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Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations?
Clin~w
Elsevier
Chmiu
Acto.
Biomedical
126 (1982)
193
193- 195
Press
CCA 2320
Brief technical
note
Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? H.G. van Eijk ‘.*, W.L. van Noort and C. van der Heul h ’ Dcpwtmermof”
Chemrcol Pathologv
md h Huemotolo~s,
Mrdrcal
P. 0. Box 1738. 3000 DR Rorterdum
(Received
Fuwlt!.
Erusmus
(ir~rcer~r~v Rotterdtrr,,,
(The Nrtherlumls)
May 28th. 19X2)
Introduction The microheterogeneity of transferrin in serum is well-known and is not only caused by the appearance of apo-, monoferricand diferric-transferrin, but also by the presence of small amounts of transferrin molecules with a different content of sialic acid, penta-, tri-, di-, mono- and asialo transferrin, besides the bulk of tetrasialo transferrin [1,2]. In a previous study we described these minor components, of which especially the disialo transferrin is present at higher concentrations in sera of alcoholics [3,4]. As the charge of the sialic acid groups is of importance in the electroimmunodiffusion (EID) method of Laurell, we compared results from the radial immunodiffuwith the EID. Such a sion technique of Mancini (RID) and the Ezxo determinations, comparison has recently been published for cY,-acid glycoproteins [5]. This protein is underestimated in the EID. The results for transferrin, a glycoprotein with 6% carbohydrate and an M, - 80000, will be presented in this paper. Materials and methods Humun
sera from healthy
donors
were used.
Transferrin fractions with different sialic acid content were isolated from serum as previously described [3]. These fractions are not artificially generated by interaction with ampholine, because they appeared as single fractions in a second and third run on isoelectric focusing. Sialic acid was determined
* To whom correspondence
OOOS-898l/82/0000-0000/$02.75
following
the method
of Warren
should be addressed
0 1982 Elsevier Biomedical
Press
[6].
194
Asialo transferrin
was prepared
as described
previously
[3].
Electroimmunodiffusion (EID) and radial immunodiffusion (RID) were carried out in a 1% agarose gel (Miles Laboratories Ltd, U.K.. lot. no. 7028) prepared in a buffer containing 37 mmol Tris, 12 mmol 5,5_diethylbarbituric acid and 0.16 mmol calcium lactate per litre, titrated to pH 8.6 with 1 mol/l HCl. The gel contained 11 ~_ll antitransferrin per ml (Dako-immunoglobulins A/S, lot. no. 01 lB, titre 750). Samples of 5 ~1 were applied with a microlitre syringe (702-N. Hamilton Company, Reno, Nevada, U.S.A.). The electrophoresis was carried out on a Multiphor 2117 (LKB, Sweden) at 12’C for 3 h at 22-25 V/cm in the same buffer. The radial immunodiffusion was performed for 48 h at 20°C in separate gels of the same composition. Then the gels were washed for 48 h in 0.15 mol/l NaCl and stained with Amido Black. Based on the E,,,- value of 14.0 for a 1% solution of all diferric-transferrins. appropriate dilutions were made for the EID and RID. Results
We investigated the estimation of transferrins by EID as a function of the sialic acid content. Based on the estimation in the RID and an E,,, determination, we subjected equal concentrations of penta-, tetra-, tri-, di- and asialo-diferric-transferrin to the EID. In Fig. 1 we see the results of 7 experiments with from left to right penta-, tetra-, tri-, di- and asialo-transferrin. The maximum difference in peak height we found was 30%. The same phenomenon was found for apo-, monoferric-A or -B
Fig. 1. EID and RID estimations of residues, 4.6; 2, tetra-sialo Tf.ZFe di-sialo Tf.ZFe sialic acid residues, Equal concentrations based on RID
sialic transferrins. From left to right: 1, penta-sialo Tf.ZFe sialic acid sialic acid residues, 4; 3, tri-sialo Tf.2Fe sialic acid residues. 3; 4, 2; 5, asialo Tf.ZFe sialic acid residues, 0. Top: EID; below: RID. and E,,,.
195
and diferric-transferrins, all of the tetrasialic acid type. The maximum between apo- and diferric-transferrin was 7%.
difference
Conclusion Preparations of diferric-transferrin with equal concentration in the RID, differ in the EID as a function of the sialic acid content. The same holds true for tetrasialo transferrins differing in Fe-content. Addition of Fe to saturate the transferrin eliminates this difference. In normal serum 80% or more is present as tetrasialo transferrin. The presence of sialo transferrins, with a different peak height in the EID, other than tetrasialo transferrin, hardly influences the result of the EID-determination, because of their low percentage contribution. References 1 Bezkorovany A. Chemistry and metabolism of the transferrins. In: Biochemistry of nonheme iron. Chapter III. New York, London: Plenum Press, 1981. 2 Wong K-L, Regoeczi E. Some observations on the carbohydrate composition of purified transferrin. Int J Peptide Protein Res 1977; 9: 241-248. 3 van Eijk HG. van Noort WL. Kroos MJ, van der Heul C. The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels: no functional difference of the fractions in vitro. Clin Chim Acta 1982; 121: 209-216. 4 Stibler H, Sydow 0, Berg S. Quantitative estimation of abnormal microheterogeneity of serum transferrin in alcoholics. Pharmacol Biochem Behaviour 1980; 13: Suppl. 1, 47-5 1. 5 Borda MC. Bion DR, Feger JM, Durand GM, Joziasse DH, van den Eijnden DH. Involvement of sialic acid residues in electro immuno diffusion of a,-acid glycoproteins. A method for determining the degree of sialylation of serum glycoproteins. Clin Chim Acta 1981: 116: 17-24. 6 Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234: 1971-1975.
Elsevier
Chmiu
Acto.
