Eijk, H. G. van

Heul, C. van der

Kroos, M. J.

Noort, W. L. van

Clinical Chemistry and Laboratory Medicine (CCLM)

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Isolation of the Two Monoferric Human Transferrins by Preparative Isoelectric Focussing

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van Eijk, van Noort, Kroos and van der Heul: Monoferric human transferrins 563: ji J. Clin. Chem. Clin. Biochem.• \ Vol. 18, 1980, pp. 563-566Isolation of the Two Monoferric Human Transferrins by Preparative Isoelectric FocussingBy H. G. van Eijk, W. L. van Noort.M. J. Kroos and C. van der Heul\ Departments of Chemical Pathology and Hematology, Medical Faculty, Erasmus University, Rotterdam,\ The Netherlands(Received January 30, 1980)Summary: 1. Human transferrin was labelled with Fe(III) in nitrilo triacetate buffer at one of its two metal bindingsites by variation of the pH. The -site was preferentially labelled at pH 7.2, the B-site at pH 9.2.2. The monoferric transferrin preparations were subjected to preparative isoelectric focussing, isolated from thegranulated gels, and checked for homogeneity and purity on an analytical scale by isoelectric focussing.3. The technique for obtaining stable monoferric transferrin (Fe)(A) and transferrin (Fe)(B) preparations is describedin detail.Isolierung von Transferrin (Fe)(A) und Transferrin (Fe)(B) des Menschen durch präparative isoelektrische FokussierungZusammenfassung: 1. Transferrin vom Menschen wurde mit Fe(III) in Nitrilotriacetatpuffer durch Variation des pH aneiner seiner zwei Metallbindungsstellen markiert. Bindungsstelle A wird Vorzugsweise bei pH 7,2, Bindungsstelle B beipH 9,2 markiert.2. Die jeweils ein Eisen enthaltenden Transferrin-Präparationen wurden der präparativen isoelektrischen Fokussierungunterworfen, aus dem granulierten Gel isoliert und durch analytische isoelektrische Fokussierung auf ihre Homogenitätund Reinheit geprüft.3. Die Technik für die Darstellung stabiler, ein Eisen enthaltender Transferrine wird im Detail beschrieben.IntroductionTransferrin is a ß-glpbulin responsible for carryingiron in the plasma. This protein has a of 81000,consists of a single polypeptide chain and has two metalbinding sites. There is still doubt whether the twobinding sites, called the B-site and -site, located inthe terminal domain, C terminal domain (1), respect-ively fulfill different physiological roles (2,3). In aprevious paper we showed that these two binding siteshave different affinities for Fe(HI) at various pH values,and that the isoelectric points of the two monoferrictransferrins differ by 0,1 pH unit (4). This was deter-mined by the elegant flat bed isoelectric focussingtechnique in polyaciylamide gel.In this paper we present a preparative method, a flat-bedisoelectrie focussing technique in granulated gels, whichenables us to separate and isolate each monoferric trans-ferrins on a 10 mg level. By incubating individual trans-ferrins with erythroid and parenchymal cells, it is possibleto investigate the question (suggested by FletcherScHuehns over a decade ago) as to whether these twobinding sites of transferrin have distinctive roles in irontransport and metabolism (5).Materials and MethodsHuman transferrin was obtained from Kabi (Sweden) and usedwithout further purification.Apotransferrin was prepared as described earlier (6) by dialysisagainst EDTA. A nitrilo triacetate (NTA) pH-buffer was madeby titration of Na3NTA with NTA (Sigma) until the desired pHwas obtained.Chemicals were of analytical reagent grade.AmpholinespH 5-7 and granulated gel (Ultrodex) were obtainedfrom LKB (Sweden). 