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High-performance liquid chromatography of α-amylases from germinating wheat and complexes with the α-amylase inhibitor from barley
Journal of Cereal Science 12 (1990) 73-81
High-performance Liquid Chromatography of IXAmylases from Germinating Wheat and Complexes with the IX-Amylase Inhibitor from Barley V. G. BATTERSHELL and R. J. HENRY
Queensland Wheat Research Institute PO Box 2282, Toowoomba, Qld 4350, Australia Received 11 December 1989
Wheat (X-amylases were separated into about eight major components in less than 10 min by high-performance liquid chromatography (HPLC) on non-porous ion exchange columns. This allowed rapid quantitative estimation of (X-amylase isoenzyme groups. The proportions of group 1 (low pI) and group 2 (high pI) isoenzymes changed very little from 2 days (74 % group 2) to 4 days (83 % group 2) of germination at 20°C despite a five-fold increase in total ex-amylase activity. Addition of the (X-amylase inhibitor from barley resulted in an apparent increase in the molecular weight of wheat (X-amylase on gel filtration. Wheat ex-amylases eluted from the HPLC earlier in the presence of the barley inhibitor probably because of the formation of enzyme/inhibitor complexes with higher pI. This suggests that the barley (X-amylase inhibitor may be useful in the utilization of wheat damaged by preharvest sprouting. This technique has potential for use in rapid quantitative analysis of genetic variations in (X-amylases and (X-amylase/inhibitor complexes.
Introduction ex-Amylase (EC 3.2.1.1) is produced in germinating grain and is associated with a serious loss of quality in wheat subjected to pre-harvest sprouting. The lX-amylase I group (pI 4·7 to 5'2) is associated with the ex-amy 2 genes on chromosomes 7A, 7B and 7D and the iX-amylase 2 group (pI 6·0 to 6'2) with the ex-amy I genes on the chromosomes 6A, 6B and 6D 1 ,2. The different forms of ex-amylase found in germinating wheat are usually separated by isoelectric focusing 3 • These isoenzymes have also been separated by liquid chromato~ graphy on ion-exchange columns 4 and more recently by chromatofocusing 5 • ex-Amylases from barley have been separated by HPLC on non-porous anion-exchange columns 6 • In this investigation HPLC has been applied to the rapid separation of ex-amylases from wheat and to quantitative determination of changes in specific isoenzyme groups during germination. Barley contains an endogenous ex-amylase inhibitor protein 7 that has a less effective analogue in wheatS. Zawistowska 9 suggested that the barley ex-amylase inhibitor may be Abbreviations used: HPLC
= high-performance liquid chromatography; IEF = isoe1ectricfocusing.
0733-5210/90/040073 +09 $03.00/0 4
© 1990 Academic Press Limited CER 12
74
V. G. BATIERSHELL AND R. J. HENRY
highly suited to the control of excess a-amylase in flour from sprouted wheat because it meets the requirement for a non-toxic a-amylase inhibitor that will not alter the essential breadmaking properties of the gluten 1o . The a-amylase inhibitor from barley has been shown to be an inhibitor of a-amylase from wheat 3 • 8 • The interaction between wheat aamylase and the barley a-amylase inhibitor has now been investigated by applying the HPLC technique to the analysis of complexes between wheat a-amylase and the barley a-amylase inhibitor.
Experimental Germination of wheat Wheat (cv. Hartog) was germinated at 20°C for up to 7 days in the germination cabinet of a Seeger micro-malting plantl l• In some experiments microbial growth was eliminated by surface sterilization and germination in the presence of antibiotics 12 . In these experiments the grain was treated with 0·1 % silver nitrate for 20 min, and the silver nitrate then removed by washing with sterile 0'5 M sodium chloride followed by sterile distilled water. The grain was then steeped in sterile distilled water containing kanamycin (100 Ilgjml) chloramphenicol (l00 ).lg/ml) penicillin (100 units/ml) and nystatin (100 units/ml); (all antibiotics were from Sigma, St Louis, U.S.A.). Germination was continued on filter paper in covered germination trays with addition of sterile distilled water when necessary. The germinated grain was frozen by immersion in liquid nitrogen and freeze-dried.
Extraction of (X-amylase The freeze-dried grain was ground to pass a 0·8 mm sieve using a laboratory hammer mill (Falling Number 3100). a-Amylase was extracted by suspending ground grain (0'5 g) in 50 mM sodium hydrogen malate buffer pH 5·2 (5 ml containing 2 mM CaCI 2 and 50 mM NaCI). The mixture was agitated by occasional vortex mixing over a period of 10 min and then centrifuged for 10 min at 2000 g.
