ANALYTICAL BIOCHEMISTRY 93, 261-266 (1979)
Combined Immunoabsorption and Isoelectric Focusing of Barley and Malt Amylases in Polyacrylamide Gel t J. DAUSSANT AND A. W. MACGREGOR2 Laboratoire de Physiologie des Organes Vkgktaux, C.N.R.S., 4 ter route des Gardes, 92190 Meudon, France Received July 5, 1978 A technique combining immunoabsorption, isoelectric focusing, and enzymatic characterization in the same polyacrylamide gel is described. The a- and/3-amylases from barley seeds, an immune serum induced in rabbits by barley malt a-amylase, the immunoglobulin G (IgG) of the immune serum, and the IgG purified from a nonimmunized animal were used. The application of this technique in physiological and genetical studies to the identification of amylolytic enzymes which cannot be distinguished by existing chromogenic reactions and which have similar isoelectric points is discussed.
Ontogenical changes in o~-and/3-amylases as well as the genetic variation of these enzymes in cereals have been the object of several electrophoretic studies [(1) for review]. Isoelectric focusing, a very sensitive technique for detecting small charge differences in enzymes, has also been used successfully for such investigations (2,3). Nevertheless, because of the large number of amylases extracted from germinated cereal grains and detected by isoelectric focusing it is still difficult to distinguish between a- and r-amylase constituents. A specific substrate such as amylopectin r-limit dextrin may be used to detect oz-amylases but there is no specific substrate available for /3-amylases. In addition, several t~- and B-amylase constituents may have very similar isoelectric points and so some important B-amylase constituents may not be identified after isoelectric focusing. During studies on the ontogenical evolution of a- and r-amylases in barley seeds the possibility of adapting to isoelectric focusing Paper No. 399 of the Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada. 2 Present address: Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada R3C 3G9.
analysis the immunoabsorption technique described for electrophoresis in agarose gel (4) was investigated. The aim of the technique was to specifically eliminate all ~amylases in malted barley without disturbing the r-amylase pattern.
MATERIALS AND METHODS The six-rowed barley cultivar, Conquest, was used in this study. Malt was prepared from Conquest barley as described previously (5). One gram of seed flour was ground at room temperature with 6 ml of 0.05 M sodium barbital buffer, pH 8.6, containing 0.2 M NaC1 and 0.001 M CaC12 and maintained in contact for 30 min. The extracts were then centrifuged for 30 min at 30,000 rpm and dialyzed for 1 h against the extracting buffer diluted twice but containing no NaC1. An immune serum induced in rabbits with o~-amylases purified from germinated barley seeds was used in this study (6). The immunoglobulin fractions (IgG) 3 from the immune serum and from a serum of a nonimmunized rabbit were prepared by amAbbreviation used: IgG, immunoglobulin G.
261
0003-2697/79/040261-06502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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DAUSSANT AND MACGREGOR
monium sulfate precipitation and DEAEcellulose chromatography (7) using a 0.0175 M potassium phosphate buffer, pH 6.55. The IgG fractions contained 2 mg protein/ml. The antigen-binding capacity of the IgG fraction was compared with that of the immune serum by line immunoelectrophoresis (8). The IgG solution was equivalent to the immune serum diluted fourfold. Sample deposition. For isoelectric focusing, pieces of Whatman No. 1 filter paper (7 x 10 mm) were inbibed with the sample solutions and deposited on the gel. For immunoabsorption, a large paper (10 x 12 mm) was imbibed with the IgG solutions or with immune serum and deposited on the gel. A second paper (7 x 10 mm) imbibed with the sample solution was then put on top of the first one and electrophoresis was started immediately thereafter. The small papers contained 12/zl of solution and the larger papers, 20/zl. Isoelectric focusing was performed according to conditions described earlier (3). The LKB Multiphor system was used for isoelectric focusing experiments. The gel mixture, containing acrylamide (10 ml, 40%), N,N'-methylenebisacrylamide (10 ml, 0.9%), Ampholine (3 ml, pH 3-10), and sucrose (7.5 g in 37 ml of water), was deaerated, mixed with riboflavin (1.4 ml, 0.004%), sandwiched between two glass plates (125 x 260 mm), and polymerized overnight under a fluorescent lamp. The electrophoresis was carried out in a cold room (4°C) with water at 0°C used as refrigerant. a-Amylase characterization was carried out as described previously (9) but the concentration of dextrin used was increased two-fold. An identical technique using starch (Merk) was used to detect both a- and r-amylase enzymes. RESULTS AND DISCUSSION
