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Wheat varietal identification by rapid ion-exchange chromatography of gliadins
Journal of Cereal Science 2 (1984) 241-248
Wheat Varietal Identification by Rapid Ion-exchange Chromatography of Gliadins I. L. BATEY
C.S./.R.O. Wheat Research Unit, Private Mail Bag, P.O., North Ryde, N.S.w. 2113, Australia Received 9 April 1984
A high performance liquid chromatography procedure, using anion exchange chromatography of the gliadin proteins at pH 10·4, has been devised as an alternative to gel electrophoresis for identification of wheat varieties. Varietal identification was, thus, possible in less than one hour. Sample preparation and extraction took about 30 min. The sample for chromatography could be prepared directly by extraction of flour with the starting buffer, indicating that a lengthy purification of the gliadin fraction is not necessary. Chromatography required approximately 20 min, and regeneration of the column was achieved in 5-10 min. Chromatographic separation of the gliadin proteins was lesseffective at pH values below 10·4. In initial experiments, 13 genotypes, including two varieties that are difficult to differentiate by electrophoresis, wereexamined and each was readily differentiated by the technique. Analysis of both purified gliadins and total flour extracts indicated that the results were independent of environmental factors.
Introduction Accurate and reliable identification of wheat varieties by wheat producers, traders and users is important for the maintenance and testing of wheat quality'. Currently, separation of the gliadin fraction of wheat by electrophoresis on either polyacrylamide or starch gels is the main laboratory method for varietal identification in most countries" It suffers from the disadvantage of being rather slow, labour intensive and reasonably expensive in the use of consumable materials. On the other hand, the apparatus required is cheap, simple and readily available, and the method has found ready acceptance because of its reliability. Column chromatography is an alternative method, by which proteins may be separated. Conventional chromatography is slow, requiring times ranging from several hours for size exclusion procedures to several days for some ion exchange separations. Recently, high performance liquid chromatography (HPLC) of proteins has become increasingly popular and the use of a variety of packing materials for columns has permitted more rapid separation of proteins, often from quite complex mixtures't-j. Fractionation of wheat proteins by reversed-phase HPLC has been reported, and good separations of the components of the gliadin fraction have been obtained", Varietal identification by this chromatographic technique has been proposed", and has been demonstrated recently". Abbreviations used: FPLC - fast prote in liquid chromatography; HPLC - high performance liquid chromatography ; TRIS - tris-(hydroxymethyl)-aminomethane. 0733-5210/84/040241 +08 $03.00/0
© 1984 Academic Press Inc. (London ) Limited
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A rapid ion-exchange chromatography system for proteins has also become available. This system, known as ' Fast Protein Liquid Chromatography' (FPLC) utilises cation or anion exchange columns , or a chromatofocusing column. The separation of proteins on these ion-exchange columns is claimed to occur solely on the basis of charge , and not to involve other interactions with the column support material. Such chromatographic methods, because of their speed and convenience, have a considerable potential as practicable alternatives to electrophoresis for grain varietal identification , especially as automatic sample loading and data handling techniques are available. The use of one of the FPLC anion exchange columns, the Mono-Q column, for the separation of the components of wheat gliadin is reported here and its applicability to varietal identification is discussed.
Experimental Materials Wheat of the soft variety, Egret, was grown at three locations, Moombooldool and Temora in southern New South Wales, and Dooen in Victoria. The hard wheat, Songlen, was grown at Narrabri, North Star and Myall Vale in northern New South Wales. Timgalen, another hard variety, was grown at North Star , Myall Vale and Moree. Other registered varieties tested were Durati (a durum wheat) and the hard wheat varieties Banks, Condor, Cook, Eagle, Gatcher, Halberd and Kite. Two breeders' lines were also examined. Of these varieties, some had been grown at a single location, while others comprised bulked samples grown at a number of sites. The protein content of the grain samples ranged from 10% to 17% on an 'as is' basis.
Sample extraction The gliadin fraction was isolated from flour by aqueous ethanol (70% , vIv) extraction after preliminary extraction of the water- and salt-soluble components with 10% (wIv) sodium chloride using the method of Baldo and Wrigley", The gliadin samples were prepared for chromatography by dispersing them in starting buffer (5 mg gliadin/ml), The samples were stirred for 16 h at room temperature (c. 20-25°C) and centrifuged at 8000 g. For direct extraction of gliadins from straight-run (76% extraction) flour, samples were extracted by stirring for 30 min at 20-25°C in starting buffer, using 100 mg flour/ml of buffer. The suspension was then centrifuged at 8000 g. Immediately prior to application to the column, each gliadin or flour extract was filtered through a 0·22 urn Millipore filter (type GS).
