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A simple method for the separation and quantitation of neutral carbohydrates of glycoproteins in the one nanomole range on an adapted amino acid analyzer
187
eirnica Chimica Acra, 139 (1984) 187-193 Elsevier
CGA
02854 Brief technical
note
A simple method for the separation and quantitation of neutral carbohydrates of glycoproteins in the one nanomole range on an adapted amino acid analyzer H.G. van Eijk ‘,*, W.L. van Noort a, C. Dekker a and C. van der Heul b Deparrments of ’ Chemical Pathology and h Hematology, Medicul Faculty, Erasmus Uniuerscty, Rotterdam (The Netherlands) (Received
December
19th, 1983)
Kq words: Neutral carboh.~drafes; ~iyco~rote~ns; Adapted amino acid analyzer
Introduction
Our interest in the function of transferrin [l-3], a glycoprotein with a molecular mass of 79550 and a carbohydrate content of about 6%, a molecule with two domains, each with a binding site for Fe(III), prompted us to study the carbohydrate moiety of this protein. In the C-terminal domain of the molecule two bi-antennary, one bi-antennary and one tri-antennary or two tri-antennary carbohydrate chains are bound [4,5]. The ~croheterogeneity in transferrin preparations isolated from normal sera, but especially in preparations isolated from pathological sera, caused by differences in the carbohydrate chain, forced us to look for a simple method to quantitate sugars in this glycoprotein. A variety of methods has been described, including some which use an animo acid analyzer [6-111. However, all the methods described have disadvantages, such as a complicated manipulation, a low sensitivity, the use of a corrosive color reagent, the need for a fluorimetric system and the need for high pressure resistant columns and pumps. In 1972, Mopper and Degens [12] described the use of ethanol as an eluant for the separation of neutral mono- and di-saccharides on a cation exchanger by partition chromatography in combination with p-anisyl tetrazolium blue as color reagent, to obtain a high sensitivity. Hobbs and Lawrence [13] improved the separation by application of trimethylammonium as counter ion. In the Beckman Information Report l/74 [14] technical details are given to change an amino acid analyzer simply and reversibly into a carbohydrate analyzer. However, these instructions are not complete; we had to modify them in order to quantitate with a better sensitivity 6-8 neutral sugars within 2 h [12,14]. * To whom
corres~ndence
mO9-8981/84/%03.@3
should
be addressed.
@ 1984 Elsevier Science Publishers
B.V.
188
This paper gives a description of this modified procedure, with as an example the analysis of the carbohydrate of three different transferrin preparations, isolated by preparative isoelectric focusing [ 151. Materials and methods The instrument we adapted is a Multichrom M (Beckman, Munich, FRG). The adaptations necessary are the following: - a reaction coil of 5 meters - instead of 30 meters - with a diameter of 0.75 mm; - a 30-meter teflon coil at the photometer outlet as back pressure coil to prevent air bubbles. However, the eluant should be degassed before use; - the reaction bath temperature is lowered to 80 ‘C by a variable resistance. The plexiglass mixing manifold and in- and outlet of the cuvette holder are replaced by components made of Delrin; - the resin we used is Aminex A-6 (Bio-Rad, Richmond, CA, USA), which is converted to the trimethylammonium form by treating the resin with a 2 molar solution, pH = 9 [13]; - the column dimensions are 60 cm long and 0.4 cm diameter; - the eluant composition is ethanol/water in a ratio 89/11 (v/v), the eluant flow rate is 25 ml/h at 75 ‘C, or 16 ml/h at 60 o C; - color reagent composition: 0.02% (w/v) p-anisyl tetrazoliumchloride (Fluka, Buchs, Switzerland; No. 88190) in 0.18 mol/l NaOH with weekly fresh reagent. The reagent flow rate is 4 ml/h; - the column eluate is measured at 570 nm, in a lo-mm cuvette at a recorder range of 0.1 A, the recorder chart speed is 75 mm/h and the peak area is determined by an Infotronics CRS-210 Integrator (Shannon, Ireland); - the sample volume applied is 0.250 ml. Hydrolysis procedure 50-300 pg protein, containing 2.5-25 nmol of every carbohydrate in 550 ~1 aqua dest. is put into a hydrolysis tube. The exact concentration of the protein is determined by a