The Technique Results presented in Fig. 1 show that the double paper procedure used for the
combined immunoabsorption and isoelectric focusing technique did not disturb the zymogram of barley malt a-amylase (compare Figs. la and lb). The presence of IgG from a nonimmunized animal also did not interfere with the a-amylase pattern (compare Figs. la and lc). However, the use of IgG of the immune serum resulted in the complete disappearance of all t~-amylase constituents (Fig. ld). Results indicate that under these conditions the electroimmunoabsorption was efficient and due only to the antigen-antibody reaction. Preliminary experiments were carried out to determine the a-amylase absorbing capacity of the lgG solution of the immune serum. With the undiluted malt extract (containing 25,000 IDC units of a-amylase/ml) the absorption was incomplete resulting in a zymogram on which some bands indeed disappeared but on which most of them remained detectable but with reduced intensity. However, complete absorption of all a-amylases was obtained with the malt extract diluted threefold. r-Amylase is known to exist under different forms of association (10) or to associate with other proteins (11). It was thus necessary to investigate whether the conditions of the immunoabsorption altered the isoelectric focusing pattern of r-amylase. An extract of mature barley containing no a-amylase was used. Results reported in Fig. 2 indicate that neither the double filterpaper procedure nor the IgG purified from the immune serum modified the /3-amylase pattern (compare Figs. 2a with b and c). In previous experiments using electroimmunoabsorption in agarose gel (4) no interference was observed between the seric proteins of whole immune serum and/3-amylase. Thus, the first experiments with isoelectric focusing were carried out with whole immune serum. However, with the double filter paper technique, the seric proteins caused changes in the/3-amylase pattern. This is visible in Fig. 2d. For this experiment, immune serum diluted fourfold was used [the diluted immune
IMMUNOABSORPTION-ISOELECTROFOCUSING
a
b
c
263
d pHlO
m
pH 3
-I-
FIG. 1. Efficiency and specificity of combined immunoabsorption and isoelectric focusing of barley malt a-amylases. Isoelectric focusing of barley malt proteins and a-amylase characterization (using B-limit dextrin as substrate). Filter papers impregnated with the malt extract diluted threefold were deposited before electrophoresis on the polyacrylamide gel under the following conditions (arrow indicates the point of sample application): (a) directly on the gel; (b) on top of a paper previously soaked in the phosphate buffer solution with which the IgG were prepared; (c) on top of a filter paper previously soaked in the IgG solution purified from the serum o f a nonimmunized animal; (d) on top of a filter paper previously soaked in the IgG solution purified from the immune serum. Note that (b) and (c) do not change the zymogram of barley malt a-amylase shown in (a) and that (d) results in the complete disappearance of a-amylase constituents.
serum, according to results obtained with line immunoelectrophoresis (8), had the same antigen-binding capacity as the IgG solution]. This change could be duplicated by soaking the first filter paper in a solution of bovine serum albumin at a concentration (20 mg/ml) similar to that of the total protein content of fourfold diluted serum (Fig. 2e). This latter result suggests that the albumin fraction of the serum may play the major role in this pattern modification. The technique was also successful with another pH range (4 to 8). Moreover, it was
possible to use two filter papers soaked in the IgG solution instead of one, without modifying the /3-amylase pattern. To sum up, although it is necessary to use the IgG preparation and not the whole immune serum, the immunoabsorption by itself involving a double filter paper application on the gel is easily carried out and the consumption of the IgG solution is small (about 20/zl).
