Electrophoresis zyxwvutsrqponml 1989, 10,23-29 High performance capillary isoelectric focusing of transferrin 23 z Ferenc Kilar Stellan HjertCn Institute of Biochemistry University, Uppsala 1 Introduction Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries zyxw , Uppsala Human serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron-free and iron-complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many compo- nents in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron-free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15-25 min) of small amounts of samples (less than 1 kg protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron-free, monoferric and differic com- plexes) can easily be identified. Both steps of isoelectric focusing in capillaries zy (i. e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e. g., carrier ampholytes, nitrilotriacetate) may release iron from micro- syringes having metal pistons causing the formation of iron-transferrin complexes. Serum transferrin is an iron-transport protein with different isoforms* containing two binding sites for iron(II1). The iso- forms can be separated by isoelectric focusing [ 1-31. Eachiso- form may exist in iron-free and iron-complexed molecular form. The four molecular forms of transferrin, i. e. iron-free or apotransferrin (Tf), two types of monoferric-transferrins (TfFec and FeNTf) having iron bound at the C or N-ter- minal binding site, respectively, and the diferric-transferrin (FeNTfFec) can also be separated by isoelectric focusing. The p l value of the iron-free transferrin decreases on binding iron(II1) [4,51. The four molecular forms ofthe major isoform containing 4 sialic acids in normals [3] are characterized by the plvalues 6.1,5.8-5.9,5.8,5.5-5.6, respectively [ 1,51. The iron-transferrin complexes are formed only with the participa- tion of an anion (e. g., bicarbonate or carbonate in vivo 161, or other anions with synergistic binding [71). The interest in analyzing the transferrin content of biological fluids has grown considerably in the last years, which is revealed by the increasing number of publications in this area. Such analyses are of importance for anthropological and genetic studies, for following the characteristic changes in the carbohydrate moiety of transferrin in diseases, and for investigation of the iron binding capacity in blood [2,3,8- 151. The techniques to detect the above transferrin forms are based on conventional analytical isoelectric focusing techniques [ 1-4, 9-121 com- bined with radioactive detection [ 16, 171 or with immunolog- ical assays IS, 18-20]. The new capillary technique for iso- electric focusing (21-251 in the high performance electro- phoresis apparatus [261 provides a rapid and easy separation of small amounts of proteins. The duration of an experiment can be as short as 10-30 min, including the detection of the focused protein zones. This focusing technique was employed in our study for analysis of the different forms in transferrin Correspondence: Dr. Ferenc Kilar, CentralResearch Laboratory, Medical University of Pecs, Szigeti lit 12, H-7643 Ptcs, Hungary Abbreviations: AU, absorption unit; NTA, nitrilotriacetic acid; p2, isoelectric point; Tf, transferrin; TWw, FeNTf, monoferric transferrim; FeNTfFec, diferric-transferrin preparations. In addition, some methodological aspects of isoelectric focusing in capillaries will also be discussed. 2 Materials and methods 2.1 Transferrin preparations Iron-free transferrin was obtained from Behring Werke AG (Marburg, FRG) and was used without further purification. Solutions containing zyxwv 50 mg transferrin/mL were prepared in 20 mM zyxwv N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer containing 20 mM NaHCO,. The pH was adjusted with NaOH to pH 7.5 (samples a-e) or 9.0 (sample zyxwv f). Iron powder from Baker Chemical Co. (Philips- burg, New Jersey, USA) was dissolved in 6 M HCI to get iron(II1)-chloride. Two types of iron-chelate solutions were used for the iron-saturation of transferrin. Iron-nitrilotriace- tate (Fe-NTA) solution with a Fe:NTA ratio of 1:2 was made from the iron(II1)-chloride solution and a nitrilotriacetic acid (NTA) (Fluka, Buchs, Switzerland) solution (prepared in 1 M NaOH). Iron-citrate (Fe-citrate) solution with an Fe:citrate ratio of 1:4 was made similarly from citric acid (KEBO Lab AB, Stockholm, Sweden). The pH of both iron-chelate solu- tions was adjusted to 3-4 with NaOH if necessary. The con- centration of the iron(II1) was determined by the method of White and Flashka [27]. Iron-transferrin complexes were prepared using the Fe-NTA (samples b,c,f) and Fe-citrate (samples d,e) solutions. Iron-chelate solutions were added to the above iron-free transferrin solution (50 mg protein/mL) to get the desired percentage of iron-saturation. 100 % satura- tion degree represents a sample with diferric-transferrin mol- ecules. The solutions of the iron-free transferrin (sample a) and the completely or partially iron-saturated transferrin samples (samoles b-f) were then incubated at room tempera- ture for 2 hand dialyzed against 20 mM NaHCO,, adjusted to pH 7.5 with HCl (8 "C, overnight). An iron-complexed transferrin sample from KabiVitrum (Stockholm, Sweden) * Theterms isoform and molecular form refer to the transferrin molecules with different carbohydrate content and molecules with different iron content, respectively. zyxwv 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 0173-08351'89/0101-023 %02.50/0