Apparatus Separation was effected on a Pharmacia Mono-Q anion exchange column (Pharmacia (South Seas) Pty Ltd, North Ryde, N.S.W.). Column dimensions were 50 x 5 mm. The solvent delivery system was a Milton-Roy single piston positive displacement pump , with a linear gradient being formed using two chambers ofa gradient maker from a Technicon AA-l amino acid analyser (Technicon Instruments, North Ryde, N.S.W.). The detector was a Pharmacia UV-l single path UV monitor with an 8 III flowcell. The wavelength for detection was 280 nm.
Chromatographic conditions The medium for chromatography was 1 M urea containing 0·01 M buffer with a linear gradient of 0-0·5 M Na acetate. Buffers used were as follows: pH 8'0, tris-(hydroxymethyI)-aminomethane (TRIS); pH 9,0, triethanolamine ; pH 9·5, 2-(cyclohexylamino)-ethane-sulphonic acid and pH 10'4,
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3-(cyclohexylamino)-propane-sulphonic acid. For the TRIS and triethanolamine buffers, the final pH was achieved by adjustment with 5 M acetic acid, while the other buffers were adjusted with I M NaOH. In order to minimise corrosion of stainless steel fittings in the chromatographic system, acetate was chosen in preference to chloride as the counter ion for the buffers and the salt gradient. Flow rate was 1·2 ml/min, resulting in a back pressure of about 2·5 kPa. The column temperature was controlled at either 20 or 25 "C. The sample volume was generally 0·1-0·2 ml. After each analysis, the column was washed with I M Na acetate (2 ml) at the appropriate pH , followed by regeneration with starting buffer.
Results Choice of chromatographic conditions
The elution pattern for isolated gliadins from the variety, Timgalen, after chromatography on a Mono-Q column is shown in Fig. 1. Elution patterns for four particular pH values are shown, and other buffers within the pH range 4·0-10· 5 have been examined. Despite the large number of components shown by electrophoresis, chromatographic separation ( a)
( b)
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FIGURE 1. Elution pattern of gIiadins extracted from the cultivar Timgalen after chromatography on a Mono-Q column in I M urea containing (a) 0·01 M tris(hydroxymethyl)-aminomethane, pH 8·0; (b) 0·01 M triethanolamine, pH 9·0; (c) 0·01 M 2-(cyclohexylamino)-ethane-sulphonic acid, pH 9·5 and (d) 0·01 M 3(cyclohexylamino)-propane-sulphonic acid, pH 10·4. In each case a linear gradient (20 ml) of 0-0·5 M sodium acetate was used. Sample volume in each case was O' 5 ml and the temperature of the column was 20°C.
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was poor unless buffers of quite high pH were used. Other varieties gave similar results, and pH 10·4 was chosen as the optimum pH for chromatography on Mono-Q. This high pH is necessary due to the low proportion of negatively-charged amino acids and the relatively high proportion of arginine residues, which remain positively-charged until quite a high pH. The shift of peaks to longer elution times as pH increased from 8 to 10·4 is due, presumably, to the amino group of lysine (pK 9) losing its positive charge. Increasing the pH further should result in even longer elution times as the guanidino group of the arginine residues (pK 12'5) becomes uncharged. Distinction between varieties
Chromatographic profiles at pH 10-4 of isolated gliadins from the varieties Egret, Condor, Durati and Eagle are shown in Fig. 2. These may also be compared with the profile for Timgalen in Fig. l(d). Each variety could be differentiated readily. The
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FIGURE 2. Elution pattern, after chromatography on a Mono-Q column at pH 10,4, of gliadins from the cultivars (a) Condor, (b) Durati, (c) Egret and (d) Eagle. The elution buffer contained in 1 M urea, 0·01 M 3-(cyclohexylamino)-propane-sulphonic acid and the proteins were eluted with a gradient of 0-0·5 M sodium acetate. Column temperature was 25°C.