Application o~-Amylases are easily identified in the presence of/3-amylases by using the specific
264
DAUSSANT AND MACGREGOR
substrate amylopectin B-limit dextrin (Fig. 3A, a). However, B-amylase bands are difficult to identify in the presence of a-amylases because of the occurrence of a large number of bands in the same area of the pattern (Fig. 3B, a) and because some /3amylase constituents are hidden by a-amylase constituents (Fig. 3B, a and a'). The immunoabsorption, which removed all aamylases from the pattern (Fig. 3A, a') enabled a clear-cut pattern for B-amylases to appear (Fig. 3B, a'). Thus, the results show clearly the advantage of combining the specificity of the immunoabsorption with the high resolving power of isoelectric focusing in studies on B-amylases when a-
a m
-I-
b
c
amylases are present. So, it became possible to detect great differences in the B-amylases extracted from barley and from malt: Germination results in disappearance of constituents in the electrophoretogram parts corresponding to acidic pH and occurrence or increase in amounts of other constituents in parts of the electrophoretogram corresponding to more basic pH. In preliminary experiments carried out with extracts of developing barley seeds, it was possible to absorb completely the a-amylase activity with the IgG purified from the immune serum used in this study. Although antigenical differences were reported between a-amylases of developing and germinated barley seeds (12)
d
e pHlO
pH 3
FIG. 2. Interference of the immunoabsorption procedure with the isoelectric focusing of B-amylase. Isoelectric focusing of barley proteins followed by a- and B-amylase characterization (using starch as substrate). Filter papers impregnated with the barley extract were deposited before electrophoresis on the polyacrylamide gel under the following conditions (arrow indicates the point of sample application): (a) directly on the gel; (b) on top of a filter paper previously soaked in the phosphate buffer solution in which the IgG were prepared; (c) on top of a filter paper previously soaked in the IgG solution of the immune serum; (d) on top of a filter paper previously soaked in the immune serum diluted fourfold; (e) on top of a filter paper previously soaked in a serum albumin solution (20 mg/ml). Note that the/3-amylase pattern of the barley extract (a) is not altered by the double paper procedure (b) nor by the presence of the IgG of the immune serum (c). However, the immune serum diluted fourfold (d) and a bovine serum albumin solution at 20 mg/ml (e) did modify the zymogram.
265
IMMUNOABSORPTION-ISOELECTROFOCUSING
a
a"
a
b
a" pHlO
pH3
-IA
B
FIG. 3. Application of the electroimmunoabsorption technique to the isoelectric focusing of/3-amylases in malt extracts where a- and r-amylases are present: (A) t~-amylase characterization after isoelectric focusing (substrate: r-limit dextrin); (B) a- and r-amylase characterization after isoelectric focusing (substrate: starch). Before electrophoresis the following samples were applied to the gel (arrows indicate the points of sample application); (a) filter paper soaked in the threefold diluted malt extract and directly applied to the gel; (a') as for (a) and applied to the top of a filter paper impregnated with the IgG solution purified from anti c~-amylase immune serum; (b) filter paper soaked in the barley extract and directly applied to the gel. Note that by comparing Fig. 3A (a) and 3B (a) it is impossible to identify malt r-amylases in the area shown by the bracket. It is worth recalling that identical amounts of malt extract were deposited on both parts of the electrophoretogram and assayed for c~-amylase only in 3A (a), for a- and r-amylases in 3B (a). The immunoabsorption, however, which absorbs all c~-amylase (Aa') provides a clear-cut pattern for r-amylase (Ba').
the results indicate that there are, nevertheless, some antigenical relationships between them. So, the technique and this immune serum will be useful in studies concerning the evolution of fi-amylases during grain development and germination since a-amylases are present during both physiological stages. Furthermore there are antigenic relationships between c~-amylases of different cereals (13,14). Therefore the use of the technique for physiological studies on fi-amylases from several cereals is probably possible by using the immune serum of only
one of these cereals thus sparing the necessity of preparing a specific immune serum for each cereal to be studied (6). In genetic studies, B-amylase may be considered as a varietal marker in some cereals (1). During germination, however, the enzymes undergo drastic changes which result in modification of the whole electrophoretic pattern as shown in Fig. 3. It then becomes questionable whether the enzyme constituents of germinated seeds could further serve as genetic markers. If the modified B-amylases still retain characteristics of
266
DAUSSANT AND MACGREGOR
varietal origin, the test could then be used for contributing to check the varietal origin of malts. This technique, by eliminating completely a-amylases without modifying the isoelectric focusing pattern of/3-amylases, provides a convenient tool for such studies. ACKNOWLEDGMENTS The authors thank Miss C. Mayer and Mrs. D. Bureau for technical assistance. This study was supported in part by the Union G6n6rale de la Brasserie Fran~aise.
