3 18
M. Kane et. al.
Electrophoresis 199O,lI, 318-321
allele frequencies between our sample and other US white populations [5-6, 131 provides further support in favor of the reliability and specificity of this method. The plasma samples we have analyzed in this investigation were collected in 1984 and have been frozen andthawedrepeatedly over this five year period for other genetic marker studies. We have used only 5 pL plasma samples for the dilution purpose. However, even a 2 pL sample is enough for a single I E F run. This method is suitable for large-scale population and epidemiological studies because only a small amount of sample is needed and no sophisticated laboratory equipment is required. To the best of our knowledge the method presented here is the first to use agarose rather than polyacrylamide as a supporting medium to detect APO E phenotypic variation. EIecause of the simple protocol presented here, IEF in agarose may prove to be the method of choice for APO E phenotyping. The present method together with our previously described procedure [ 131 provides a simple screening tool for investigation of the APO E polymorphism on a worldwide basis and the validity ofthis argument has been confirmed in our laboratory by screening several thousand plasma samples (unpublished data).
5 References [ 11 Mahley, R. W., Science 1988,240, 622-630. 121 Myklebost, 0. and Rogne, S., Hum. Genet. 1988, 78,244-247. I31 Zannis,V.l.,Just,P.W.andBreslow,J.L.,Am.J.HuniGenet.1981, 33, 11-24. 141 Wardell. M . R., Brennan, S. O., Janus, E. D., Fraser, R. and Carrell. R . W.. J . Clin. Invest. 1987. 80. 483-490. 151 Davignon, J., Gregg, R. E. and Sing, C. F.,Arteriosclerosis 1988.8. 1-21. I6 I Kamboh, M. I., Sepehrnia, B. and Ferrell, R. E., Dis.Markers 1989,7, 49-55. 171 Mahley, R. W. and Innerarity, T. L., Biochirn. Biophys. Aria. 1983, 737, 197-222. 181 Gregg, R. E., Zech, L. A., Schaefer, E. J., Stark, D., Wilson, D. and Brewer, H. B., Jr., J. Clin. Invest. 1986, 78, 815-821. 191 Utermann, G.,Arn. Heart J. 1987,113,433-440. [ 101 Menzel,H.J.andUtermann,G., Electrophoresis 1986,7,492-495. II 11 Havekes, L. M., deKnijff, P., Beisiegel, U., Havinga, J., Smith, M. and Klasen, E., J. LipidRes. 1987,28,455-463. [ 121 Steinmetz, A., J. LipidRes. 1987,28, 1364-1370. [ 131 Kamboh, M. I., Ferrell, R. E. and Kottke, B., J . Lipid Res. 1988,29, 1535-1 543. 1
This work was supported in part by N.I. H . Grants HL39107 and HL24489. We thank Jeanette Norbut for secretarial assistance. Received October 4, 1989
Masateru Kane '. Yoshio Yamamoto2 Mitsuko Yamada2 Tatsushige Fukunaga2 Yoahitsugu Tatsuno2
Phenotyping of erythrocyte acid phosphatase and esterase D by high field strength isoelectric focusing on cellulose acetate membrane
'Forensic Science Laboratory, Shiga Prefectural Police Headquaters,Ohtsu 'Department of Legal Medicine, Shiga University of Medical Science, Ohtsu
Phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (ESD) by cellulose acetate membrane isoelectric focusing (CAM-IEF) under a nonequilibrium condition is described. In an attempt to improve the method of CAM-IEF, we shortened the electrode distance to provide a higher field strength at a given (low) voltage. Various carrier ampholytes for EAP typing and various chemical separators for ESD typing were also tested. Good separations were obtained after 30 rnin IEF for EAP typing and 25 min for ESD typing. When applied to blood stains and stored for various periods at room temperature, the stains up to 8 months old could still be phenotyped for EAP and those up to 4 weeks old for ESD. CAM-IEF is suitable for routine forensic work of EAP and ESD phenotyping.
