A Discontinuous Buffer System for Paper Electrophoresis of Human Hemoglobins

A Discontinuous Buffer System for Paper Electrophoresis of Human Hemoglobins C. A. J. Goldberg NEW BUFFER SYSTEM has recently been introduced for the separation of proteins (1) and hemoglobins (2) on starch gel. It differs from the conventional barbital system most frequently used heretofore in that the buffer used for the supporting medium differs from that used in the buffer vessels. For this reason the buffer system is referred to as discontinuous. The discontinuous buffer system appears to be superior not only for electrophoretic separations using starch gel as a supporting medium (2) but for electrophoresis on paper as well. A method for paper electrophoresis of hemoglobins will be described, in which Tris-EDTA-borate buffer is used for the immersion of the paper and barbital buffer is used in the buffer vessels. MATERIALS AND METHODS Two types of electrophoresis equipment were used, one of the pressure plate type described elsewhere (3), and one featuring the suspension of the paper at its center over a glass rod (4). The procedure used with both types of apparatus was the same. REAGENTS 1. Tris-EDTA-boric acid buffer described by Aronsson and Gr#{246}nwall (5), diluted to give a final molarity of 0.12. It will be referred to as TEB buffer. 6.050 Gm. tris(hydroxymethyl)aminomethane (Sigma 121), 0.780 Gm. disodium ethylenediaminetetraacetate, dihydrate, reagent grade, and 0.460 Gm. boric acid, reagent grade, are Prom the William Pepper Laboratory of Clinical Medicine, University of Pennsylvania, Philadelphia, Pa. Received for publication March 21, 1959. 446

Vol. 5, No. 5, 1959 DISCONTINUOUS BUFFER SYSTEM 447 dissolved in double-distilled water. The volume is adjusted to 500 ml. The ph is 9.1. 2. Barbital buffer, ph 8.6, 0.058 M. 30.90 Gm. sodium barbital and 4.20 Gm. diethylbarbituric acid are dissolved in double-distilled water. The volume is adjusted to 3 liters. PROCEDURE The buffer vessels of the electrophoresis chamber are filled with barbital buffer. Paper strips appropriate for the type of apparatus used are immersed in the TEB buffer, blotted very lightly, and placed in the electrophoresis chamber. About 8 p.1. of buffered hemoglobin solution (3) are applied at the mid-point of the paper. Electrophoresis is carried out overnight, at 150 volts, corresponding to a current density of approximately 0.15 ma. per cm. of strip width. When electrophoresis is completed, the patterns are dried, either at room temperature or at 60#{176}. Quantitative evaluation of the patterns containing abnormal hemoglobins is carried out as described earlier (3) by densitometry and subsequent planimetry. The patterns showing hemoglobin A only are stained with Amido Black lob to expose the hemoglobin A2 component for inspection. EXPERIMENTAL Twelve hemolysates, 6 normal controls and 6 containing respectively A-A2 (thalassemia intermedia), A-S, S-S, A-C, S-C, and C-C, were used to establish the optimal concentration of the TEB buffer. The following molarities were investigated: 0.03, 0.06, 0.12, and 0.24. The concentration of the barbital buffer was not altered. In a buffer system containing a TEB concentration of 0.24 M. the patterns were short with little displacement between the hemoglobins. With TEB concentrations of 0.12 M. and less, satisfactory total migration of the hemoglobins was achieved. The resolution was found to be proportional to the concentration of the buffer. The distance between the various hemoglobins was very small in the buffer of the lowest molarity and increased with increasing concentration of the buffer. Optimal resolution and total migration was obtained in TEB buffer of 0.12 M. concentration (Fig. 1). Patterns 1 and 3 were obtamed with the conventional continuous barbital buffer system, patterns 2 and 4 to 8 were obtained with the discontinuous buffer system described above. Patterns 1 and 2 were obtained from the same hemolysate. Pattern 1 illustrates how the hemoglobin A2 component

448 GOLDBERG Clinical Chemistry Fig. 1. Paper eleetrophoresis of human hemoglobins in a continuous barbital buffer system (as shown in patterns 1 and 3) and in a discontinuous buffer system of Tris- EDTA-borate (TEB) and barbital (see text) as shown in patterns 2 and 4 to 8. The migration of the hemoglobins is anodic in both systems. The site of sample application is at the top of the patterns, and is visible in patterns 1 and 3. The sample of hemoglobin H trait was received through the courtesy of Dr. D. A. Rigas, University of Oregon Medical School, Portland, Ore. may be completely unresolved when small sample sizes are used in the conventional barbital system. There is, however, excellent resolution between hemoglobins A and A2 in the discontinuous system (patterns 2, 7 and 8), making this buffer system particularly useful for the detection of the hemoglobin A2 component. An interesting phenomenon is the effect of the TEB-barbital system on the electrophoretic patterns of aged hemoglobin samples. It has not been possible in our experience to obtain good resolution of hemoglobins by paper electrophoresis in a continuous barbital system if the hemoglobins are more than 1 month old. The sample used for patterns 3 and 4, containing hemoglobins A and H, had been frozen and thawed twice. It was 1 month old at the time of the experiment.* Again, there is a striking difference between the pat- *preezing and thawing of this sample for a third time resulted in complete denaturation of the abnormal hemoglobin. Rigas et at. (11) observed that almost complete denaturation and precipitation of hemoglobin H occurred in aqueous hemolysates upon freezing and thawing once.