REFERENCES 1. Scandalios, J. G. (1974) Annu. Rev. Plant Physiol. 25, 225. 2. Kruger, J. E. (1976)Cereal Res. Commun. 4, 187. 3. MacGregor, A. W. (1976) Cereal Chem. 53, 792.
4. Daussant, J., and Carfantan, N. (1975)J. lmmunol. Methods 8, 373. 5. Bettner, R. E., Meredith, W. O. S., and Andersen, J. A. (1962)Amer. Soc. Brew. Chem. Proc. 5. 6. Daussant, J. (1978)Ann. Immunol. (Inst. Pasteur) 129e, 215. 7. Williams, C. A., and Chase, M. W. (1967) in Methods in Immunology and Immunochemistry, Vol. 1, p. 321, Academic Press, New York. 8. Kr¢ll, J. (1973) Scand. J. Immunol. 2 (suppl. 1), 61. 9. MacGregor, A. W., Thompson, R. G., and Meredith, W. O. S. (1974) J. Inst. Brew. 80, 181. 10. Niku-Paavola, M. L., Skakoun, A., Nummi, M., and Daussant, J. (1973)Biochim. Biophys. Aeta 322, 181. 11. Heigaard, J., and Carlsen, S. (1977)J. Sci. Food Agr. 28, 900. 12. Daussant, J., Skakoun, A., and Niku-Paavola, M. L. (1974)J. Inst. Brew. 80, 55. 13. Daussant, J., and Grabar, P. (1966) Ann. Inst. Pasteur 110, 79. 14. Alexandrescu, V., and Mihailescu, F. (1973) Rev. Roum. Biochim. 10, 89.
Combined Immunoabsorption and Isoelectric Focusing of Barley and Malt Amylases in Polyacrylamide Gel t J. DAUSSANT AND A. W. MACGREGOR2 Laboratoire de Physiologie des Organes Vkgktaux, C.N.R.S., 4 ter route des Gardes, 92190 Meudon, France Received July 5, 1978 A technique combining immunoabsorption, isoelectric focusing, and enzymatic characterization in the same polyacrylamide gel is described. The a- and/3-amylases from barley seeds, an immune serum induced in rabbits by barley malt a-amylase, the immunoglobulin G (IgG) of the immune serum, and the IgG purified from a nonimmunized animal were used. The application of this technique in physiological and genetical studies to the identification of amylolytic enzymes which cannot be distinguished by existing chromogenic reactions and which have similar isoelectric points is discussed.
Ontogenical changes in o~-and/3-amylases as well as the genetic variation of these enzymes in cereals have been the object of several electrophoretic studies [(1) for review]. Isoelectric focusing, a very sensitive technique for detecting small charge differences in enzymes, has also been used successfully for such investigations (2,3). Nevertheless, because of the large number of amylases extracted from germinated cereal grains and detected by isoelectric focusing it is still difficult to distinguish between a- and r-amylase constituents. A specific substrate such as amylopectin r-limit dextrin may be used to detect oz-amylases but there is no specific substrate available for /3-amylases. In addition, several t~- and B-amylase constituents may have very similar isoelectric points and so some important B-amylase constituents may not be identified after isoelectric focusing. During studies on the ontogenical evolution of a- and r-amylases in barley seeds the possibility of adapting to isoelectric focusing Paper No. 399 of the Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada. 2 Present address: Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada R3C 3G9.
analysis the immunoabsorption technique described for electrophoresis in agarose gel (4) was investigated. The aim of the technique was to specifically eliminate all ~amylases in malted barley without disturbing the r-amylase pattern.