1 Introduction
phenotypes are usually discriminated by either conventional electrophoresis or isoelectric focusing (IEF), broader bands In 1963 Hopkinson et al. I 1 1 first described genetic polymor- resulting from diffusion during isozyme visualization ocphism of erythrocyte acid phosphatase (EAP). Although six casionally causes mistyping. Destro-Bisol and Ranalletta 121 showed a sharper isozyme pattern by using hydrophilic Correspondence: Dr. Masateru Kane. Department of Legal Medicine, cellophane film soaked in prewarmed substrate solution at Shiga University of Medical Science, Seta-Tsukinowa-cho, Ohtsu 520-2 1. 50 OC to shorten the incubation time. Esterase D (ESD) was Japan first phenotyped by Hopkinson et al. [3] in 1973. Two rare Abbreviations: BES, N,N-bis(2-hydroxyethy1)-2-aminoethanesulfonic variants, ESD*5 and ESD*7, are not separated from three acid; CAM, cellulose acetate membrane; DTr, dithiothreitol; EAP, common phenotypes by conventional electrophoresis but are erythrocyte acid phosphatase; EPPS,N-(2-hydroxyethyl)-piperazine-N'- well separated by I E F [4,5], although the discrimination ofthe three common phenotypes by I E F is unsatisfactory. Yuasa et 3-propanesulfonic acid; ESD, esterase D; IEF, isoelectric focusing; Vh, volt x hour al. [ 61 reported that low voltage I E F under a nonequilibrium 0VCH Verlagsgesellschaft mbH. D-6940 Weinheim, I990
0173-0835/90/0404-03 18$2.50/0
Electrophoresis 1990,II, 318-321
condition overcame this problem. Budowle [71 described the use ofcarrier ampholytes ofpH 4.5-5.4 and aseparatorunder equilibrium conditions. Recently, Weidinger and Henke [ 81 showed a good separation of isozymes in agarose gel IEF. The advantage of cellulose acetate membrane (CAM) lies in operational simplicity. In IEF. however. CAMs are not widely used, because drying of CAMs during high-voltage I E F results in poor reproducibilty. Recently, Toda et al. [91 developed an improved apparatus for high-voltage CAM-IEF and applied it to an analysis of serum proteins with favorable results. In this study, we shortened the electrode distance from the normal length of 10 cm to 6 cm. O n applications of the same low voltage the shorter electrode distance provides higher field strength and also prevents the CAMsfromdrying. Furthermore, EAP and ESD isozymes can be phenotyped under nonequilibrium conditions and the isozymes can be visualized on the membranes directly, without transfer. The modified CAM-IEF technique was applied to an analysis of EAP and ESD phenotypes.
Phenotyping of erythrocyte acid phosphatase and esterase D
3 19
distance of 1 cm from the anode for EAP typing and at a distance of 0.5 cm from the cathode for ESD typing, with sample tabs (7 x 5 mm in size, Whatman No. 1). Theelectrode solutions were 0.2 M sodium hydroxide at the cathode and 1 M phosphoric acid containing 30 % sucrose at the anode. Sample tabs were removed after 5 min. The minimum volthour (Vh) for optimal separation was established in preliminary tests.
2.4 Isozyme visualization Following IEF, CAMs were stained with 4-methylumbelliferylphosphate for EAP typing and with 4-methylumbelliferylacetate for ESD typing. A piece of filter paper soaked in the staining solution was placed on the focused C A M and excess solution was removed by blotting. After incubation of the membrane for 5-10 rnin at 37 "C, the isozymes were visualized under 365 nm UV light after removing the filter paper.
2 Materials and methods 2.1 Sample preparation Erythrocyte lysates were prepared from saline-washed, centrifuged red cells by freezing and thawing. Blood stains were obtained on filter paper, air dried and stored at room temperature. The lysates were diluted 4 times with 0.05 M dithiothreitol (DTT) and kept for 15 rnin at room temperature before typing. The stain extracts were prepared from 5 x 5 mmpieces of blood stain materials stored at room temperature. They were extracted in one drop of 0.05 M DTT solution for 30 min. A blood stain of a rare ESD phenotype, ESD7-1, stored at -85 "C, was treated with the 0.05 M DTT solution as mentioned above.
3 Results and discussion 3.1 EAP phenotyping
The isozyme patterns obtained with various carrier ampholytes for EAP typing under the same I E F conditions were different. As shown in Figs. l a and lb, with the pH 5-6.5 carrier ampholytes, A , and A, were separated, whereas the distance between A, and B, became smaller. With pH 5-8 carrier ampholytes the opposite result was obtained. On the other hand, with p H 5-7 carrier ampholytes, A,, B and B, could be adequately separated, but A, and A, were not resolved (data not shown). The results were similar to those reported by Budowle using a p H range of 5-7 [ 101. When p H 5-8 carrier ampholytes were added to the pH 5-6.5 carrier ampholytes, the distance between A, and B, became wider with A, and A, 2.2 CAM preparation separated. Figure lc shows EAP patterns with a 1:5 mixture of p H 5-8 and p H 5-6.5 ampholytes. Whereas after applying