Eleclrophoresis 1991,12,313-314
Isoelectric focusing of alloalbumins
3 13
Short communications Jorge Rocha' Jost Kompf2 Nuno Ferrand3 Antonio Amorim' Horst €titter2 'Instituto de Antropologia,Faculdade de CiCncias, Universidade do Porto *Institutf i r Anthropologie und Humangenetik der Universitat Tubingen 31nstitutode Zoologia, Faculdade de Ci&ncias,Universidade do Porto
Separation of human alloalbumin variants by isoelectric focusing A technique for the separation of human alloalbumin variants by means ofisoelectric focusing in the presence of 8111 urea and 60 mM L-serine is described. The potential usefulness ofthis technique in the detection and classification ofgenetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.
Analysis of serum alloalbumins by means of electrophoresis has allowed the description of several genetic variants in different human populations. Attempts to standardize the discrimination and classification methods of these albumin types were made by Weitkamp el al. [ I , 21 and Fine et al. [31. The technical approaches that were developed by these authors to maximize the capacity of variant discrimination rely essentially on the sequential use of three different electrophoretic buffer systems (pH 5.0, 6.9, and 6.0 or 8.6) with starch or cellulose acetate as separation media. In contrast to the relative simplicity of the electrophoretic analyses of human serum albumin, some major difficulties were reported to be associated with its separation by isoelectric focusing (IEF) [3-51. These are thought to be due to the ability ofthe albumin molecule to bind an extensive array of ligands, leading to several types of nongenetic heterogeneities that obscure the definition of its IEF patterns. Basu et al. 141 and Gianazza et aE. 151show the importance ofthe association of some ofthese ligands with the albumin molecule in determining both its isoelectric point and the degree of heterogeneity of tlie patterns displayed. Basu et al. [41 demonstrated that a shift from a heterogeneous pattern, centered around p1 4.8, to a more homogeneous pattern, centered around p I 5.6, could be observed by fatty acid removal from albumin either by defatting the molecule prior to IEF analysis or by increasing the duration of the separation experiments. Gianazza et al. 151 also performed time-course monitoring during focusing and confirmed the tendency for a progressive displacement of the molecule towards a more basic pH range. Saturation experiments, on the other hand, showed an increasing heterogeneity of the IEF pattern and a shift from p15.8 to p14.9 after addition of fatty acids to albumin. Despite these attempts, Fine et al. [3] concluded "that high resolutive systems such as isoelectric focusing cannot be employed" for separation of allobumins. We describe an IEF technique that seems to constitute a sensitive and reliable method for alloalbumin separation. Its potential usefulness for the detection and classification of genetic heterogeneity on the human albumin locus (ALB) is demonstrated by the differentiation of three variants of European origin.
Correspondence: Dr. Jorge Rocha, Instituto de Antropologia, Faculdade de Cisncias, P-4000 Porto, Portugal Abbreviations: IEF, isoelectric focusing; ALB, albumin; TEMED,
N,N,N',N'-tetramethylethylenediamine 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Serum or EDTA-plasma samples, from families in whom alloalbumin variants were detected by electrophoretic screening for clinical purposes, were kindly provided by Prof. Ott and Dr. Bohner. Samples were stored at -20 "Cuntil use. IEF was performed in polyacrylamide gels (5 %T, 3 %C; 235 x 100 x 0.3 mm) in the presence of 8 M urea. The pH gradient was established using 6.25 % v/v carrier ampholytes (1 : 1 mixture of pH 5-6 and pH 5-8, Pharmacia). I n order to increase separation in the plateau region (pH 5-6) of the gradient, L-serine (60 mM) was also added to the gel [61. Gel polymerization was achieved in 1 h at room temperature by adding 0.7 % v/v of a0.4 M ammonium persulfate solution and 0.1 % of N,N,N',N'-tetramethylethylenediamine(TEMED). Electrode solutions were 0.04 M aspartic acid and 1 M NaOH at the anode and cathode, respectively. Gels were prefocused for 1 h at 1500 V and 25 mA (maximum settings) with the power limits being fixed at 0.7 W (30 rnin), 2 W (15 min) and 3 W (15 min). After prefocusing, 7 pL of serum or plasma samples, diluted 1 : 50 in distilled water, were applied 1.5 cm from the cathode, using a silicon rubber application strip (Desaga). Focusing was performed with the same voltage and current settings of prefocusing and the power limits were fixed at 3 W (3 h), 3.6 W (30 mia) and 4.2 W until constant voltage was reached (ca. 30 min). The application strip was removed after the first hour of focusing. At the end of IEF the gels were placed in a 10 5% w/v solution of trichloroacetic acid for 10 min. For protein staining aO. 1 % w/v solution of Kenacid Blue R-250 (British Drug House) in ethanol: water (1 :2, v/v) was used. Destaining was performed with ethanob'acetic acid/ water 3 : 1 : 8 v/v. Alternatively, staining can also be carried out according to Vesterberg et al. 171. Figure 1 depicts the patterns of human serum albumin after separation by IEF under the conditions described above. Patterns in lanes 1, 2, 6, and 7 represent homozygotes for the normal albumin gene product (ALB*A). They show one major band and up to two anodal minor bands. The variants recognized in our family material are expressed in a heterozygous condition. They exhibit cathodal positions with respect to the ALB*A gene product and can be grouped into three distinct types (lanes 3,4, and 5). They each show amajor band and two anodal minor bands, which can be clearly seen in the pattern of lane 5 and which seem to overlap with the minor fractions associated with ALB*A in the other patterns (lanes 3 and 4). Variants in lanes 4 and 5 were classified by Prof. Ott as being heterozygotes for ALB*B and ALB*B', respectively. The pattern in lane 4 elucidates the separation that can be 0173-0835/91/0404-03 13 %3.50+.25/0
3 14
Electrophoresis 1991,12,313-314
J . Rocha et al.