E,,,-estimation, E$i’ cm apo Tf = 10.9, diferric Tf = 14.0. Then 100 ~1 100% tri~uoracetic acid (Merck, Darmstadt, FRG) is added, the tube is evacuated at - 30°C and sealed. After a hydrolysis of 2 h at 100 ‘C the tube is opened and the contents are quantitatively transferred into a tube of a vacuum rotary evaporator, and evaporated to dryness at 30-50 OC, dissolved in 100 ~1 aqua dest. and then 1.00 ml absolute ethanol is added. All standards used are of analytical reagent grade. The three different human transferrin preparations with different sialic acid content, 4.3, 3.9 and 2.8 mol sialic acid per mol protein, and tentatively called penta-, tetra- and disialo transferrin [l] were hydrolyzed and used especially for the galactose estimation. For clarity the structure of the bi-antennary saccharide chain is given: NANA-Gal-GlcNac-Man
I
NANA-Gal-GlcNac-Man
7
Man-GlcNac-GlcNac-Asn
189
Calculations and accuracy A calibration mixture (Fig. 1) was analyzed standard
deviation
$
on 4 successive
. 100% for six sugars studied
varied between
days. The relativ 0.9 and 1.5%.
Fig. 1. Separation of six sugars, present in glycoproteins, following the slow program (18 ml/h at 60 o C). Retention time galactose: 108 min. 6, galactose (2.97 nmol); 5, glucose (1.54 nmol); 4, mannose (1.55 nmol); 3, arabinose (5.37 nmol); 2, xylose (1.75 nmol); 1, fucose (1.45 nmol).
190
The coefficient of determination is calculated from the formula:
(r’),
if different
amounts
of sugars are applied,
-aI* r2= [qx-T)‘][8(y-jq2] [Q
- X)(Y
with x = nmol sugar y = integration units. The coefficient of determination (r*) for mannose is 0.998, for galactose 0.999, for fucose 0.998. Regeneration of the resin needed after 50 runs is simple, following the BioRad instructions. Results In Fig. 1 is given the separation of the six principal sugars present in glycoproteins, following the slow program (18 ml/h at 6O’C). Good separation is obtained. In Fig. 2 the separation diagrams of mannose and galactose under different conditions are shown. Diagram A is obtained with 0.2% tetrazolium blue and an eluant/reagent ratio of 2.4 as described by Mopper and Degens [12]; diagram B under our modified conditions 0.02% tetrazolium blue and an eluant/reagent ratio
A
i
1
i/
Fig. 2. Separation of mannose/galactose following the fast program (25 ml/h, 75 o C). Retention time 48 and 54 min. respectively. (A) 0.2$ tetrazolium and an eluant/reagent ratio 2.4 with decreasing base line. (B) 0.02% tetrazolium and an eluant/reagent ratio 6.3 with a flat base line: 1, mannose, 2, galactose.
191 C
A
Fig. 3. Analysis of equal amounts of transfertin fractions. In the order A, B and C they contain increasing amounts of sialic acid, 2.8, 3.9 and 4.3 sialic acid residues per mol protein, but also increasing amounts of galactose, 3.0, 3.9 and 4.3 residues per mol protein. This indicates heterogeneity of the fractions. 1, mannose; 2, glucose (as impurity); 3, galactose.
of 6.3 with 1.6 nmol of each sugar recorded at 0.1 A. Diagram B shows a flat base line. In Fig. 3 the sugar diagrams of three different transferrin preparations [1,15] are given. Equal amounts of protein are applied. In the order A, B and C increasing amounts of sialic acid were found, causing a difference in the isoelectric point of the proteins. However, a heterogeneity with respect to their antennary structures would also differ in galactose content. From the Figs. it is clear that the fractions A, B and C differ in galactose content. Discussion The application of p-anisyl tetrazolium blue as a color reagent for the quantitation of reducing sugars, as proposed by Mopper and Degens, is not commonly in use, in spite of several attractive properties. The reagent is not corrosive, permits a high sensitivity, linearity, reproducibility and is not expensive. The stability is limited, but with weekly fresh solutions no problems are encountered. Although Mopper and Degens [12] claimed a sensitivity of lOPi mol, only 10e8 mol could be quantitatively analyzed. To obtain reliable sensitivity in the 1 nmol range with a flat base line we had to decrease the reagent concentration 10 times and to increase the eluant/reagent ratio 2-3 times. Using tetrazolium blue as color reagent one can only apply ethanol as eluant since ethanol prevents the precipitation of the reaction product diformazon [12].