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varieties Egret and Condor are difficult to distinguish by electrophoresis, and failure to do so may cause problems for wheat users because they are wheats of different quality types. The elution profiles of gliadins isolated from these two varieties showed marked differences on chromatography on the Mono-Q column at pH 10-4 and, thus, they may be readily differentiated by this chromatographic procedure. A total of 13 wheat genotypes, both commercial varieties and breeders' lines, have also been tested and all were distinguishable from each other. Direct extraction of gliadins from flour
Flour from the variety, Songlen, was extracted with starting buffer and the proteins in this extract were subjected to chromatography on the Mono-Q column. The elution profile of the extract (Fig. 3(a» differed from that of the isolated gliadins (Fig. 3(d» mainly in the appearance of peaks that were eluted before and after those for isolated gliadins. Chromatography of an albumin and globulin preparation indicated that the material that was eluted early from the column was water- and/or salt-soluble protein. The broad peaks that were eluted after the gliadin peaks (c. 13-20 min) are presumed ( 0)
( b)
(c)
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FIGURE 3(a)-(c). Elution pattern of an extract of Songlen flour milled from wheat grown at three locations: (a) Narrabri (irrigated), (b) North Star (dry-land) and (c) Myall Vale (irrigated). The flour was extracted with I M urea containing 0·01 M 3-(cyclohexylamino j-propane-sulphonic acid(pH I0'4).(d) Elutionpattern of purified gliadins from flour (a). Chromatographic conditions were as described in Fig. 2.
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to be glutenin components that were extracted in the starting buffer. As there was no interference with the peaks corresponding to the gliadin proteins, analysis of proteins extracted directly from flour proved as satisfactory for varietal identification as analysis of isolated gliadins with respect to the profiles obtained, but superior in terms of convenience and speed. Effect of growth conditions
Samples from the variety Egret, grown at three different locations, were examined to determine whether or not variations in the FPLC profiles due to growing conditions might affect the usefulness of this form of chromatography as a method for varietal identification. As shown in Fig. 4, the profiles for isolated gliadins were identical with respect to the peaks present although there were small variations in the relative heights of some peaks. The variety Timgalen also showed no site-dependent difference in the chromatographic profile for gliadins. When direct extracts of flour were examined for Songlen from different locations, only minor differences were observed in the elution profiles (Fig. 3(a)-(c)); as with the isolated gliadins from the variety Egret, small variations in the relative heights of some peaks were observed.
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FIGURE 4. Elution pattern of isolated gliadins from the cultivar, Egret, grown at three locations: (a) Dooen, (b) Moombooldool and (c) Temora. Chromatographic conditions were as described in Fig. 2.
Discussion The results presented here indicate that rapid anion exchange chromatography has potential as a method for varietal identification. In its ability to differentiate between the closely related varieties Condor and Egret, it is better than gel electrophoresis. This, of course, may not apply to all closely related varieties. At this stage, gliadin composition, as observed by FPLC, appears to be independent of growth environment, although more extensivepractical experiencewith the technique willbe required to establish unequivocally independence from environmental effects. However, since the same protein fraction (gliadin) is being examined as in electrophoretic identification, it seems reasonable to expect that the absence of environmental effects on the actual components present in electrophoretic patterns--" (as opposed to quantitative differences in the relative proportions of different components) will also hold for FPLC.
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The rather lengthy extraction procedure needed for the isolation of gliadins from flour makes this sample preparation method unsuitable for varietal identification. The direct extraction procedure, however, offers a practicable alternative as there is no interference from the non-gliadin proteins extracted with the gliadins from the flour. Although minor differences in peak height in profiles ofsamples from different locations may be associated with this simple extraction procedure (Fig. 3(a)--(cj), they are unlikely to pose a significant problem for either manual or machine (computer) evaluation of chromatographic profiles. Minor quantitative variations, which may also occur in electrophoretic analysis, might not be detected by visual examination of gels. Differences can be detected more readily after densitometric scanning of gels, but this is riot applied routinely. Obvious quantitative variations in electrophoretic patterns have been reported due to sulphur deficiency", though even this effect has not proved to be a practical difficulty in varietal identification by electrophoresis-. A more serious problem with evaluation of the chromatograms could occur with changing elution times. This has not proved to be a problem with one column with over 200 injections, but using a replacement column could result in the peaks of the chromatograms being shifted to either shorter or longer elution times. Care