5^Fe(III) citrate was obtained from Radio-chemical Centre, Amersham, Bucks, United Kingdom and wasdiluted about five fold with freshly prepared 56Fe(III) citrate(0,6 g/1). To this solution NaHCO3 was added in a molar ratioFe/HC03= 1/50.0340-076X/80/0018-0563$02.00© by Walter de Gruyter & Co. · Berlin · New York564 van Eijk, van Noort, Kroos and van der Heul: Monoferric human transferrinsPreparation of monoferric transferrins A and BTo a solution of human apotransferrin (ca. 60 g/1) 10 vol%1 mol/1 NTA buffer was added of either pH = 7.2 (transferrin(Fe)(A)) or 9.2 (transferrin(Fe)(B)).Then the apotransferrin solutions were brought to a 0.60 Fesaturation with the mixed 56Fe/59Fe citrate solution (about1 mmol/1 Fe).The Fe-transferrin solutions were kept overnight at room temper-ature, then dialysed for 24 hours at 4 °C against 1 litre 0.1 mol/1Tris pH = 7 (A), or pH = 9 (B). After dialysis the Fe-transferrinsolutions were used for preparative isoelectric focussing whichwas carried out according to the Application Note 198 (LKB)with the LKB 2117 Multiphor, but with a few minor adapta-tions.Instead of Sephadex G75, a new granulated gel, Ultrodex, wasused. Four (4) grams of the freeze dried powder was suspendedin 100 ml of a 20 g/l ampholine pH 5-7 solution. The focussingwas carried out over the broad-short region instead of the narrowlong region. Between the gel bearing glass plate and the coolingglass plate two sheets of wetted filter paper were placed to makeit easier to distinguish the transferrin-Fe bands after focussing.Before applying the sample, the gel was prerun for 60-90 minutes.Instead of the low power and time-consuming adjustment givenby LKB, we started the prerun with 500 V and 50 mA, and thesevalues automatically changed during focussing to 900 V and35 mA. Maxima of 1200 V, 30 W and 50 mA were adjusted. Afast, high power focussing is necessary because the relative lowstability of transferrin(Fe)(B). Instead of mixing the samplewith the gel, it was absorbed into a dry electrode strip (ca.70 μΐ/cm strip), then the strip was carefully pressed into thegel parallel to the electrode strips at a distance of 1 cm fromthe cathode.The focussing was performed in 100-140 minutes until sharpbands had been formed (visible from their pink color).Next with a glass spatula the protein containing gel (zones) werescooped into special elution columns (LKB, 2117-502). Trans-ferrin(Fe)(A) and transferrin(Fe)(B) were eluted with 0.5 mol/1Tris pH 7 or 8, respectively. The eluates were dialyzed over-night at 4 °C against 11 each of a 0.02 mol/1 Tris buffer pH = 7or 8, respectively. Elution and dialysis buffer were made Fe-freeby passing the solution through a small column of Chelex 100(Biorad).Finally the fractions were tested for homogeneity on a poly-acrylamide gel on an analytical scale (4) and used for incubationstudies with rat reticulocy tes and bone marrow cells, as well aswith human bone marrow cells. These experiments are describedelsewhere (5).Θ .TransferrinTransferrin (Fe) (A)Transferrin (Fe)(B)Transferrin (2Fe)ΦFig. 1. Preparative isoelectric focussing.Transferrin(Fej(A) (Fe N-terminal left) and transferrin-(Fe)(B) (Fe C-terminal right) as well as transferrin(2Fe)are visible.of transferrin(Fe)(B) and some transferrin(2Fe) wasobtained in the focussing (left); if the sample was notdialysed (i.e. the chelatprs were not removed) most ofthe transferrin(Fe)(B) was converted to transferrin-(Fe)(A) during the focussing (fig. 3 right). Surprisingly,this conversion was not observed on the analytical gel(fig. 1 right). The transferrin(Fe)(A) and transferrin-(Fe)(B) preparations obtained from the preparative gelswere focussed again on an analytical scale. This resultis shown in figure 4.ResultsIn figure 1 a typical diagram of the separation of speci-fically labelled monoferric transferrin(Fe)(A) and trans-ferrin(Fe)(B) is shown. On the left is a labelling at pH 7.2,mainly Α-site, and in the middle a labelling at pH 9.2,mainly the B-site. The labelling occurred in a NTA buffer,the separation of 70 Mg samples on polyacrylamide gelswith isoelectric focussing as has been described earlier(4). In figures 2 and 3 are