NaOH were used as anode and cathode electrodes, respectively; the other conditions regarding gel composition and electric settings remained as described above. In this case, all albumin types presented highly heterogeneous patterns without any clear discrimination among them. These results indicate that the presence of 8 M urea not only displaces albumin towards a more basic pH range but also increases its homogeneity, resulting in better defined IEF patterns. This is similar to the effect of defatting reported by Basu et al. 141and is probably due to an action of urea in the dissociation of the complexes formed between albumin and its ligands.
Figure 1. Albumin patterns obtained after IEF in the presence of 8M urea and 6 0 m L-serine ~ in the pH range of 5-8. The major band of each gene product is indicated by an arrow. Lanes (I), (2), (6), and (7) homozygotes ALB A; (3) unidentified variant; (4) heterozygote ALB A-B; (5) heterozygote ALB A-B’.
achieved for a 2-unit charge difference because the ALB*B gene product is characterized by a mutation involving a 570 Glu-Lys amino acid substitution [81.The phenotype shown in lane 3 remains to be classified since it has not yet been compared with a more extensive panel of reference samples. ALB*B was the most common variant gene product among the families in whom segregation was observed. The results clearly indicate that the genetic heterogeneity at the albumin locus can be demonstrated by means of IEF in the pH range of 5-8 in the presence of 8 M urea and using 60 mMLserine as separator. Without L-serine, bands sharpness decreased and the IEF protein patterns were not reproducible. When IEF was performed without urea, a clear shift of albumin to the most acidic region of the gel was observed. For a more detailed evaluation of the behavior of albumin in this more acid pH region, IEF was also performed, with 13 % saccharose as the sole additive, in a pH gradient established with a 1 : 1 mixture of pH 4-6 (LKB) and p H 4.5-5.4 (Pharmacia) carrier ampholytes. Saturated aspartic acid and 0 . 2 ~
Our method for the separation of human albumin therefore seems to overcome some of the major difficulties associated with the differentiation of its allotypes by IEF. Further work is in progress with known referenced samples in order to evaluate the extension of the variant discrimination capacity of this method. The authors are indebted to Pro$ Ott and Dr. Bohner for providing the samples from families with albumin variants. We also wish to thank three anonymous referees whose criticism considerably improved the content of this paper. Received August 20, 1990
References Ill Weitkamp, L. R., Salzano, F. M., Neel, J. V., Porta F., Geerdink, R. A.
andTarnoky, A. L.,Ann. Hum. Genet. 1973,36,381-392. L21 Weitkamp, L. R.,McDermid, E. M., Neel,J. V., Fine, J. M., Petrini, C., Bonazzi L., Ortali, V., Porta, F.,Tanis, R., Harris, D. J., Peters, T., Ruffini, G. and Johnston, E., Ann. Hum. Genet. 1973,37, 219-226. [31 Fine, J . M., Marneux,M. and Rochu,D., Am.J. Hum.Genet. 1987,40, 278-286. 14J Basu, S. P., Rao, S. N. and Hartsuck, J . A., Biochim. Biophys. Acta 1978,533,66-73. [51 Gianazza, E., Frigerio, A., Astrua-Testori, S. and Righetti, P. G., Electrophoresis 1984,5, 3 10-3 12. [61 Caspers, M. L., Posey Y. and Brown, R. K.,Anal. Biochem. 1977,79, 166-180. 171 Vesterberg, O., Hansen, L. and Sjosten, A., Biochim. Biophys. Actu 1977,491, 160-166. [Sl Winter, W. P., Weitkamp, L. R. and Rucknagel, D. L., Biochemistry 1972,II, 889-896.