Carrier ampholytes, p H 5-6.5 (LKB, Bromma), p H 5-7 (LKB) and pH 5-8 (Pharmacia, Uppsala), were used in 300 Vh (18 rnin of IEF) a poorly separated pattern was obthis study. Carrier ampholytes with different pH ranges were served, well resolved patterns were obtained after applying diluted 10 times with 10 o/o w/v sucrose solution, The fol- 550 Vh (30 min of IEF). Optimal conditions for EAP typing lowing separators were tested for the separation of ESD were found for a 1:5 mixture of p H 5-8 and pH 5-6.5 carrier phenotypes: N,N-bis(2-hydroxyethyl)-2-aminoethanesulfon- ampholytes and 30 rnin offocusing. Although agradient of pH ic acid (BES), N-(2-hydroxyethyl)piperazine-N'-2-ethanesul- 4-8 (a 1:l mixture of pH 4-6 and p H 6-8 range carrier fonic acid (HEPES) and N-(2-hydroxyethyl)piperazine-N'-3- ampholytes) has been used in several reports [ 11-14], it takes propanesulfonic acid (EPPS). Each separator was added to more than 75 rnin to achieve a sufficient separation ofbands in the pH 5-6.5 carrier ampholyte solution at aconcentrations of this p H range. With our method, a good separation of bands 2.5 96w/v. CAMs (7 x 10 cm, Separax EF, Fuji Film, Tokyo) was obtained in 30 rnin with the relative intensities ofthe bands were floated on the carrier ampholyte solution for 30 s and similar to those obtained in a wider p H gradient [ l 1-141. placed on a glass plate cooled on a flat bed (Pharmacia FEB However, a broader band appeared occasionally as shown in 3000) at 4 OC, and excess solution was removed by blotting Fig. 2a when erythrocyte lysates were diluted 4 times with DTT. Distro-Bisol and Ranalletta [21 showed a sharper with filter paper. isozyme pattern by using hydrophilic cellophane film soaked in prewarmed substrate solution at 50 OC, whereby diffusion 2.3 IEF of the developed bands was minimized by shortening the incubation time. In an experiment to obtain sharper bands by I E F was performed using a Pharmacia system (constant diluting erythrocyte lysates with DTT, eight-fold dilution was power supply ECPS 3000/150 and volthour integrator VH- found to give a result comparable to that of Distro-Bisol and 1). An initial voltage of 900V (150 V/cm) was applied under a Ranalletta [21(Fig. 2b). Our method was also successfully apconstant power (4W) and unlimited current, and the maximum plied to an analysis of blood stains stored for up to 8 months voltage was set at 1200 V. Samples were applied to C A M at a (Fig. 2c).
,
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M. Kane et. al.
3.2 ESD phenotyping CAM-IEF with pH 5-6.5 carrier ampholytes failed to resolve the three common phenotypes (ESD 1,2 and 2- 1) after applying 300 Vh (18 min of focusing) as shown in Fig. 3a. Ofthree different separators examined to improve the patterns, the addition of 2.5 % w/v EPPS resulted in a glood separation of the three common phenotypes after 300 Vh, whereas a rare ESD 7- I could not be discriminated (Fig. 3b). Good separation of the three common phenotypes and also the rare ESD 7-1 phenotype was obtained after applying 450 Vh (25 rnin) as shown in Fig. 3c. Yuasa et al. [6] reported that the good resolution of ESD phenotypes results fro'm charge differences under nonequilibrium conditions rather than from differences in p1 values. Gill and Sattun I 151 reported that the addition of EPPS resulted in flattening the pH gradient between pH 5.6 to
Elecfrophoresis 1990,II, 318-321
5.9. The p l values ofESD phenotypes(ESD 1,2,2-1 and 7-1) range from pH 5.02-5.33 I5 1. These two lines of evidence indicate that the addition of EPPS increases the charge differences among the isozymes under nonequilibrium conditions, widening the distance between the isozyme bands. Narrow p H range carrier ampholytes are often used as an alternative for ESD typing. Budowle [71 found that carrier ampholytes with a pH range 4.5-5.4, combined with BES, allowed sufficient resolution on IEF under equilibrium conditions. Recently, Weidinger and Henke [81 reported the use of an agarose gel with a p H 4.5-5.4 range of carrier ampholyte. However, focusing by these procedures took more than 95 min [ 7,8]. CAM-IEF requires neither preparation of gels nor prefocusing, and completed the separation in only 25 min. Such a focusing time is much shorter than any previously reported for the separationofESD isozymes by IEF. When we
Figure 1. EAP isozyme patterns with various carrier ampholytes on CAM-IEF. Electrode distance, 6 cm; focusing time, 30 min, 550 Vh. (a) pH 5-6.5; (b) pH 5-8; (c) pH 5-8: pH 5-6.5, 1 5 .
Figure 2. Analysis of erythorocyte lysates and blood stains. (a)Lysates diluted 4 times with DTT, arrows showing broader bands; (b) lysates diluted 8 times with DTT; (c) blood stains stored for 8 months.
Figure 3. ESD isozyme patterns on CAMIEF. Electrode distance, 6 cm. (a) pH 5-6.5, 18 niin of focusing, 300 Vh; (b) pH 5-6.5 containing 2.5 %) EPPS, 18 min of focusing, 300 Vh; (c) pH 5-6.5 containing 2.5 % EPPS, 25 min of focusing, 450 Vh.