must also be exercised in the preparation of the buffer solutions in order to obtain consistent results. The use of internal reference proteins run concurrently with each sample might be necessary to overcome possible problems arising from variations between columns or buffers. Despite there being over 50 components in the gliadin fraction of wheat, less than a dozen are resolved by chromatography on a Mono-Q column. Anion exchange chromatography separates on the basis ofnegative charge, whilein most gelelectrophoresis procedures used for cereal proteins separation is on the basis of positive charge. It appears that there is more homogeneity in the net negative charge of wheat gliadins than there is in the net positive charge. Although fewer gliadin components are resolved by this ion-exchange system than by cathodic gel electrophoresis- or reversed-phase HPLC~, 6, this is not necessarily a disadvantage as very complex patterns or profiles can be difficult to compare. Furthermore, the FPLC profiles provide intervarietal distinctions on the basis of differences in the major components of the chromatogram. This feature is an advantage over reversed-phase HPLC, which relies for varietal identification on less obvious differences in profile. There is, however, the potential for more differences to be observed in the reversed-phase system because of the larger number of components that are resolved. The limitations that may be imposed by the small number of peaks in the ion-exchange chromatograms may make the procedure impracticable for varietal identification of the large range of varieties that are grown commercially. Both FPLC and reversed-phase HPLC procedures have the potential for modification so as to accentuate differences that may be shown in any specific part of the profile. Both ion-exchange and reversed-phase HPLC offer many advantages for routine varietal identification. Such methods are rapid, and can be used at a high sensitivity, permitting the identification of variety in samples of considerably less material than that in a single grain. This has been demonstrated in the case of reversed-phase HPLC6 and the sensitivity of the detector in the experiments described here may be increased by a factor of 10. Another advantage of modern chromatographic systems using any type of column is their capacity for automated sample handling. The incorporation of an 17
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automatic sampler into a system would enable sequential analyses to be run continuously for 24 h or more, a feature which electrophoresis does not allow. Currently available microprocessor hardware also offers the advantage that profiles ofauthentic samples may be stored for comparison with test samples. Although liquid chromatographic systems have a high initial capital cost, their throughput over a long period should repay the initial investment for any laboratory engaged in large numbers of varietal identifications. The possibility of using HPLC for a wide range of analyses makes it more versatile than electrophoresis. With increasing sophistication of chromatographic and data-handling systems, HPLC in its various forms promises to become a powerful tool for varietal identification. I wish to thank Dr David Tomlinson ofPharmacia (South Seas) Pty Ltd for providing a Mono-Q column for this work, Miss Robyn Smith and Mrs Shana Lennox for gliadin preparations and Dr Colin Wrigley for helpful discussions in the course of this work.
References 1. Wrigley, C. W. Food Technol. Aust. 32 (1980) 508-511. 2. Wrigley, C. W., Autran, J. C. and Bushuk, W. In 'Advances in Cereal Science and Technology' Vol. 5 (Y. Pomeranz, ed.), American Association of Cereal Chemists, St Paul (1982) pp 211-259. 3. Regnier, F. E. and Gooding, K. M. Anal. Biochem. 103 (1980) 1-25. 4. Kopaciewicz, W. and Regnier, F. E. Anal. Biochem. 133 (1983) 251-259. 5. Bietz, J. A. J. Chromatogr. 255 (1983) 219-238. 6. Bietz, J. A., Burnouf, T., Cobb, L. A. and Wall, J. S. Cereal Chem. 61 (1984) 129-135. 7. Baldo, B. A. and Wrigley, C. W. Clin. Allergy 8 (1978) 109-124. 8. Wrigley, C. W. and Shepherd, K. W. Aust. J. Exp. Agric. Anim. Hush. 14 (1974) 796-804. 9. Wrigley, C. W., du Cros, D. L., Archer, M. J., Downie, P. G. and Roxburgh, C. M. Aust. J. Plant Physiol. 7 (1980) 755-766.
Note added in proof
Both electrophoresis and reversed-phased HPLC may be applied to the analysis of single kernels. This technique can present some pitfalls, particularly in ensuring that the kernel or kernels selected are representative of the total sample from which they are drawn. Nevertheless, single kernel analysis does have its role in variety identification, and it has now been determined that ion-exchange chromatography on a Mono-Q column may also be applied for this purpose, using either whole kernels from which the germ has been removed, or the distal portion of bisected grains. The lipids present in the germ make it necessary to remove this part of the kernel before extraction as these lipids have a detrimental effect on the chromatographic separation of the gliadin proteins and on the column itself. The fragment was crushed and extracted with 10 times its weight of starting buffer. After centrifugation, the sample was filtered through a Millipore Millex-HV4 filter and 25 ul was applied to the column. Detection was effected at 280 nm with the detector sensitivity set at 0·05 absorbance units (full scale deflection). Using this procedure, it was possible for several chromatographic injections to be made from a single halfkernel, while retaining the proximal section of the grain with the embryo intact for cultivation if required.