MATERIALS AND METHODS The six-rowed barley cultivar, Conquest, was used in this study. Malt was prepared from Conquest barley as described previously (5). One gram of seed flour was ground at room temperature with 6 ml of 0.05 M sodium barbital buffer, pH 8.6, containing 0.2 M NaC1 and 0.001 M CaC12 and maintained in contact for 30 min. The extracts were then centrifuged for 30 min at 30,000 rpm and dialyzed for 1 h against the extracting buffer diluted twice but containing no NaC1. An immune serum induced in rabbits with o~-amylases purified from germinated barley seeds was used in this study (6). The immunoglobulin fractions (IgG) 3 from the immune serum and from a serum of a nonimmunized rabbit were prepared by amAbbreviation used: IgG, immunoglobulin G.
261
0003-2697/79/040261-06502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
262
DAUSSANT AND MACGREGOR
monium sulfate precipitation and DEAEcellulose chromatography (7) using a 0.0175 M potassium phosphate buffer, pH 6.55. The IgG fractions contained 2 mg protein/ml. The antigen-binding capacity of the IgG fraction was compared with that of the immune serum by line immunoelectrophoresis (8). The IgG solution was equivalent to the immune serum diluted fourfold. Sample deposition. For isoelectric focusing, pieces of Whatman No. 1 filter paper (7 x 10 mm) were inbibed with the sample solutions and deposited on the gel. For immunoabsorption, a large paper (10 x 12 mm) was imbibed with the IgG solutions or with immune serum and deposited on the gel. A second paper (7 x 10 mm) imbibed with the sample solution was then put on top of the first one and electrophoresis was started immediately thereafter. The small papers contained 12/zl of solution and the larger papers, 20/zl. Isoelectric focusing was performed according to conditions described earlier (3). The LKB Multiphor system was used for isoelectric focusing experiments. The gel mixture, containing acrylamide (10 ml, 40%), N,N'-methylenebisacrylamide (10 ml, 0.9%), Ampholine (3 ml, pH 3-10), and sucrose (7.5 g in 37 ml of water), was deaerated, mixed with riboflavin (1.4 ml, 0.004%), sandwiched between two glass plates (125 x 260 mm), and polymerized overnight under a fluorescent lamp. The electrophoresis was carried out in a cold room (4°C) with water at 0°C used as refrigerant. a-Amylase characterization was carried out as described previously (9) but the concentration of dextrin used was increased two-fold. An identical technique using starch (Merk) was used to detect both a- and r-amylase enzymes. RESULTS AND DISCUSSION
The Technique Results presented in Fig. 1 show that the double paper procedure used for the
combined immunoabsorption and isoelectric focusing technique did not disturb the zymogram of barley malt a-amylase (compare Figs. la and lb). The presence of IgG from a nonimmunized animal also did not interfere with the a-amylase pattern (compare Figs. la and lc). However, the use of IgG of the immune serum resulted in the complete disappearance of all t~-amylase constituents (Fig. ld). Results indicate that under these conditions the electroimmunoabsorption was efficient and due only to the antigen-antibody reaction. Preliminary experiments were carried out to determine the a-amylase absorbing capacity of the lgG solution of the immune serum. With the undiluted malt extract (containing 25,000 IDC units of a-amylase/ml) the absorption was incomplete resulting in a zymogram on which some bands indeed disappeared but on which most of them remained detectable but with reduced intensity. However, complete absorption of all a-amylases was obtained with the malt extract diluted threefold. r-Amylase is known to exist under different forms of association (10) or to associate with other proteins (11). It was thus necessary to investigate whether the conditions of the immunoabsorption altered the isoelectric focusing pattern of r-amylase. An extract of mature barley containing no a-amylase was used. Results reported in Fig. 2 indicate that neither the double filterpaper procedure nor the IgG purified from the immune serum modified the /3-amylase pattern (compare Figs. 2a with b and c). In previous experiments using electroimmunoabsorption in agarose gel (4) no interference was observed between the seric proteins of whole immune serum and/3-amylase. Thus, the first experiments with isoelectric focusing were carried out with whole immune serum. However, with the double filter paper technique, the seric proteins caused changes in the/3-amylase pattern. This is visible in Fig. 2d. For this experiment, immune serum diluted fourfold was used [the diluted immune
IMMUNOABSORPTION-ISOELECTROFOCUSING
a
b
c
263
d pHlO
m
pH 3
-I-
FIG. 1. Efficiency and specificity of combined immunoabsorption and isoelectric focusing of barley malt a-amylases. Isoelectric focusing of barley malt proteins and a-amylase characterization (using B-limit dextrin as substrate). Filter papers impregnated with the malt extract diluted threefold were deposited before electrophoresis on the polyacrylamide gel under the following conditions (arrow indicates the point of sample application): (a) directly on the gel; (b) on top of a paper previously soaked in the phosphate buffer solution with which the IgG were prepared; (c) on top of a filter paper previously soaked in the IgG solution purified from the serum o f a nonimmunized animal; (d) on top of a filter paper previously soaked in the IgG solution purified from the immune serum. Note that (b) and (c) do not change the zymogram of barley malt a-amylase shown in (a) and that (d) results in the complete disappearance of a-amylase constituents.