Biomedical
126 (1982)
193
193- 195
Press
CCA 2320
Brief technical
note
Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? H.G. van Eijk ‘.*, W.L. van Noort and C. van der Heul h ’ Dcpwtmermof”
Chemrcol Pathologv
md h Huemotolo~s,
Mrdrcal
P. 0. Box 1738. 3000 DR Rorterdum
(Received
Fuwlt!.
Erusmus
(ir~rcer~r~v Rotterdtrr,,,
(The Nrtherlumls)
May 28th. 19X2)
Introduction The microheterogeneity of transferrin in serum is well-known and is not only caused by the appearance of apo-, monoferricand diferric-transferrin, but also by the presence of small amounts of transferrin molecules with a different content of sialic acid, penta-, tri-, di-, mono- and asialo transferrin, besides the bulk of tetrasialo transferrin [1,2]. In a previous study we described these minor components, of which especially the disialo transferrin is present at higher concentrations in sera of alcoholics [3,4]. As the charge of the sialic acid groups is of importance in the electroimmunodiffusion (EID) method of Laurell, we compared results from the radial immunodiffuwith the EID. Such a sion technique of Mancini (RID) and the Ezxo determinations, comparison has recently been published for cY,-acid glycoproteins [5]. This protein is underestimated in the EID. The results for transferrin, a glycoprotein with 6% carbohydrate and an M, - 80000, will be presented in this paper. Materials and methods Humun
sera from healthy
donors
were used.
Transferrin fractions with different sialic acid content were isolated from serum as previously described [3]. These fractions are not artificially generated by interaction with ampholine, because they appeared as single fractions in a second and third run on isoelectric focusing. Sialic acid was determined
* To whom correspondence
OOOS-898l/82/0000-0000/$02.75
following
the method
of Warren
should be addressed
0 1982 Elsevier Biomedical
Press
[6].
194
Asialo transferrin
was prepared
as described
previously
[3].
Electroimmunodiffusion (EID) and radial immunodiffusion (RID) were carried out in a 1% agarose gel (Miles Laboratories Ltd, U.K.. lot. no. 7028) prepared in a buffer containing 37 mmol Tris, 12 mmol 5,5_diethylbarbituric acid and 0.16 mmol calcium lactate per litre, titrated to pH 8.6 with 1 mol/l HCl. The gel contained 11 ~_ll antitransferrin per ml (Dako-immunoglobulins A/S, lot. no. 01 lB, titre 750). Samples of 5 ~1 were applied with a microlitre syringe (702-N. Hamilton Company, Reno, Nevada, U.S.A.). The electrophoresis was carried out on a Multiphor 2117 (LKB, Sweden) at 12’C for 3 h at 22-25 V/cm in the same buffer. The radial immunodiffusion was performed for 48 h at 20°C in separate gels of the same composition. Then the gels were washed for 48 h in 0.15 mol/l NaCl and stained with Amido Black. Based on the E,,,- value of 14.0 for a 1% solution of all diferric-transferrins. appropriate dilutions were made for the EID and RID. Results
We investigated the estimation of transferrins by EID as a function of the sialic acid content. Based on the estimation in the RID and an E,,, determination, we subjected equal concentrations of penta-, tetra-, tri-, di- and asialo-diferric-transferrin to the EID. In Fig. 1 we see the results of 7 experiments with from left to right penta-, tetra-, tri-, di- and asialo-transferrin. The maximum difference in peak height we found was 30%. The same phenomenon was found for apo-, monoferric-A or -B
Fig. 1. EID and RID estimations of residues, 4.6; 2, tetra-sialo Tf.ZFe di-sialo Tf.ZFe sialic acid residues, Equal concentrations based on RID
sialic transferrins. From left to right: 1, penta-sialo Tf.ZFe sialic acid sialic acid residues, 4; 3, tri-sialo Tf.2Fe sialic acid residues. 3; 4, 2; 5, asialo Tf.ZFe sialic acid residues, 0. Top: EID; below: RID. and E,,,.
195
and diferric-transferrins, all of the tetrasialic acid type. The maximum between apo- and diferric-transferrin was 7%.
difference
Conclusion Preparations of diferric-transferrin with equal concentration in the RID, differ in the EID as a function of the sialic acid content. The same holds true for tetrasialo transferrins differing in Fe-content. Addition of Fe to saturate the transferrin eliminates this difference. In normal serum 80% or more is present as tetrasialo transferrin. The presence of sialo transferrins, with a different peak height in the EID, other than tetrasialo transferrin, hardly influences the result of the EID-determination, because of their low percentage contribution. References 1 Bezkorovany A. Chemistry and metabolism of the transferrins. In: Biochemistry of nonheme iron. Chapter III. New York, London: Plenum Press, 1981. 2 Wong K-L, Regoeczi E. Some observations on the carbohydrate composition of purified transferrin. Int J Peptide Protein Res 1977; 9: 241-248. 3 van Eijk HG. van Noort WL. Kroos MJ, van der Heul C. The heterogeneity of human serum transferrin and human transferrin preparations on isoelectric focusing gels: no functional difference of the fractions in vitro. Clin Chim Acta 1982; 121: 209-216. 4 Stibler H, Sydow 0, Berg S. Quantitative estimation of abnormal microheterogeneity of serum transferrin in alcoholics. Pharmacol Biochem Behaviour 1980; 13: Suppl. 1, 47-5 1. 5 Borda MC. Bion DR, Feger JM, Durand GM, Joziasse DH, van den Eijnden DH. Involvement of sialic acid residues in electro immuno diffusion of a,-acid glycoproteins. A method for determining the degree of sialylation of serum glycoproteins. Clin Chim Acta 1981: 116: 17-24. 6 Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234: 1971-1975.