Haptoglobin subtyping by IEF under reducting conditions
Electrophoresis 1990, 11, 321-324
applied this technique to blood stains stored for various periods at room temperature, ESD 2- 1 up to 4 weeks old could be phenotyped. ESD 1 and ESD 2 up to 10 weeks old could also be separated sufficiently. The detection limit in analysis was similar to that in results described by Diva1 [ 161.
4 Concluding remarks We developed amethod for faster separation of EAP and ESD phenotypes by CAM-IEF. Adequate resolution was obtained after 30 min offocusing for EAP typing and 25 min offocusing for ESD typing. Such a short focusing time was possible owing to the nonrestrictive properties of CAMS as opposed to agarose or polyacrylamide gels (Nishimura, T., Fuji Film, Tokyo, pesonal communication). In addition, CAM-IEF affords the advantage of operational simplicity without preparing gels. CAM-IEF is thus useful for the routine work of EAP and ESD phenotyping in forensic investigations.
The authors thank Dr. K . Suzuki, Department of Legal Medicine, Osaka Medical University of Medical Science, for helpful suggestions and critical reading of the manuscript.
32 1
5 References Hopkinson, D., Spencer, W. and Harris, H., Nature 1963, 199, 969-97 1. Destro-Bisol, G. and Ranalletta, D., Electrophoresis 1988. 9, 106- 108. Hopkinson, D., Mestriner, M., Corntner, J. and Harris, H., Ann. Hum. Genet. 1973,37, 119-137. Olaisen, B., Siverts, A., Jonassen, R., Mevag, B. and Gedde-Dahl, T., Hum. Genet. 1981,57,351-353. Nishigaki, I. and Itoh, T., Hum. Genet. 1984,66, 92-95. Yuasa, I., Tamaki, N., Suenaga, K., Itoh, K.,lnoue,T.andOkada,K., Electrophoresis 1985,6,588-592. Budowle, B., Electrophoresis 1986, 7, 141-144. Weidinger, S. and Henke, J., Electrophoresis 1988,9,429-432. Toda, T., Sano-Shiba, K., Cho, H., Soon, P., Nakao, M. and Ohashi, M., Electrophoresis 1988, 9, 149-150. Budowle, B., Electrophoresis 1984,5,254-255. Randall, T., Harland, W. and Thorpe, J., Med. Sci. Law 1980, 20, 43-47. Divall, G., Forens. Sci. Znt. 1981,18, 67-78. Finney, S., Renshaw, N. and Werrett, D., Forens. Sci. Int. 1985,27, 237-245. Frank, W. and Stolorow, M., J. Forens. Sci. 1986,31, 1089- 1094. Gill, P. and Sutton, J., Electrophoresis 1985,6,23-26. Divall, G., Forens. Sci. Int. 1985,28, 277-285.
Received June 28, 1989; in revised form December 20, 1989
Antonio Alonso' Guillermo Visedo2 Manuel Sancho' Jose Fernandez-Piqueras2 'Instituto Nacional de Toxicologia, Madrid Wnidad de Genktica, Facultad de Ciencias, Universidad Autonoma de Madrid
Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamidegels rehydrated in presence of 2-mercaptoethanol A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 5 5 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the H p a and H p p chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp a allelic products (IS, IF, 2FS, 2SS and 2FF). A population study carried out with 250 unrelatedindividualsliving in Central Spain is also presented.
1 Introduction The haptoglobin (Hp) molecule, encoded by a locus on chromosome 16 ( 1 6 ~ 2 2 )i l l , is a polymorphic serum glycoprotein composed of two different polypeptide chains (a and 0) linked through disulfide bridges. The genetic polymorphism displayed by the Hp-a chain was first reported using Correspondence: Dr. Antonio Alonso, Seccion de Biologia, lnstituto Nacional de Toxicologia, C/Luis Cabrera 9, E-28002 Madrid, Spain Abbreviations: Hp, haptoglobin; IEF, isoelectric focusing; 2-ME, 2-mercaptoethanol; RS,reswelling solution; TEMED, N,N,N',N'-tetramethylethylenediamine
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
starch gel electrophoresis [ 21. This technique allowed the identification of three common phenotypes (Hpl, Hp2-1 Hp2). The existence of H p subtypes was first demonstrated using acidic starch gel electrophoresis in presence ofurea [3,41, and application of isoelectric focusing (IEF) to Hp subtyping further improved the separation of the different Hp-a allele products (IF, IS, 2FS, 2FF, 2SS) [Sl.At present, there are two methods available for large-scaleroutineHp subtyping: (i)partial purification, reductive cleavage and carboxymethylation of the H p molecule, followed by IEF with nonspecific protein staining [6] ;and (ii) desialylation and reductive cleavage ofthe H p molecule in whole serum, followed by IEF and immunoblotting [ 71. This article presents a fast Hp subtyping method based on IEF of Hp under reducting conditions, using dry 01 73-083519010404-0321%2.50/0