serum, according to results obtained with line immunoelectrophoresis (8), had the same antigen-binding capacity as the IgG solution]. This change could be duplicated by soaking the first filter paper in a solution of bovine serum albumin at a concentration (20 mg/ml) similar to that of the total protein content of fourfold diluted serum (Fig. 2e). This latter result suggests that the albumin fraction of the serum may play the major role in this pattern modification. The technique was also successful with another pH range (4 to 8). Moreover, it was
possible to use two filter papers soaked in the IgG solution instead of one, without modifying the /3-amylase pattern. To sum up, although it is necessary to use the IgG preparation and not the whole immune serum, the immunoabsorption by itself involving a double filter paper application on the gel is easily carried out and the consumption of the IgG solution is small (about 20/zl).
Application o~-Amylases are easily identified in the presence of/3-amylases by using the specific
264
DAUSSANT AND MACGREGOR
substrate amylopectin B-limit dextrin (Fig. 3A, a). However, B-amylase bands are difficult to identify in the presence of a-amylases because of the occurrence of a large number of bands in the same area of the pattern (Fig. 3B, a) and because some /3amylase constituents are hidden by a-amylase constituents (Fig. 3B, a and a'). The immunoabsorption, which removed all aamylases from the pattern (Fig. 3A, a') enabled a clear-cut pattern for B-amylases to appear (Fig. 3B, a'). Thus, the results show clearly the advantage of combining the specificity of the immunoabsorption with the high resolving power of isoelectric focusing in studies on B-amylases when a-
a m
-I-
b
c
amylases are present. So, it became possible to detect great differences in the B-amylases extracted from barley and from malt: Germination results in disappearance of constituents in the electrophoretogram parts corresponding to acidic pH and occurrence or increase in amounts of other constituents in parts of the electrophoretogram corresponding to more basic pH. In preliminary experiments carried out with extracts of developing barley seeds, it was possible to absorb completely the a-amylase activity with the IgG purified from the immune serum used in this study. Although antigenical differences were reported between a-amylases of developing and germinated barley seeds (12)
d
e pHlO
pH 3
FIG. 2. Interference of the immunoabsorption procedure with the isoelectric focusing of B-amylase. Isoelectric focusing of barley proteins followed by a- and B-amylase characterization (using starch as substrate). Filter papers impregnated with the barley extract were deposited before electrophoresis on the polyacrylamide gel under the following conditions (arrow indicates the point of sample application): (a) directly on the gel; (b) on top of a filter paper previously soaked in the phosphate buffer solution in which the IgG were prepared; (c) on top of a filter paper previously soaked in the IgG solution of the immune serum; (d) on top of a filter paper previously soaked in the immune serum diluted fourfold; (e) on top of a filter paper previously soaked in a serum albumin solution (20 mg/ml). Note that the/3-amylase pattern of the barley extract (a) is not altered by the double paper procedure (b) nor by the presence of the IgG of the immune serum (c). However, the immune serum diluted fourfold (d) and a bovine serum albumin solution at 20 mg/ml (e) did modify the zymogram.