Wheat Varietal Identification by Rapid Ion-exchange Chromatography of Gliadins I. L. BATEY
C.S./.R.O. Wheat Research Unit, Private Mail Bag, P.O., North Ryde, N.S.w. 2113, Australia Received 9 April 1984
A high performance liquid chromatography procedure, using anion exchange chromatography of the gliadin proteins at pH 10·4, has been devised as an alternative to gel electrophoresis for identification of wheat varieties. Varietal identification was, thus, possible in less than one hour. Sample preparation and extraction took about 30 min. The sample for chromatography could be prepared directly by extraction of flour with the starting buffer, indicating that a lengthy purification of the gliadin fraction is not necessary. Chromatography required approximately 20 min, and regeneration of the column was achieved in 5-10 min. Chromatographic separation of the gliadin proteins was lesseffective at pH values below 10·4. In initial experiments, 13 genotypes, including two varieties that are difficult to differentiate by electrophoresis, wereexamined and each was readily differentiated by the technique. Analysis of both purified gliadins and total flour extracts indicated that the results were independent of environmental factors.
Introduction Accurate and reliable identification of wheat varieties by wheat producers, traders and users is important for the maintenance and testing of wheat quality'. Currently, separation of the gliadin fraction of wheat by electrophoresis on either polyacrylamide or starch gels is the main laboratory method for varietal identification in most countries" It suffers from the disadvantage of being rather slow, labour intensive and reasonably expensive in the use of consumable materials. On the other hand, the apparatus required is cheap, simple and readily available, and the method has found ready acceptance because of its reliability. Column chromatography is an alternative method, by which proteins may be separated. Conventional chromatography is slow, requiring times ranging from several hours for size exclusion procedures to several days for some ion exchange separations. Recently, high performance liquid chromatography (HPLC) of proteins has become increasingly popular and the use of a variety of packing materials for columns has permitted more rapid separation of proteins, often from quite complex mixtures't-j. Fractionation of wheat proteins by reversed-phase HPLC has been reported, and good separations of the components of the gliadin fraction have been obtained", Varietal identification by this chromatographic technique has been proposed", and has been demonstrated recently". Abbreviations used: FPLC - fast prote in liquid chromatography; HPLC - high performance liquid chromatography ; TRIS - tris-(hydroxymethyl)-aminomethane. 0733-5210/84/040241 +08 $03.00/0
© 1984 Academic Press Inc. (London ) Limited
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I. L. BATEY
A rapid ion-exchange chromatography system for proteins has also become available. This system, known as ' Fast Protein Liquid Chromatography' (FPLC) utilises cation or anion exchange columns , or a chromatofocusing column. The separation of proteins on these ion-exchange columns is claimed to occur solely on the basis of charge , and not to involve other interactions with the column support material. Such chromatographic methods, because of their speed and convenience, have a considerable potential as practicable alternatives to electrophoresis for grain varietal identification , especially as automatic sample loading and data handling techniques are available. The use of one of the FPLC anion exchange columns, the Mono-Q column, for the separation of the components of wheat gliadin is reported here and its applicability to varietal identification is discussed.
Experimental Materials Wheat of the soft variety, Egret, was grown at three locations, Moombooldool and Temora in southern New South Wales, and Dooen in Victoria. The hard wheat, Songlen, was grown at Narrabri, North Star and Myall Vale in northern New South Wales. Timgalen, another hard variety, was grown at North Star , Myall Vale and Moree. Other registered varieties tested were Durati (a durum wheat) and the hard wheat varieties Banks, Condor, Cook, Eagle, Gatcher, Halberd and Kite. Two breeders' lines were also examined. Of these varieties, some had been grown at a single location, while others comprised bulked samples grown at a number of sites. The protein content of the grain samples ranged from 10% to 17% on an 'as is' basis.
Sample extraction The gliadin fraction was isolated from flour by aqueous ethanol (70% , vIv) extraction after preliminary extraction of the water- and salt-soluble components with 10% (wIv) sodium chloride using the method of Baldo and Wrigley", The gliadin samples were prepared for chromatography by dispersing them in starting buffer (5 mg gliadin/ml), The samples were stirred for 16 h at room temperature (c. 20-25°C) and centrifuged at 8000 g. For direct extraction of gliadins from straight-run (76% extraction) flour, samples were extracted by stirring for 30 min at 20-25°C in starting buffer, using 100 mg flour/ml of buffer. The suspension was then centrifuged at 8000 g. Immediately prior to application to the column, each gliadin or flour extract was filtered through a 0·22 urn Millipore filter (type GS).