192
Although 2 h hydrolysis is long enough for a 100% recovery of galactose, only 75% of mannose is recovered. A prolonged hydrolysis time (8 h) gave 85% recovery for mannose, but 16 h hydrolysis resulted in 75% recovery. Apparently mannose is so difficult to liberate that it is partly destroyed before the hydrolysis is complete. Differences in the mannose amounts in Fig. 3 are partly caused by differences in recovery. The existence of transferrin forms with different amounts of mannose is not known. The glucose found must be ascribed to contamination of the proteins caused by the isolation procedure. This is very often seen in analysis of glycoproteins. In conclusion With the technique described it is possible hydrolysates of glycoproteins within 2 h. It may composition of the carbohydrate moiety in order chain is degraded. This is important with respect e.g. galactose residues, with cell membranes and
to quantitate neutral sugars in be of great value to determine the to know how far the carbohydrate to the interaction of carbohydrates, lectins.
References 1 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. 2 Van Eijk HG, Van Noort WL, Van der Heul C. Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? Clin Chim Acta 1982; 126: 1933195. 3 Van Eijk HG, Van Noort WL, Dubelaar ML, Van der Heul C. The microheterogeneity of human transferrins in biological fluids. Clin Chim Acta 1983; 132: 167-171. 4 Spik G, Bayard B, Foumet B, Strecker G, Bouquelet S, Montreuil J. Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human sero transferrin. FEBS Lett 1975; 50: 196-199. 5 Hatton MWC, M;irz L, Berry LR, Debanne MT, Regoeczi E. Bi- and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins. Biochem J 1979; 181: 633-638. MM, Khorlin AY, Voelter W, Bauer H. High performance liquid chromatographic 6 Tikhomirov investigation of the amino acid, amino sugar and neutral sugar content in glycoproteins. J Chromatogr 1978; 167: 197-203. 7 Hara S, Ikegami H, Shono A, Mega T, Ikenaka T, Matsushima Y. Analysis of neutral sugars as glycamines using an amino acid analyzer. Anal Biochem 1979; 97: 166-172. of neutral and amino sugars found in glycoproteins. Anal 8 Barr J, Nordin Ph. Microdetermination Biochem 1980; 108: 313-319. 9 Kennedy JF, Fox JE. [l] Fully automatic ion-exchange chromatographic analysis of neutral monosaccharides and oligosaccharides. Methods of carbohydrate chemistry. Vol. VIII. 1980: 3-12. Eds. Whistler RL, Beuiller JN, IL. 10 Honda S, Takahashi M, Nishimura Y, Kakehi K, Ganno S. Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide. Anal Biochem 1981; 118: 162-167. analysis of amino sugars and derivatized neutral sugars. Anal 11 Perini F, Peters BP. Fluorimetric Biochem 1982; 123: 357-363. 12 Mopper K, Degens ET. A new chromatographic sugar autoanalyzer with a sensitivity of lo-i0 moles. Anal Biochem 1972; 45: 147-153.