265
IMMUNOABSORPTION-ISOELECTROFOCUSING
a
a"
a
b
a" pHlO
pH3
-IA
B
FIG. 3. Application of the electroimmunoabsorption technique to the isoelectric focusing of/3-amylases in malt extracts where a- and r-amylases are present: (A) t~-amylase characterization after isoelectric focusing (substrate: r-limit dextrin); (B) a- and r-amylase characterization after isoelectric focusing (substrate: starch). Before electrophoresis the following samples were applied to the gel (arrows indicate the points of sample application); (a) filter paper soaked in the threefold diluted malt extract and directly applied to the gel; (a') as for (a) and applied to the top of a filter paper impregnated with the IgG solution purified from anti c~-amylase immune serum; (b) filter paper soaked in the barley extract and directly applied to the gel. Note that by comparing Fig. 3A (a) and 3B (a) it is impossible to identify malt r-amylases in the area shown by the bracket. It is worth recalling that identical amounts of malt extract were deposited on both parts of the electrophoretogram and assayed for c~-amylase only in 3A (a), for a- and r-amylases in 3B (a). The immunoabsorption, however, which absorbs all c~-amylase (Aa') provides a clear-cut pattern for r-amylase (Ba').
the results indicate that there are, nevertheless, some antigenical relationships between them. So, the technique and this immune serum will be useful in studies concerning the evolution of fi-amylases during grain development and germination since a-amylases are present during both physiological stages. Furthermore there are antigenic relationships between c~-amylases of different cereals (13,14). Therefore the use of the technique for physiological studies on fi-amylases from several cereals is probably possible by using the immune serum of only
one of these cereals thus sparing the necessity of preparing a specific immune serum for each cereal to be studied (6). In genetic studies, B-amylase may be considered as a varietal marker in some cereals (1). During germination, however, the enzymes undergo drastic changes which result in modification of the whole electrophoretic pattern as shown in Fig. 3. It then becomes questionable whether the enzyme constituents of germinated seeds could further serve as genetic markers. If the modified B-amylases still retain characteristics of
266
DAUSSANT AND MACGREGOR
varietal origin, the test could then be used for contributing to check the varietal origin of malts. This technique, by eliminating completely a-amylases without modifying the isoelectric focusing pattern of/3-amylases, provides a convenient tool for such studies. ACKNOWLEDGMENTS The authors thank Miss C. Mayer and Mrs. D. Bureau for technical assistance. This study was supported in part by the Union G6n6rale de la Brasserie Fran~aise.
REFERENCES 1. Scandalios, J. G. (1974) Annu. Rev. Plant Physiol. 25, 225. 2. Kruger, J. E. (1976)Cereal Res. Commun. 4, 187. 3. MacGregor, A. W. (1976) Cereal Chem. 53, 792.
4. Daussant, J., and Carfantan, N. (1975)J. lmmunol. Methods 8, 373. 5. Bettner, R. E., Meredith, W. O. S., and Andersen, J. A. (1962)Amer. Soc. Brew. Chem. Proc. 5. 6. Daussant, J. (1978)Ann. Immunol. (Inst. Pasteur) 129e, 215. 7. Williams, C. A., and Chase, M. W. (1967) in Methods in Immunology and Immunochemistry, Vol. 1, p. 321, Academic Press, New York. 8. Kr¢ll, J. (1973) Scand. J. Immunol. 2 (suppl. 1), 61. 9. MacGregor, A. W., Thompson, R. G., and Meredith, W. O. S. (1974) J. Inst. Brew. 80, 181. 10. Niku-Paavola, M. L., Skakoun, A., Nummi, M., and Daussant, J. (1973)Biochim. Biophys. Aeta 322, 181. 11. Heigaard, J., and Carlsen, S. (1977)J. Sci. Food Agr. 28, 900. 12. Daussant, J., Skakoun, A., and Niku-Paavola, M. L. (1974)J. Inst. Brew. 80, 55. 13. Daussant, J., and Grabar, P. (1966) Ann. Inst. Pasteur 110, 79. 14. Alexandrescu, V., and Mihailescu, F. (1973) Rev. Roum. Biochim. 10, 89.