Isoelectric focusing of alloalbumins
3 13
Short communications Jorge Rocha' Jost Kompf2 Nuno Ferrand3 Antonio Amorim' Horst €titter2 'Instituto de Antropologia,Faculdade de CiCncias, Universidade do Porto *Institutf i r Anthropologie und Humangenetik der Universitat Tubingen 31nstitutode Zoologia, Faculdade de Ci&ncias,Universidade do Porto
Separation of human alloalbumin variants by isoelectric focusing A technique for the separation of human alloalbumin variants by means ofisoelectric focusing in the presence of 8111 urea and 60 mM L-serine is described. The potential usefulness ofthis technique in the detection and classification ofgenetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.
Analysis of serum alloalbumins by means of electrophoresis has allowed the description of several genetic variants in different human populations. Attempts to standardize the discrimination and classification methods of these albumin types were made by Weitkamp el al. [ I , 21 and Fine et al. [31. The technical approaches that were developed by these authors to maximize the capacity of variant discrimination rely essentially on the sequential use of three different electrophoretic buffer systems (pH 5.0, 6.9, and 6.0 or 8.6) with starch or cellulose acetate as separation media. In contrast to the relative simplicity of the electrophoretic analyses of human serum albumin, some major difficulties were reported to be associated with its separation by isoelectric focusing (IEF) [3-51. These are thought to be due to the ability ofthe albumin molecule to bind an extensive array of ligands, leading to several types of nongenetic heterogeneities that obscure the definition of its IEF patterns. Basu et al. 141 and Gianazza et aE. 151show the importance ofthe association of some ofthese ligands with the albumin molecule in determining both its isoelectric point and the degree of heterogeneity of tlie patterns displayed. Basu et al. [41 demonstrated that a shift from a heterogeneous pattern, centered around p1 4.8, to a more homogeneous pattern, centered around p I 5.6, could be observed by fatty acid removal from albumin either by defatting the molecule prior to IEF analysis or by increasing the duration of the separation experiments. Gianazza et al. 151 also performed time-course monitoring during focusing and confirmed the tendency for a progressive displacement of the molecule towards a more basic pH range. Saturation experiments, on the other hand, showed an increasing heterogeneity of the IEF pattern and a shift from p15.8 to p14.9 after addition of fatty acids to albumin. Despite these attempts, Fine et al. [3] concluded "that high resolutive systems such as isoelectric focusing cannot be employed" for separation of allobumins. We describe an IEF technique that seems to constitute a sensitive and reliable method for alloalbumin separation. Its potential usefulness for the detection and classification of genetic heterogeneity on the human albumin locus (ALB) is demonstrated by the differentiation of three variants of European origin.
Correspondence: Dr. Jorge Rocha, Instituto de Antropologia, Faculdade de Cisncias, P-4000 Porto, Portugal Abbreviations: IEF, isoelectric focusing; ALB, albumin; TEMED,
N,N,N',N'-tetramethylethylenediamine 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Serum or EDTA-plasma samples, from families in whom alloalbumin variants were detected by electrophoretic screening for clinical purposes, were kindly provided by Prof. Ott and Dr. Bohner. Samples were stored at -20 "Cuntil use. IEF was performed in polyacrylamide gels (5 %T, 3 %C; 235 x 100 x 0.3 mm) in the presence of 8 M urea. The pH gradient was established using 6.25 % v/v carrier ampholytes (1 : 1 mixture of pH 5-6 and pH 5-8, Pharmacia). I n order to increase separation in the plateau region (pH 5-6) of the gradient, L-serine (60 mM) was also added to the gel [61. Gel polymerization was achieved in 1 h at room temperature by adding 0.7 % v/v of a0.4 M ammonium persulfate solution and 0.1 % of N,N,N',N'-tetramethylethylenediamine(TEMED). Electrode solutions were 0.04 M aspartic acid and 1 M NaOH at the anode and cathode, respectively. Gels were prefocused for 1 h at 1500 V and 25 mA (maximum settings) with the power limits being fixed at 0.7 W (30 rnin), 2 W (15 min) and 3 W (15 min). After prefocusing, 7 pL of serum or plasma samples, diluted 1 : 50 in distilled water, were applied 1.5 cm from the cathode, using a silicon rubber application strip (Desaga). Focusing was performed with the same voltage and current settings of prefocusing and the power limits were fixed at 3 W (3 h), 3.6 W (30 mia) and 4.2 W until constant voltage was reached (ca. 30 min). The application strip was removed after the first hour of focusing. At the end of IEF the gels were placed in a 10 5% w/v solution of trichloroacetic acid for 10 min. For protein staining aO. 1 % w/v solution of Kenacid Blue R-250 (British Drug House) in ethanol: water (1 :2, v/v) was used. Destaining was performed with ethanob'acetic acid/ water 3 : 1 : 8 v/v. Alternatively, staining can also be carried out according to Vesterberg et al. 171. Figure 1 depicts the patterns of human serum albumin after separation by IEF under the conditions described above. Patterns in lanes 1, 2, 6, and 7 represent homozygotes for the normal albumin gene product (ALB*A). They show one major band and up to two anodal minor bands. The variants recognized in our family material are expressed in a heterozygous condition. They exhibit cathodal positions with respect to the ALB*A gene product and can be grouped into three distinct types (lanes 3,4, and 5). They each show amajor band and two anodal minor bands, which can be clearly seen in the pattern of lane 5 and which seem to overlap with the minor fractions associated with ALB*A in the other patterns (lanes 3 and 4). Variants in lanes 4 and 5 were classified by Prof. Ott as being heterozygotes for ALB*B and ALB*B', respectively. The pattern in lane 4 elucidates the separation that can be 0173-0835/91/0404-03 13 %3.50+.25/0
3 14
Electrophoresis 1991,12,313-314
J . Rocha et al.