193
13 Hobbs JS, Lawrence JG. The separation of carbohydrates on cation-exchange resin columns having organic counterions. J Chromatogr 1972; 72: 311-318. 14 Von Wilm M, SordC G. A method for the analysis of neutral mono- and di-saccharides by column chromatography with the multichrom B. Beckman Information l/1974, Geneva, Switzerland. 15 Van Eijk HG, Van Noort WL, Kroos MJ, Van der Heul C. Isolation of the two monoferric human transfertins by preparative isoelectric focusing. J Clin Chem Clin Biochem 1980; 18: 563-566.
eirnica Chimica Acra, 139 (1984) 187-193 Elsevier
CGA
02854 Brief technical
note
A simple method for the separation and quantitation of neutral carbohydrates of glycoproteins in the one nanomole range on an adapted amino acid analyzer H.G. van Eijk ‘,*, W.L. van Noort a, C. Dekker a and C. van der Heul b Deparrments of ’ Chemical Pathology and h Hematology, Medicul Faculty, Erasmus Uniuerscty, Rotterdam (The Netherlands) (Received
December
19th, 1983)
Kq words: Neutral carboh.~drafes; ~iyco~rote~ns; Adapted amino acid analyzer
Introduction
Our interest in the function of transferrin [l-3], a glycoprotein with a molecular mass of 79550 and a carbohydrate content of about 6%, a molecule with two domains, each with a binding site for Fe(III), prompted us to study the carbohydrate moiety of this protein. In the C-terminal domain of the molecule two bi-antennary, one bi-antennary and one tri-antennary or two tri-antennary carbohydrate chains are bound [4,5]. The ~croheterogeneity in transferrin preparations isolated from normal sera, but especially in preparations isolated from pathological sera, caused by differences in the carbohydrate chain, forced us to look for a simple method to quantitate sugars in this glycoprotein. A variety of methods has been described, including some which use an animo acid analyzer [6-111. However, all the methods described have disadvantages, such as a complicated manipulation, a low sensitivity, the use of a corrosive color reagent, the need for a fluorimetric system and the need for high pressure resistant columns and pumps. In 1972, Mopper and Degens [12] described the use of ethanol as an eluant for the separation of neutral mono- and di-saccharides on a cation exchanger by partition chromatography in combination with p-anisyl tetrazolium blue as color reagent, to obtain a high sensitivity. Hobbs and Lawrence [13] improved the separation by application of trimethylammonium as counter ion. In the Beckman Information Report l/74 [14] technical details are given to change an amino acid analyzer simply and reversibly into a carbohydrate analyzer. However, these instructions are not complete; we had to modify them in order to quantitate with a better sensitivity 6-8 neutral sugars within 2 h [12,14]. * To whom
corres~ndence
mO9-8981/84/%03.@3
should
be addressed.
@ 1984 Elsevier Science Publishers
B.V.
188
This paper gives a description of this modified procedure, with as an example the analysis of the carbohydrate of three different transferrin preparations, isolated by preparative isoelectric focusing [ 151. Materials and methods The instrument we adapted is a Multichrom M (Beckman, Munich, FRG). The adaptations necessary are the following: - a reaction coil of 5 meters - instead of 30 meters - with a diameter of 0.75 mm; - a 30-meter teflon coil at the photometer outlet as back pressure coil to prevent air bubbles. However, the eluant should be degassed before use; - the reaction bath temperature is lowered to 80 ‘C by a variable resistance. The plexiglass mixing manifold and in- and outlet of the cuvette holder are replaced by components made of Delrin; - the resin we used is Aminex A-6 (Bio-Rad, Richmond, CA, USA), which is converted to the trimethylammonium form by treating the resin with a 2 molar solution, pH = 9 [13]; - the column dimensions are 60 cm long and 0.4 cm diameter; - the eluant composition is ethanol/water in a ratio 89/11 (v/v), the eluant flow rate is 25 ml/h at 75 ‘C, or 16 ml/h at 60 o C; - color reagent composition: 0.02% (w/v) p-anisyl tetrazoliumchloride (Fluka, Buchs, Switzerland; No. 88190) in 0.18 mol/l NaOH with weekly fresh reagent. The reagent flow rate is 4 ml/h; - the column eluate is measured at 570 nm, in a lo-mm cuvette at a recorder range of 0.1 A, the recorder chart speed is 75 mm/h and the peak area is determined by an Infotronics CRS-210 Integrator (Shannon, Ireland); - the sample volume applied is 0.250 ml. Hydrolysis procedure 50-300 pg