M. Kane et. al.
Electrophoresis 199O,lI, 318-321
allele frequencies between our sample and other US white populations [5-6, 131 provides further support in favor of the reliability and specificity of this method. The plasma samples we have analyzed in this investigation were collected in 1984 and have been frozen andthawedrepeatedly over this five year period for other genetic marker studies. We have used only 5 pL plasma samples for the dilution purpose. However, even a 2 pL sample is enough for a single I E F run. This method is suitable for large-scale population and epidemiological studies because only a small amount of sample is needed and no sophisticated laboratory equipment is required. To the best of our knowledge the method presented here is the first to use agarose rather than polyacrylamide as a supporting medium to detect APO E phenotypic variation. EIecause of the simple protocol presented here, IEF in agarose may prove to be the method of choice for APO E phenotyping. The present method together with our previously described procedure [ 131 provides a simple screening tool for investigation of the APO E polymorphism on a worldwide basis and the validity ofthis argument has been confirmed in our laboratory by screening several thousand plasma samples (unpublished data).
5 References [ 11 Mahley, R. W., Science 1988,240, 622-630. 121 Myklebost, 0. and Rogne, S., Hum. Genet. 1988, 78,244-247. I31 Zannis,V.l.,Just,P.W.andBreslow,J.L.,Am.J.HuniGenet.1981, 33, 11-24. 141 Wardell. M . R., Brennan, S. O., Janus, E. D., Fraser, R. and Carrell. R . W.. J . Clin. Invest. 1987. 80. 483-490. 151 Davignon, J., Gregg, R. E. and Sing, C. F.,Arteriosclerosis 1988.8. 1-21. I6 I Kamboh, M. I., Sepehrnia, B. and Ferrell, R. E., Dis.Markers 1989,7, 49-55. 171 Mahley, R. W. and Innerarity, T. L., Biochirn. Biophys. Aria. 1983, 737, 197-222. 181 Gregg, R. E., Zech, L. A., Schaefer, E. J., Stark, D., Wilson, D. and Brewer, H. B., Jr., J. Clin. Invest. 1986, 78, 815-821. 191 Utermann, G.,Arn. Heart J. 1987,113,433-440. [ 101 Menzel,H.J.andUtermann,G., Electrophoresis 1986,7,492-495. II 11 Havekes, L. M., deKnijff, P., Beisiegel, U., Havinga, J., Smith, M. and Klasen, E., J. LipidRes. 1987,28,455-463. [ 121 Steinmetz, A., J. LipidRes. 1987,28, 1364-1370. [ 131 Kamboh, M. I., Ferrell, R. E. and Kottke, B., J . Lipid Res. 1988,29, 1535-1 543. 1
This work was supported in part by N.I. H . Grants HL39107 and HL24489. We thank Jeanette Norbut for secretarial assistance. Received October 4, 1989
Masateru Kane '. Yoshio Yamamoto2 Mitsuko Yamada2 Tatsushige Fukunaga2 Yoahitsugu Tatsuno2
Phenotyping of erythrocyte acid phosphatase and esterase D by high field strength isoelectric focusing on cellulose acetate membrane
'Forensic Science Laboratory, Shiga Prefectural Police Headquaters,Ohtsu 'Department of Legal Medicine, Shiga University of Medical Science, Ohtsu
Phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (ESD) by cellulose acetate membrane isoelectric focusing (CAM-IEF) under a nonequilibrium condition is described. In an attempt to improve the method of CAM-IEF, we shortened the electrode distance to provide a higher field strength at a given (low) voltage. Various carrier ampholytes for EAP typing and various chemical separators for ESD typing were also tested. Good separations were obtained after 30 rnin IEF for EAP typing and 25 min for ESD typing. When applied to blood stains and stored for various periods at room temperature, the stains up to 8 months old could still be phenotyped for EAP and those up to 4 weeks old for ESD. CAM-IEF is suitable for routine forensic work of EAP and ESD phenotyping.