NaOH were used as anode and cathode electrodes, respectively; the other conditions regarding gel composition and electric settings remained as described above. In this case, all albumin types presented highly heterogeneous patterns without any clear discrimination among them. These results indicate that the presence of 8 M urea not only displaces albumin towards a more basic pH range but also increases its homogeneity, resulting in better defined IEF patterns. This is similar to the effect of defatting reported by Basu et al. 141and is probably due to an action of urea in the dissociation of the complexes formed between albumin and its ligands.
Figure 1. Albumin patterns obtained after IEF in the presence of 8M urea and 6 0 m L-serine ~ in the pH range of 5-8. The major band of each gene product is indicated by an arrow. Lanes (I), (2), (6), and (7) homozygotes ALB A; (3) unidentified variant; (4) heterozygote ALB A-B; (5) heterozygote ALB A-B’.
achieved for a 2-unit charge difference because the ALB*B gene product is characterized by a mutation involving a 570 Glu-Lys amino acid substitution [81.The phenotype shown in lane 3 remains to be classified since it has not yet been compared with a more extensive panel of reference samples. ALB*B was the most common variant gene product among the families in whom segregation was observed. The results clearly indicate that the genetic heterogeneity at the albumin locus can be demonstrated by means of IEF in the pH range of 5-8 in the presence of 8 M urea and using 60 mMLserine as separator. Without L-serine, bands sharpness decreased and the IEF protein patterns were not reproducible. When IEF was performed without urea, a clear shift of albumin to the most acidic region of the gel was observed. For a more detailed evaluation of the behavior of albumin in this more acid pH region, IEF was also performed, with 13 % saccharose as the sole additive, in a pH gradient established with a 1 : 1 mixture of pH 4-6 (LKB) and p H 4.5-5.4 (Pharmacia) carrier ampholytes. Saturated aspartic acid and 0 . 2 ~
Our method for the separation of human albumin therefore seems to overcome some of the major difficulties associated with the differentiation of its allotypes by IEF. Further work is in progress with known referenced samples in order to evaluate the extension of the variant discrimination capacity of this method. The authors are indebted to Pro$ Ott and Dr. Bohner for providing the samples from families with albumin variants. We also wish to thank three anonymous referees whose criticism considerably improved the content of this paper. Received August 20, 1990
References Ill Weitkamp, L. R., Salzano, F. M., Neel, J. V., Porta F., Geerdink, R. A.
andTarnoky, A. L.,Ann. Hum. Genet. 1973,36,381-392. L21 Weitkamp, L. R.,McDermid, E. M., Neel,J. V., Fine, J. M., Petrini, C., Bonazzi L., Ortali, V., Porta, F.,Tanis, R., Harris, D. J., Peters, T., Ruffini, G. and Johnston, E., Ann. Hum. Genet. 1973,37, 219-226. [31 Fine, J . M., Marneux,M. and Rochu,D., Am.J. Hum.Genet. 1987,40, 278-286. 14J Basu, S. P., Rao, S. N. and Hartsuck, J . A., Biochim. Biophys. Acta 1978,533,66-73. [51 Gianazza, E., Frigerio, A., Astrua-Testori, S. and Righetti, P. G., Electrophoresis 1984,5, 3 10-3 12. [61 Caspers, M. L., Posey Y. and Brown, R. K.,Anal. Biochem. 1977,79, 166-180. 171 Vesterberg, O., Hansen, L. and Sjosten, A., Biochim. Biophys. Actu 1977,491, 160-166. [Sl Winter, W. P., Weitkamp, L. R. and Rucknagel, D. L., Biochemistry 1972,II, 889-896.
![[Characterization of genetic variants of human albumin by isoelectric focusing].](/assets/img/hold.png)