protein, containing 2.5-25 nmol of every carbohydrate in 550 ~1 aqua dest. is put into a hydrolysis tube. The exact concentration of the protein is determined by a E,,,-estimation, E$i’ cm apo Tf = 10.9, diferric Tf = 14.0. Then 100 ~1 100% tri~uoracetic acid (Merck, Darmstadt, FRG) is added, the tube is evacuated at - 30°C and sealed. After a hydrolysis of 2 h at 100 ‘C the tube is opened and the contents are quantitatively transferred into a tube of a vacuum rotary evaporator, and evaporated to dryness at 30-50 OC, dissolved in 100 ~1 aqua dest. and then 1.00 ml absolute ethanol is added. All standards used are of analytical reagent grade. The three different human transferrin preparations with different sialic acid content, 4.3, 3.9 and 2.8 mol sialic acid per mol protein, and tentatively called penta-, tetra- and disialo transferrin [l] were hydrolyzed and used especially for the galactose estimation. For clarity the structure of the bi-antennary saccharide chain is given: NANA-Gal-GlcNac-Man
I
NANA-Gal-GlcNac-Man
7
Man-GlcNac-GlcNac-Asn
189
Calculations and accuracy A calibration mixture (Fig. 1) was analyzed standard
deviation
$
on 4 successive
. 100% for six sugars studied
varied between
days. The relativ 0.9 and 1.5%.
Fig. 1. Separation of six sugars, present in glycoproteins, following the slow program (18 ml/h at 60 o C). Retention time galactose: 108 min. 6, galactose (2.97 nmol); 5, glucose (1.54 nmol); 4, mannose (1.55 nmol); 3, arabinose (5.37 nmol); 2, xylose (1.75 nmol); 1, fucose (1.45 nmol).
190
The coefficient of determination is calculated from the formula:
(r’),
if different
amounts
of sugars are applied,
-aI* r2= [qx-T)‘][8(y-jq2] [Q
- X)(Y
with x = nmol sugar y = integration units. The coefficient of determination (r*) for mannose is 0.998, for galactose 0.999, for fucose 0.998. Regeneration of the resin needed after 50 runs is simple, following the BioRad instructions. Results In Fig. 1 is given the separation of the six principal sugars present in glycoproteins, following the slow program (18 ml/h at 6O’C). Good separation is obtained. In Fig. 2 the separation diagrams of mannose and galactose under different conditions are shown. Diagram A is obtained with 0.2% tetrazolium blue and an eluant/reagent ratio of 2.4 as described by Mopper and Degens [12]; diagram B under our modified conditions 0.02% tetrazolium blue and an eluant/reagent ratio
A
i
1
i/
Fig. 2. Separation of mannose/galactose following the fast program (25 ml/h, 75 o C). Retention time 48 and 54 min. respectively. (A) 0.2$ tetrazolium and an eluant/reagent ratio 2.4 with decreasing base line. (B) 0.02% tetrazolium and an eluant/reagent ratio 6.3 with a flat base line: 1, mannose, 2, galactose.
191 C
A
Fig. 3. Analysis of equal amounts of transfertin fractions. In the order A, B and C they contain increasing amounts of sialic acid, 2.8, 3.9 and 4.3 sialic acid residues per mol protein, but also increasing amounts of galactose, 3.0, 3.9 and 4.3 residues per mol protein. This indicates heterogeneity of the fractions. 1, mannose; 2, glucose (as impurity); 3, galactose.
of 6.3 with 1.6 nmol of each sugar recorded at 0.1 A. Diagram B shows a flat base line. In Fig. 3 the sugar diagrams of three different transferrin preparations [1,15] are given. Equal amounts of protein are applied. In the order A, B and C increasing amounts of sialic acid were found, causing a difference in the isoelectric point of the proteins. However, a heterogeneity with respect to their antennary structures would also differ in galactose content. From the Figs. it is clear that the fractions A, B and C differ in galactose content. Discussion The application of p-anisyl tetrazolium blue as a color reagent for the quantitation of reducing sugars, as proposed by Mopper and Degens, is not commonly in use, in spite of several attractive properties. The reagent is not corrosive, permits a high sensitivity, linearity, reproducibility and is not expensive. The stability is limited, but with weekly fresh solutions no problems are encountered. Although Mopper and Degens [12] claimed a sensitivity of lOPi mol, only 10e8 mol could be quantitatively analyzed. To obtain reliable sensitivity in the 1 nmol range with a flat base line we had to decrease the reagent concentration 10 times and to increase the eluant/reagent ratio 2-3 times. Using tetrazolium blue as color reagent one can only apply ethanol as eluant since ethanol prevents the precipitation of the reaction product diformazon [12].