Apparatus Separation was effected on a Pharmacia Mono-Q anion exchange column (Pharmacia (South Seas) Pty Ltd, North Ryde, N.S.W.). Column dimensions were 50 x 5 mm. The solvent delivery system was a Milton-Roy single piston positive displacement pump , with a linear gradient being formed using two chambers ofa gradient maker from a Technicon AA-l amino acid analyser (Technicon Instruments, North Ryde, N.S.W.). The detector was a Pharmacia UV-l single path UV monitor with an 8 III flowcell. The wavelength for detection was 280 nm.
Chromatographic conditions The medium for chromatography was 1 M urea containing 0·01 M buffer with a linear gradient of 0-0·5 M Na acetate. Buffers used were as follows: pH 8'0, tris-(hydroxymethyI)-aminomethane (TRIS); pH 9,0, triethanolamine ; pH 9·5, 2-(cyclohexylamino)-ethane-sulphonic acid and pH 10'4,
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3-(cyclohexylamino)-propane-sulphonic acid. For the TRIS and triethanolamine buffers, the final pH was achieved by adjustment with 5 M acetic acid, while the other buffers were adjusted with I M NaOH. In order to minimise corrosion of stainless steel fittings in the chromatographic system, acetate was chosen in preference to chloride as the counter ion for the buffers and the salt gradient. Flow rate was 1·2 ml/min, resulting in a back pressure of about 2·5 kPa. The column temperature was controlled at either 20 or 25 "C. The sample volume was generally 0·1-0·2 ml. After each analysis, the column was washed with I M Na acetate (2 ml) at the appropriate pH , followed by regeneration with starting buffer.
Results Choice of chromatographic conditions
The elution pattern for isolated gliadins from the variety, Timgalen, after chromatography on a Mono-Q column is shown in Fig. 1. Elution patterns for four particular pH values are shown, and other buffers within the pH range 4·0-10· 5 have been examined. Despite the large number of components shown by electrophoresis, chromatographic separation ( a)
( b)
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FIGURE 1. Elution pattern of gIiadins extracted from the cultivar Timgalen after chromatography on a Mono-Q column in I M urea containing (a) 0·01 M tris(hydroxymethyl)-aminomethane, pH 8·0; (b) 0·01 M triethanolamine, pH 9·0; (c) 0·01 M 2-(cyclohexylamino)-ethane-sulphonic acid, pH 9·5 and (d) 0·01 M 3(cyclohexylamino)-propane-sulphonic acid, pH 10·4. In each case a linear gradient (20 ml) of 0-0·5 M sodium acetate was used. Sample volume in each case was O' 5 ml and the temperature of the column was 20°C.
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was poor unless buffers of quite high pH were used. Other varieties gave similar results, and pH 10·4 was chosen as the optimum pH for chromatography on Mono-Q. This high pH is necessary due to the low proportion of negatively-charged amino acids and the relatively high proportion of arginine residues, which remain positively-charged until quite a high pH. The shift of peaks to longer elution times as pH increased from 8 to 10·4 is due, presumably, to the amino group of lysine (pK 9) losing its positive charge. Increasing the pH further should result in even longer elution times as the guanidino group of the arginine residues (pK 12'5) becomes uncharged. Distinction between varieties
Chromatographic profiles at pH 10-4 of isolated gliadins from the varieties Egret, Condor, Durati and Eagle are shown in Fig. 2. These may also be compared with the profile for Timgalen in Fig. l(d). Each variety could be differentiated readily. The
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FIGURE 2. Elution pattern, after chromatography on a Mono-Q column at pH 10,4, of gliadins from the cultivars (a) Condor, (b) Durati, (c) Egret and (d) Eagle. The elution buffer contained in 1 M urea, 0·01 M 3-(cyclohexylamino)-propane-sulphonic acid and the proteins were eluted with a gradient of 0-0·5 M sodium acetate. Column temperature was 25°C.