1 Introduction
phenotypes are usually discriminated by either conventional electrophoresis or isoelectric focusing (IEF), broader bands In 1963 Hopkinson et al. I 1 1 first described genetic polymor- resulting from diffusion during isozyme visualization ocphism of erythrocyte acid phosphatase (EAP). Although six casionally causes mistyping. Destro-Bisol and Ranalletta 121 showed a sharper isozyme pattern by using hydrophilic Correspondence: Dr. Masateru Kane. Department of Legal Medicine, cellophane film soaked in prewarmed substrate solution at Shiga University of Medical Science, Seta-Tsukinowa-cho, Ohtsu 520-2 1. 50 OC to shorten the incubation time. Esterase D (ESD) was Japan first phenotyped by Hopkinson et al. [3] in 1973. Two rare Abbreviations: BES, N,N-bis(2-hydroxyethy1)-2-aminoethanesulfonic variants, ESD*5 and ESD*7, are not separated from three acid; CAM, cellulose acetate membrane; DTr, dithiothreitol; EAP, common phenotypes by conventional electrophoresis but are erythrocyte acid phosphatase; EPPS,N-(2-hydroxyethyl)-piperazine-N'- well separated by I E F [4,5], although the discrimination ofthe three common phenotypes by I E F is unsatisfactory. Yuasa et 3-propanesulfonic acid; ESD, esterase D; IEF, isoelectric focusing; Vh, volt x hour al. [ 61 reported that low voltage I E F under a nonequilibrium 0VCH Verlagsgesellschaft mbH. D-6940 Weinheim, I990
0173-0835/90/0404-03 18$2.50/0
Electrophoresis 1990,II, 318-321
condition overcame this problem. Budowle [71 described the use ofcarrier ampholytes ofpH 4.5-5.4 and aseparatorunder equilibrium conditions. Recently, Weidinger and Henke [ 81 showed a good separation of isozymes in agarose gel IEF. The advantage of cellulose acetate membrane (CAM) lies in operational simplicity. In IEF. however. CAMs are not widely used, because drying of CAMs during high-voltage I E F results in poor reproducibilty. Recently, Toda et al. [91 developed an improved apparatus for high-voltage CAM-IEF and applied it to an analysis of serum proteins with favorable results. In this study, we shortened the electrode distance from the normal length of 10 cm to 6 cm. O n applications of the same low voltage the shorter electrode distance provides higher field strength and also prevents the CAMsfromdrying. Furthermore, EAP and ESD isozymes can be phenotyped under nonequilibrium conditions and the isozymes can be visualized on the membranes directly, without transfer. The modified CAM-IEF technique was applied to an analysis of EAP and ESD phenotypes.
Phenotyping of erythrocyte acid phosphatase and esterase D
3 19
distance of 1 cm from the anode for EAP typing and at a distance of 0.5 cm from the cathode for ESD typing, with sample tabs (7 x 5 mm in size, Whatman No. 1). Theelectrode solutions were 0.2 M sodium hydroxide at the cathode and 1 M phosphoric acid containing 30 % sucrose at the anode. Sample tabs were removed after 5 min. The minimum volthour (Vh) for optimal separation was established in preliminary tests.
2.4 Isozyme visualization Following IEF, CAMs were stained with 4-methylumbelliferylphosphate for EAP typing and with 4-methylumbelliferylacetate for ESD typing. A piece of filter paper soaked in the staining solution was placed on the focused C A M and excess solution was removed by blotting. After incubation of the membrane for 5-10 rnin at 37 "C, the isozymes were visualized under 365 nm UV light after removing the filter paper.
2 Materials and methods 2.1 Sample preparation Erythrocyte lysates were prepared from saline-washed, centrifuged red cells by freezing and thawing. Blood stains were obtained on filter paper, air dried and stored at room temperature. The lysates were diluted 4 times with 0.05 M dithiothreitol (DTT) and kept for 15 rnin at room temperature before typing. The stain extracts were prepared from 5 x 5 mmpieces of blood stain materials stored at room temperature. They were extracted in one drop of 0.05 M DTT solution for 30 min. A blood stain of a rare ESD phenotype, ESD7-1, stored at -85 "C, was treated with the 0.05 M DTT solution as mentioned above.
3 Results and discussion 3.1 EAP phenotyping
The isozyme patterns obtained with various carrier ampholytes for EAP typing under the same I E F conditions were different. As shown in Figs. l a and lb, with the pH 5-6.5 carrier ampholytes, A , and A, were separated, whereas the distance between A, and B, became smaller. With pH 5-8 carrier ampholytes the opposite result was obtained. On the other hand, with p H 5-7 carrier ampholytes, A,, B and B, could be adequately separated, but A, and A, were not resolved (data not shown). The results were similar to those reported by Budowle using a p H range of 5-7 [ 101. When p H 5-8 carrier ampholytes were added to the pH 5-6.5 carrier ampholytes, the distance between A, and B, became wider with A, and A, 2.2 CAM preparation separated. Figure lc shows EAP patterns with a 1:5 mixture of p H 5-8 and p H 5-6.5 ampholytes. Whereas