192
Although 2 h hydrolysis is long enough for a 100% recovery of galactose, only 75% of mannose is recovered. A prolonged hydrolysis time (8 h) gave 85% recovery for mannose, but 16 h hydrolysis resulted in 75% recovery. Apparently mannose is so difficult to liberate that it is partly destroyed before the hydrolysis is complete. Differences in the mannose amounts in Fig. 3 are partly caused by differences in recovery. The existence of transferrin forms with different amounts of mannose is not known. The glucose found must be ascribed to contamination of the proteins caused by the isolation procedure. This is very often seen in analysis of glycoproteins. In conclusion With the technique described it is possible hydrolysates of glycoproteins within 2 h. It may composition of the carbohydrate moiety in order chain is degraded. This is important with respect e.g. galactose residues, with cell membranes and
to quantitate neutral sugars in be of great value to determine the to know how far the carbohydrate to the interaction of carbohydrates, lectins.
References 1 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. 2 Van Eijk HG, Van Noort WL, Van der Heul C. Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? Clin Chim Acta 1982; 126: 1933195. 3 Van Eijk HG, Van Noort WL, Dubelaar ML, Van der Heul C. The microheterogeneity of human transferrins in biological fluids. Clin Chim Acta 1983; 132: 167-171. 4 Spik G, Bayard B, Foumet B, Strecker G, Bouquelet S, Montreuil J. Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human sero transferrin. FEBS Lett 1975; 50: 196-199. 5 Hatton MWC, M;irz L, Berry LR, Debanne MT, Regoeczi E. Bi- and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins. Biochem J 1979; 181: 633-638. MM, Khorlin AY, Voelter W, Bauer H. High performance liquid chromatographic 6 Tikhomirov investigation of the amino acid, amino sugar and neutral sugar content in glycoproteins. J Chromatogr 1978; 167: 197-203. 7 Hara S, Ikegami H, Shono A, Mega T, Ikenaka T, Matsushima Y. Analysis of neutral sugars as glycamines using an amino acid analyzer. Anal Biochem 1979; 97: 166-172. of neutral and amino sugars found in glycoproteins. Anal 8 Barr J, Nordin Ph. Microdetermination Biochem 1980; 108: 313-319. 9 Kennedy JF, Fox JE. [l] Fully automatic ion-exchange chromatographic analysis of neutral monosaccharides and oligosaccharides. Methods of carbohydrate chemistry. Vol. VIII. 1980: 3-12. Eds. Whistler RL, Beuiller JN, IL. 10 Honda S, Takahashi M, Nishimura Y, Kakehi K, Ganno S. Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide. Anal Biochem 1981; 118: 162-167. analysis of amino sugars and derivatized neutral sugars. Anal 11 Perini F, Peters BP. Fluorimetric Biochem 1982; 123: 357-363. 12 Mopper K, Degens ET. A new chromatographic sugar autoanalyzer with a sensitivity of lo-i0 moles. Anal Biochem 1972; 45: 147-153.
193
13 Hobbs JS, Lawrence JG. The separation of carbohydrates on cation-exchange resin columns having organic counterions. J Chromatogr 1972; 72: 311-318. 14 Von Wilm M, SordC G. A method for the analysis of neutral mono- and di-saccharides by column chromatography with the multichrom B. Beckman Information l/1974, Geneva, Switzerland. 15 Van Eijk HG, Van Noort WL, Kroos MJ, Van der Heul C. Isolation of the two monoferric human transfertins by preparative isoelectric focusing. J Clin Chem Clin Biochem 1980; 18: 563-566.