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varieties Egret and Condor are difficult to distinguish by electrophoresis, and failure to do so may cause problems for wheat users because they are wheats of different quality types. The elution profiles of gliadins isolated from these two varieties showed marked differences on chromatography on the Mono-Q column at pH 10-4 and, thus, they may be readily differentiated by this chromatographic procedure. A total of 13 wheat genotypes, both commercial varieties and breeders' lines, have also been tested and all were distinguishable from each other. Direct extraction of gliadins from flour
Flour from the variety, Songlen, was extracted with starting buffer and the proteins in this extract were subjected to chromatography on the Mono-Q column. The elution profile of the extract (Fig. 3(a» differed from that of the isolated gliadins (Fig. 3(d» mainly in the appearance of peaks that were eluted before and after those for isolated gliadins. Chromatography of an albumin and globulin preparation indicated that the material that was eluted early from the column was water- and/or salt-soluble protein. The broad peaks that were eluted after the gliadin peaks (c. 13-20 min) are presumed ( 0)
( b)
(c)
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FIGURE 3(a)-(c). Elution pattern of an extract of Songlen flour milled from wheat grown at three locations: (a) Narrabri (irrigated), (b) North Star (dry-land) and (c) Myall Vale (irrigated). The flour was extracted with I M urea containing 0·01 M 3-(cyclohexylamino j-propane-sulphonic acid(pH I0'4).(d) Elutionpattern of purified gliadins from flour (a). Chromatographic conditions were as described in Fig. 2.
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to be glutenin components that were extracted in the starting buffer. As there was no interference with the peaks corresponding to the gliadin proteins, analysis of proteins extracted directly from flour proved as satisfactory for varietal identification as analysis of isolated gliadins with respect to the profiles obtained, but superior in terms of convenience and speed. Effect of growth conditions
Samples from the variety Egret, grown at three different locations, were examined to determine whether or not variations in the FPLC profiles due to growing conditions might affect the usefulness of this form of chromatography as a method for varietal identification. As shown in Fig. 4, the profiles for isolated gliadins were identical with respect to the peaks present although there were small variations in the relative heights of some peaks. The variety Timgalen also showed no site-dependent difference in the chromatographic profile for gliadins. When direct extracts of flour were examined for Songlen from different locations, only minor differences were observed in the elution profiles (Fig. 3(a)-(c)); as with the isolated gliadins from the variety Egret, small variations in the relative heights of some peaks were observed.
Ec o
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e., o
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FIGURE 4. Elution pattern of isolated gliadins from the cultivar, Egret, grown at three locations: (a) Dooen, (b) Moombooldool and (c) Temora. Chromatographic conditions were as described in Fig. 2.
Discussion The results presented here indicate that rapid anion exchange chromatography has potential as a method for varietal identification. In its ability to differentiate between the closely related varieties Condor and Egret, it is better than gel electrophoresis. This, of course, may not apply to all closely related varieties. At this stage, gliadin composition, as observed by FPLC, appears to be independent of growth environment, although more extensivepractical experiencewith the technique willbe required to establish unequivocally independence from environmental effects. However, since the same protein fraction (gliadin) is being examined as in electrophoretic identification, it seems reasonable to expect that the absence of environmental effects on the actual components present in electrophoretic patterns--" (as opposed to quantitative differences in the relative proportions of different components) will also hold for FPLC.
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The rather lengthy extraction procedure needed for the isolation of gliadins from flour makes this sample preparation method unsuitable for varietal identification. The direct extraction procedure, however, offers a practicable alternative as there is no interference from the non-gliadin proteins extracted with the gliadins from the flour. Although minor differences in peak height in profiles ofsamples from different locations may be associated with this simple extraction procedure (Fig. 3(a)--(cj), they are unlikely to pose a significant problem for either manual or machine (computer) evaluation of chromatographic profiles. Minor quantitative variations, which may also occur in electrophoretic analysis, might not be detected by visual examination of gels. Differences can be detected more readily after densitometric scanning of gels, but this is riot applied routinely. Obvious quantitative variations in electrophoretic patterns have been reported due to sulphur deficiency", though even this effect has not proved to be a practical difficulty in varietal identification by electrophoresis-. A more serious problem with evaluation of the chromatograms could occur with changing elution times. This has not proved to be a problem with one column with over 200 injections, but using a replacement column could result in the peaks of the chromatograms being shifted to either