 7.4).Enzyme-linked immunosorbent assayELISAwas conducted using bovine lactoferrin (bLf) ELISAkit (Alpha Diagnostic International, San Antonio, TX,USA). A standard curve was prepared with the supplied bLf standard with concentration ranging from 10 to140 ng/mL. The tested samples were diluted 500,000 and1,000,000 times in a sample diluent buffer and the test  performed according to the manufacturer  ’ s protocol. ELISA plates were read using Tecan Infinite 200 Reader plate at 450 nm to determine Lf concentration. Measurement at 630 nm was performed for background subtraction. Testswere performed for the samples with the lowest  A 280 /   A 466 ratio as well as partially saturated lactoferrin samples, withthe results used for calibration curve preparation.Inductively coupled plasma  –  mass spectrometryThe total iron content in lactoferrin was measured by theapplication of ICP-MS. For ICP-MS measurements,lactoferrin samples with the lowest values of  A 280 /   A 466 and partially saturated lactoferrin preparations were chosen. Prior to the determination of iron content, 100 μ  L of the sampleswasdiluted with 6mLofconcentratedHNO 3 and mineralizedusing Microwave Digestion System: Multiwave 3000 (AntonPaar) by employing 1,000-W power, 15-min ramp, followed by a 20-min hold with a rate of 0.4 bar s − 1 up to 50 bar, at 213  –  230 °C. Iron concentration was determined in thesesampleswiththeICP-MStechniqueperformedwithanELANDRCe Perkin Elmer spectrometer, employing the followingconditions: plasma gas (Ar), 15 L min − 1 ; Rf power, 1,270 W;nebulizer gas flow, 0.99 L min − 1 ; auxiliary gas flow,1.20Lmin − 1 .The 57 Fe isotope wasusedforthequantificationof iron content in samples. Results and discussion Purity check In order to verify the information provided by the manufac-turers concerning the purity of lactoferrin preparations, twocomplementary techniques were exploited. First, denaturingelectrophoresis in polyacrylamide gel was performed, yield-ing one distinct band of molecular weight approx. 80 kDa(Fig.1a) for the DMV International and Sigma lactoferrinsamples. Few lower-molecular-weight bands were alsodetected for both commercially available lactoferrin prepa-rations. The presence of these bands was previouslyreported [38] and identified as degraded forms of lactoferrin.The purity of lactoferrin supplied by DMV International wasdensitometrically estimated as over 90 % and was higher than the purity of Lf preparation purchased from Sigma-Aldrich. The second approach involved the chromatograph-ic separation of lactoferrin preparations on the gel filtrationcolumn. In order to overcome the problem of the unspecific binding of protein to the dextran resin observed when performing separation at pH ranging from 7.0 to 8.0, a G. Majka et al.
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