after applying Carrier ampholytes, p H 5-6.5 (LKB, Bromma), p H 5-7 (LKB) and pH 5-8 (Pharmacia, Uppsala), were used in 300 Vh (18 rnin of IEF) a poorly separated pattern was obthis study. Carrier ampholytes with different pH ranges were served, well resolved patterns were obtained after applying diluted 10 times with 10 o/o w/v sucrose solution, The fol- 550 Vh (30 min of IEF). Optimal conditions for EAP typing lowing separators were tested for the separation of ESD were found for a 1:5 mixture of p H 5-8 and pH 5-6.5 carrier phenotypes: N,N-bis(2-hydroxyethyl)-2-aminoethanesulfon- ampholytes and 30 rnin offocusing. Although agradient of pH ic acid (BES), N-(2-hydroxyethyl)piperazine-N'-2-ethanesul- 4-8 (a 1:l mixture of pH 4-6 and p H 6-8 range carrier fonic acid (HEPES) and N-(2-hydroxyethyl)piperazine-N'-3- ampholytes) has been used in several reports [ 11-14], it takes propanesulfonic acid (EPPS). Each separator was added to more than 75 rnin to achieve a sufficient separation ofbands in the pH 5-6.5 carrier ampholyte solution at aconcentrations of this p H range. With our method, a good separation of bands 2.5 96w/v. CAMs (7 x 10 cm, Separax EF, Fuji Film, Tokyo) was obtained in 30 rnin with the relative intensities ofthe bands were floated on the carrier ampholyte solution for 30 s and similar to those obtained in a wider p H gradient [ l 1-141. placed on a glass plate cooled on a flat bed (Pharmacia FEB However, a broader band appeared occasionally as shown in 3000) at 4 OC, and excess solution was removed by blotting Fig. 2a when erythrocyte lysates were diluted 4 times with DTT. Distro-Bisol and Ranalletta [21 showed a sharper with filter paper. isozyme pattern by using hydrophilic cellophane film soaked in prewarmed substrate solution at 50 OC, whereby diffusion 2.3 IEF of the developed bands was minimized by shortening the incubation time. In an experiment to obtain sharper bands by I E F was performed using a Pharmacia system (constant diluting erythrocyte lysates with DTT, eight-fold dilution was power supply ECPS 3000/150 and volthour integrator VH- found to give a result comparable to that of Distro-Bisol and 1). An initial voltage of 900V (150 V/cm) was applied under a Ranalletta [21(Fig. 2b). Our method was also successfully apconstant power (4W) and unlimited current, and the maximum plied to an analysis of blood stains stored for up to 8 months voltage was set at 1200 V. Samples were applied to C A M at a (Fig. 2c).
,
320
M. Kane et. al.
3.2 ESD phenotyping CAM-IEF with pH 5-6.5 carrier ampholytes failed to resolve the three common phenotypes (ESD 1,2 and 2- 1) after applying 300 Vh (18 min of focusing) as shown in Fig. 3a. Ofthree different separators examined to improve the patterns, the addition of 2.5 % w/v EPPS resulted in a glood separation of the three common phenotypes after 300 Vh, whereas a rare ESD 7- I could not be discriminated (Fig. 3b). Good separation of the three common phenotypes and also the rare ESD 7-1 phenotype was obtained after applying 450 Vh (25 rnin) as shown in Fig. 3c. Yuasa et al. [6] reported that the good resolution of ESD phenotypes results fro'm charge differences under nonequilibrium conditions rather than from differences in p1 values. Gill and Sattun I 151 reported that the addition of EPPS resulted in flattening the pH gradient between pH 5.6 to
Elecfrophoresis 1990,II, 318-321
5.9. The p l values ofESD phenotypes(ESD 1,2,2-1 and 7-1) range from pH 5.02-5.33 I5 1. These two lines of evidence indicate that the addition of EPPS increases the charge differences among the isozymes under nonequilibrium conditions, widening the distance between the isozyme bands. Narrow p H range carrier ampholytes are often used as an alternative for ESD typing. Budowle [71 found that carrier ampholytes with a pH range 4.5-5.4, combined with BES, allowed sufficient resolution on IEF under equilibrium conditions. Recently, Weidinger and Henke [81 reported the use of an agarose gel with a p H 4.5-5.4 range of carrier ampholyte. However, focusing by these procedures took more than 95 min [ 7,8]. CAM-IEF requires neither preparation of gels nor prefocusing, and completed the separation in only 25 min. Such a focusing time is much shorter than any previously reported for the separationofESD isozymes by IEF. When we
Figure 1. EAP isozyme patterns with various carrier ampholytes on CAM-IEF. Electrode distance, 6 cm; focusing time, 30 min, 550 Vh. (a) pH 5-6.5; (b) pH 5-8; (c) pH 5-8: pH 5-6.5, 1 5 .
Figure 2. Analysis of erythorocyte lysates and blood stains. (a)Lysates diluted 4 times with DTT, arrows showing broader bands; (b) lysates diluted 8 times with DTT; (c) blood stains stored for 8 months.
Figure 3. ESD isozyme patterns on CAMIEF. Electrode distance, 6 cm. (a) pH 5-6.5, 18 niin of focusing, 300 Vh; (b) pH 5-6.5 containing 2.5 %) EPPS, 18 min of focusing, 300 Vh; (c) pH 5-6.5 containing 2.5 % EPPS, 25 min of focusing, 450 Vh.