shorter or longer elution times. Care must also be exercised in the preparation of the buffer solutions in order to obtain consistent results. The use of internal reference proteins run concurrently with each sample might be necessary to overcome possible problems arising from variations between columns or buffers. Despite there being over 50 components in the gliadin fraction of wheat, less than a dozen are resolved by chromatography on a Mono-Q column. Anion exchange chromatography separates on the basis ofnegative charge, whilein most gelelectrophoresis procedures used for cereal proteins separation is on the basis of positive charge. It appears that there is more homogeneity in the net negative charge of wheat gliadins than there is in the net positive charge. Although fewer gliadin components are resolved by this ion-exchange system than by cathodic gel electrophoresis- or reversed-phase HPLC~, 6, this is not necessarily a disadvantage as very complex patterns or profiles can be difficult to compare. Furthermore, the FPLC profiles provide intervarietal distinctions on the basis of differences in the major components of the chromatogram. This feature is an advantage over reversed-phase HPLC, which relies for varietal identification on less obvious differences in profile. There is, however, the potential for more differences to be observed in the reversed-phase system because of the larger number of components that are resolved. The limitations that may be imposed by the small number of peaks in the ion-exchange chromatograms may make the procedure impracticable for varietal identification of the large range of varieties that are grown commercially. Both FPLC and reversed-phase HPLC procedures have the potential for modification so as to accentuate differences that may be shown in any specific part of the profile. Both ion-exchange and reversed-phase HPLC offer many advantages for routine varietal identification. Such methods are rapid, and can be used at a high sensitivity, permitting the identification of variety in samples of considerably less material than that in a single grain. This has been demonstrated in the case of reversed-phase HPLC6 and the sensitivity of the detector in the experiments described here may be increased by a factor of 10. Another advantage of modern chromatographic systems using any type of column is their capacity for automated sample handling. The incorporation of an 17
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automatic sampler into a system would enable sequential analyses to be run continuously for 24 h or more, a feature which electrophoresis does not allow. Currently available microprocessor hardware also offers the advantage that profiles ofauthentic samples may be stored for comparison with test samples. Although liquid chromatographic systems have a high initial capital cost, their throughput over a long period should repay the initial investment for any laboratory engaged in large numbers of varietal identifications. The possibility of using HPLC for a wide range of analyses makes it more versatile than electrophoresis. With increasing sophistication of chromatographic and data-handling systems, HPLC in its various forms promises to become a powerful tool for varietal identification. I wish to thank Dr David Tomlinson ofPharmacia (South Seas) Pty Ltd for providing a Mono-Q column for this work, Miss Robyn Smith and Mrs Shana Lennox for gliadin preparations and Dr Colin Wrigley for helpful discussions in the course of this work.
References 1. Wrigley, C. W. Food Technol. Aust. 32 (1980) 508-511. 2. Wrigley, C. W., Autran, J. C. and Bushuk, W. In 'Advances in Cereal Science and Technology' Vol. 5 (Y. Pomeranz, ed.), American Association of Cereal Chemists, St Paul (1982) pp 211-259. 3. Regnier, F. E. and Gooding, K. M. Anal. Biochem. 103 (1980) 1-25. 4. Kopaciewicz, W. and Regnier, F. E. Anal. Biochem. 133 (1983) 251-259. 5. Bietz, J. A. J. Chromatogr. 255 (1983) 219-238. 6. Bietz, J. A., Burnouf, T., Cobb, L. A. and Wall, J. S. Cereal Chem. 61 (1984) 129-135. 7. Baldo, B. A. and Wrigley, C. W. Clin. Allergy 8 (1978) 109-124. 8. Wrigley, C. W. and Shepherd, K. W. Aust. J. Exp. Agric. Anim. Hush. 14 (1974) 796-804. 9. Wrigley, C. W., du Cros, D. L., Archer, M. J., Downie, P. G. and Roxburgh, C. M. Aust. J. Plant Physiol. 7 (1980) 755-766.
Note added in proof
Both electrophoresis and reversed-phased HPLC may be applied to the analysis of single kernels. This technique can present some pitfalls, particularly in ensuring that the kernel or kernels selected are representative of the total sample from which they are drawn. Nevertheless, single kernel analysis does have its role in variety identification, and it has now been determined that ion-exchange chromatography on a Mono-Q column may also be applied for this purpose, using either whole kernels from which the germ has been removed, or the distal portion of bisected grains. The lipids present in the germ make it necessary to remove this part of the kernel before extraction as these lipids have a detrimental effect on the chromatographic separation of the gliadin proteins and on the column itself. The fragment was crushed and extracted with 10 times its weight of starting buffer. After centrifugation, the sample was filtered through a Millipore Millex-HV4 filter and 25 ul was applied to the column. Detection was effected at 280 nm with the detector sensitivity set at 0·05 absorbance units (full scale deflection). Using this procedure, it was possible for several chromatographic injections to be made from a single halfkernel, while retaining the proximal section of the grain with the embryo intact for cultivation if required.