Vol. 5, No. 5, 1959 DISCONTINUOUS BUFFER SYSTEM 449 tern obtained with a continuous barbital buffer system and that obtained with the discontinuous buffer system. The hemoglobin sample shown in pattern 6, containing hemoglobins A and C, was 2#{189} months old. It was frozen and thawed twice and had been refrigerated for 14 days. Pattern 8 shows the increase in hemoglobin A2 associated with the presence of thalassemia. Here (the sample used for this pattern was 1 month old) hemoglobin A2 migrates more nearly like hemoglobin C, whereas in fresh samples it has a slightly more rapid migration rate (7, 8). A group of 18 hemolysates, obtained from patients heterozygous for an abnormal hemoglobin, was studied by electrophoresis on paper and starch gel. The results obtained with both supporting media are listed in Table 1. DISCUSSION From the results in Table 1, it appears that there is very little interaction between hemoglobin and the paper in the presence of TEB Table 1. Paper eleetrophorecie Starch gel electrophoreaie Sample %HbA %HbS %HbC %Hb 4. %HbS %MbO %HbA2 Di8continuoua TEB-barbito2 aojetem Discontinuous TEB-arbiia1 syetem F.F. 54 46 55 41 3.7.T. 61 39 58 40 2.3 W.Z. 53 47 55 42 3.2 E.W. 64 36 63 35 2.4 B.B. 63 37 64 34 2.4 G.E. 55 45 54 42 3.5 H.C. 63 37 60 36 3.8 P.L. 62 38 63 33 3.9 R.L.*+ 83 17 79 17 4.5 M.W.* 66 34 63 32 5.0 H.J.* 62 38 66 30 4.0 Contintsot,s barbitaz syetem R.w. 57 43 65 31 4.0 5.K. 25 39 36 33 37 30 M.D. 55 45 65 35 P.L.4 49 51 63 33 3.9 R.L.*+ 65 35 79 17 4.5 M.W.* 54 46 63 32 5.0 H.J.* 57 43 66 30 4.0 Same patients studied by both methods. +After multiple transfusions.

450 GOLDBERG Clinical Chemistry buffer ions. As a result, close agreement exists between the hemoglobin values obtained on paper and on starch gel. On the other hand, the marked interaction between protein and cellulose fibers in the presence of barbital ions is responsible for the formation of a trail of the more rapidly moving hemoglobin, resulting in falsely low values for this component and correspondingly increased values for the abnormal hemoglobins which migrate more slowly (Table 1). Especially in the presence of an amount of abnormal hemoglobin approaching 50 per cent, densitometric values obtained from barbital patterns may give the misleading impression that the abnormal hemoglobin is present in an amount exceeding 50 per cent. This might lead to an erroneous assumption of the presence of thalassemia. The true values for abnormal hemoglobins obtained in the TEB-barbital buffer system avoid pitfalls of this kind. The discontinuous buffer system described was found to be supei-ior not only to a continuous barbital buffer system but to a continuous Tris-EDTA-borate buffer system of 0.12 M. as well. Use of the latter resulted in patterns of equally good resolution, but diffusion and electroendosmotic flow were markedly increased. The superior electrophoretic separation of hemoglobins in the discontinuous buffer system probably is due largely to the low conductivity of the TEB buffer. As we have pointed out previously (3), the displacement between 2 different hemoglobins during electrophoresis is inversely proportional to current density. The potential required to achieve a comparable distance of migration in a continuous barbital system is twice that used in the discontinuous buffer system, and the current density in the barbital system at that potential is 4 times as high as that encountered in the discontinuous buffer system. However, other factors besides conductivity and decreased interaction between protein and cellulose appear to be involved as well. As may be seen from pattern 7 (Fig. 1), hemoglobin S migrates slightly faster than midway between hemoglobins A and C. The slightly faster migration rate of hemoglobin S may be further increased by using a TEB buffer of 0.06 M. If the migration of hemoglobin S depended solely on the current effect of the discontinuous buffer system, it would be expected to migrate halfway between hemoglobins A and C as befits its isoelectric point (9, 10). This indicates that buffer-ion displacement and perhaps the p11 gradient between the buffer boundaries may play roles as well.

Vol. 5, No. 5, 1959 DISCONTINUOUS BUFFER SYSTEM 451 SUMMARY A method for paper electrophoresis of hemoglobins in a discontinuous buffer system is presented. Tris-EDTA-borate buffer 0.12 M., ph 9.1, is applied to the paper. Barbital buffer 0.06 M., ph 8.6, is used in the buffer vessels. The discontinuous buffer system enables the detection of hemoglobin A2 in small samples of hemolysate. Resolution of abnormal hemoglobins is superior to that in barbital or Tris-EDTA-borate buffer alone. It offers special advantage for the examination of aged samples of hemoglobin. Reasons for the behavior of the hemoglobins in this buffer system are discussed. 1. Poulik, M. D., Nature 180, 1477 (1957). REFERENCES 2. Goldberg, C. A. J., Clin. Chem. 4, 484 (1958). 3. Goldberg, C. A. J., Clin. Chem. 3, 1 (1957). 4. Durrum, E. L., J. Am. Chem. Soc. 72, 2943 (1950). 5. Aronseon, T., and Gr#{246}nwall, A., J. Lab. Clin. Invest. 9, 338 (1957). 6. von Prijtag Drabbc, C. A. J., and Reinhold, J. G., Ana& Chern. 27, 1090 (1955). 7. Goldberg, C. A. J. Unpublished data. 8. Ku#{241}kel,H. G., Ceppellini, B., Mtiller-Eberhard, U., and Wolf, J., J. Clin. Invest. 36, 1615 (1957). 9. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C., Science 110, 543 (1949). 10. Itano, H. A., Science 117, 89 (1953). 11. Rigas, D. A., Koler, B. D., and Osgood, E. E., J. Lob. 4. CUn. Med. 47, 51 (1956).