Haptoglobin subtyping by IEF under reducting conditions
Electrophoresis 1990, 11, 321-324
applied this technique to blood stains stored for various periods at room temperature, ESD 2- 1 up to 4 weeks old could be phenotyped. ESD 1 and ESD 2 up to 10 weeks old could also be separated sufficiently. The detection limit in analysis was similar to that in results described by Diva1 [ 161.
4 Concluding remarks We developed amethod for faster separation of EAP and ESD phenotypes by CAM-IEF. Adequate resolution was obtained after 30 min offocusing for EAP typing and 25 min offocusing for ESD typing. Such a short focusing time was possible owing to the nonrestrictive properties of CAMS as opposed to agarose or polyacrylamide gels (Nishimura, T., Fuji Film, Tokyo, pesonal communication). In addition, CAM-IEF affords the advantage of operational simplicity without preparing gels. CAM-IEF is thus useful for the routine work of EAP and ESD phenotyping in forensic investigations.
The authors thank Dr. K . Suzuki, Department of Legal Medicine, Osaka Medical University of Medical Science, for helpful suggestions and critical reading of the manuscript.
32 1
5 References Hopkinson, D., Spencer, W. and Harris, H., Nature 1963, 199, 969-97 1. Destro-Bisol, G. and Ranalletta, D., Electrophoresis 1988. 9, 106- 108. Hopkinson, D., Mestriner, M., Corntner, J. and Harris, H., Ann. Hum. Genet. 1973,37, 119-137. Olaisen, B., Siverts, A., Jonassen, R., Mevag, B. and Gedde-Dahl, T., Hum. Genet. 1981,57,351-353. Nishigaki, I. and Itoh, T., Hum. Genet. 1984,66, 92-95. Yuasa, I., Tamaki, N., Suenaga, K., Itoh, K.,lnoue,T.andOkada,K., Electrophoresis 1985,6,588-592. Budowle, B., Electrophoresis 1986, 7, 141-144. Weidinger, S. and Henke, J., Electrophoresis 1988,9,429-432. Toda, T., Sano-Shiba, K., Cho, H., Soon, P., Nakao, M. and Ohashi, M., Electrophoresis 1988, 9, 149-150. Budowle, B., Electrophoresis 1984,5,254-255. Randall, T., Harland, W. and Thorpe, J., Med. Sci. Law 1980, 20, 43-47. Divall, G., Forens. Sci. Znt. 1981,18, 67-78. Finney, S., Renshaw, N. and Werrett, D., Forens. Sci. Int. 1985,27, 237-245. Frank, W. and Stolorow, M., J. Forens. Sci. 1986,31, 1089- 1094. Gill, P. and Sutton, J., Electrophoresis 1985,6,23-26. Divall, G., Forens. Sci. Int. 1985,28, 277-285.
Received June 28, 1989; in revised form December 20, 1989
Antonio Alonso' Guillermo Visedo2 Manuel Sancho' Jose Fernandez-Piqueras2 'Instituto Nacional de Toxicologia, Madrid Wnidad de Genktica, Facultad de Ciencias, Universidad Autonoma de Madrid
Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamidegels rehydrated in presence of 2-mercaptoethanol A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 5 5 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the H p a and H p p chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp a allelic products (IS, IF, 2FS, 2SS and 2FF). A population study carried out with 250 unrelatedindividualsliving in Central Spain is also presented.
1 Introduction The haptoglobin (Hp) molecule, encoded by a locus on chromosome 16 ( 1 6 ~ 2 2 )i l l , is a polymorphic serum glycoprotein composed of two different polypeptide chains (a and 0) linked through disulfide bridges. The genetic polymorphism displayed by the Hp-a chain was first reported using Correspondence: Dr. Antonio Alonso, Seccion de Biologia, lnstituto Nacional de Toxicologia, C/Luis Cabrera 9, E-28002 Madrid, Spain Abbreviations: Hp, haptoglobin; IEF, isoelectric focusing; 2-ME, 2-mercaptoethanol; RS,reswelling solution; TEMED, N,N,N',N'-tetramethylethylenediamine
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
starch gel electrophoresis [ 21. This technique allowed the identification of three common phenotypes (Hpl, Hp2-1 Hp2). The existence of H p subtypes was first demonstrated using acidic starch gel electrophoresis in presence ofurea [3,41, and application of isoelectric focusing (IEF) to Hp subtyping further improved the separation of the different Hp-a allele products (IF, IS, 2FS, 2FF, 2SS) [Sl.At present, there are two methods available for large-scaleroutineHp subtyping: (i)partial purification, reductive cleavage and carboxymethylation of the H p molecule, followed by IEF with nonspecific protein staining [6] ;and (ii) desialylation and reductive cleavage ofthe H p molecule in whole serum, followed by IEF and immunoblotting [ 71. This article presents a fast Hp subtyping method based on IEF of Hp under reducting conditions, using dry 01 73-083519010404-0321%2.50/0