shown the separation of about7 mg samples in granulated gels (Ultrodex). The gels arestained for iron, so apotransferrin is not visible. On theleft in figure 2 is shown transferrin(Fe)(A), and trans-ferrin(Fe)(B) is on the right. In figure 3 is shown theresult of dialysing the sample after labelling the trans-ferrin with Fe(III). If the sample of transferrin(Fe)(B)was dialysed in order to remove chelators a sharp bandDiscussionWith the method, described here in detail, it appearspossible to separate and isolate monoferric humantransferrins A and B. The yield is at least 20% underfavourable conditions.We wish to draw attention on the conditions necessaryfor optimal preparative focussing.1. To obtain pure transferrin(Fe)(B) it is absolutelynecessary to remove NTA and citrate by dialysis orgel filtration before transferrin(Fe)(B) solutions aresubjected to preparative focussing. If this step is° omitted a considerable amount of transferrin(Fe)(B)is converted into transferrin(Fe)(A) during focussingand transferrin(Fe)(B) preparations are than con-taminated with transferrin(Fe)(A).J. Clin. Chem. Clin. Biochem. / Vol. 18,1980 / No. 9van Eijk, van Noort, Kroos and van der Heul: Monofeiric human transferrins 565ΘTransferrinTransferrin (Fe)(A)Transferrin (Fe)(B)Fig. 2. The same solutions as in fig. 1, but on analytical gels.Transferrin (2Fe)ΘTransferrinTransferrin (Fe)(A)Transferrin (Fe)(B)Transferrin (2Fe)θFig. 3. Isoelectric focussing of (a) solutions which contain chelators, not removed before the focussing technique (left) and (b) solu-tions from which chelators were removed before the focussing (right), in this case only transferrin(Fe)(B) is seen and nottransferrin(Fe)(A).Transferrin^^^WSFTransferrin (Fe)(A)Transferrin (Fe)(B)Transferrin 12Fe)θMoreover the yield is diminished. Removing chelatorsfrom transferrin(Fe)(A) solutions before focussingresulted in sharper migration.2. The optimal and most reproducible ratio of trans-ferrin(Fe)(A) to transferrin(Fe)(B) was found whenthe labelling of apotransferrin with Fe was carried outin a NTA buffer, with an excess of HCO3~, so thatHC03" and not NTA is bound as an anion (7).3. A solution of high concentration, at least 30 g/1, isnecessary in order to work with sample volumes of1.5—2 ml and to obtain a reasonable amount of pro-tein.4. To prove that the ampholine had no harmful effecton the nature of the protein we carried out severalincubation experiments with reticulocytes and trans-ferrin-solutions, treated or non-treated with ampho-line. No differences were observed in iron uptake (5).Fig. 4. Isoelectric focussing on an analytical scale of samplesprepared by the preparative technique.The pure transferrin(Fe)(A)(left) and t_ransferrin(Fe)(B)-(right) obtained by preparative isoelectric focussing,dialysed as described and ready for incubation experi-ment.J. Clin. Chem. din. Biochem. / Vol. 18,1980 / No. 9566 van Eijk, van Noort, Kroos and van der Heul: Monofemc human transferrinsReferences1. Evans, R. W. & Williams, J. (1978), Biochem. J. 173, 5. Van der Heul, C., Krops, M. J., Van Noort, W. L. & Van Eijk,543-552. H. G., Clin. Science, accepted for publication.2. Fletcher, J. & Huehns, E. R. (1968), Nature 218,1211-1214. 6. Verhoef, N. J. & Van EJjki H. G. (1975), Clin. Sei. Mol. Med.3. Fletcher, J. & Huehns, E. R. (1967), Nature 275,584-586. 48, 335-340.4. Van Eijk, H. G., Van Noort, W. L., Kroos, M. J. & Van der 7. Schlabach, M. R. & Bates, G. W. (1975), J. Biol. Chem. 250,Heul, C. (1978), J. Clin. Chem. Clin. Biochem. 16, 557-560. 2182-2188.Prof. Dr. H. G; vail EijkDept. of Chemical PathologyErasmus University RotterdamPostbus 1738RotterdamJ. Clin. Chem. Clin. Biochem. / Vol. 18,1980 / No. 9

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Isolation of the Two Monoferric Human Transferrins by Preparative Isoelectric Focussing

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