Preparation for HPLC The supernatant following centrifugation was filtered through a 0'22 ).lm filter (Millipore). The extract was desalted on a 16 mm x 50 mm column of Bio-Gel P-6 (Bio-Rad) equilibrated with 5 mM Tris-HCI pH 8·6 containing 1 mM calcium chloride.
HPLC The HPLC procedure was similar to that previously described 6 • The desalted sample (1 ml) was loaded onto a 50 x 7·8 mm MA7P (Bio-Rad) anion exchange column. rx-Amylases were eluted by applying a gradient of sodium chloride in the 5 mM Tris-HCI buffer pH 8·6 containing 1 mM calcium chloride. Protein was monitored at 280 nm and ex-amylase was determined on fractions collected.
(X-Amylase assay a-Amylase was determined by dilution of an aliquot (0'05 ml) of each fraction to 1 ml with the original extraction (50 mM sodium malate, pH 5'2,50 mM sodium chloride, 2 mM calcium chloride) and assayed using dye-labelled substrate tablets (phadebas Pharmacia, Uppsala, Sweden)13.
HPLC OF WHEAT ex-AMYLASE
75
Isoelectric fact/sing (/EF) This was performed on Ampholine PAG (polyacrylamide gel) plate gels pH 3·5 to 9'5 (LKB). Gels were run for 1'25 h at 30 W. The pH gradient was determined using pI markers (Pharmacia) and staining with Coomassie Blue. a-Amylase was detected by incubating the IEF gels in a solution of I % soluble starch (American Society of Brewing Chemists, St Paul, Minnesota, U.S.A.) in 50 mM NaCI, 2 mM CaCI 2 , 50 mM sodium hydrogen malate buffer pH 5·2 at 40°C for 15 min. The starch was dissolved by bringing the solution to 95 °C and then cooling slowly to 40°C. Amylase bands were revealed as colourless bands on a blue background by staining with a solution containing 12 (0,04 %) and KI (0,4 %) in 0'5 MHCI. In some experiments pinkish bands were detected, probably due to the presence of ~-amylase.
Isolation of barley a-amylase inhibitor Barley (Hordeum l'ulgare cv. Grimmett) was pearled in a Strong-Scott barley pearIer to remove approximately 5 % of the grain weight. The pearled barley was ground to pass a 0·8 mm sieve using a Falling Number 3100 hammer mill. The ground barley (l kg) was extracted with 20 mM MOPS (morpholino propane sulphonic acid) buffer pH 7·0 (3 I containing 20 mM NaCI) with stirring for 20 min at 4°C. The extract was centrifuged for 5 min at 3000 g and filtered through Whatman No. I filter paper. The pH was adjusted to 7'0 with dilute NaOH. The extract was then passed through beds of DEAE-cellulose (DE52 Whatman) and CM-cellulose (CM52 Whatman) equilibrated with the extraction buffer. The ion-exchange chromatography was performed with the media in sintered glass funnels allowing acceleration of flow by application of vacuum. The solution was made 100 % saturated with ammonium sulphate (2280 g). The solids were collected following flocculation and resuspended in 5 mM Tris HCI pH 8·6 (500 ml containing 1 mM CaCI 2) and dialysed against 5 I of the same buffer with three changes of buffer. The dialysed solution was stored frozen as a crude inhibitor preparation.
Gel filtration Wheat a-amylase and a-amylase/inhibitor complexes were analysed by gel filtration on a TSK-50 (S) gel filtration column (100 x 1'6 cm) in 5 mM Tris pH 8'6 containing I mM CaCI 2 • Equal volumes of the wheat IX-amylase and crude barley IX-amylase inhibitor preparations were mixed and incubated at room temperature for 30 min to allow complex formation. In all experiments 0·5 ml of sample was loaded and 1'3 ml fractions were collected. a-Amylase was determined following the addition of 0·5 ml of the a-amylase assay buffer to 0·5 ml of each fraction.
Results and Discussion Separation of wheat a-amylases by HPLC Ion-exchange chromatography at a flow rate of 1·5 ml/min allowed the separation shown in Fig. 1 in less than 10 min. The profile is similar to that reported by Marchylo and Kruger 5 for the separation of a-amylases from hard red spring wheat by chromatofocusing. The HPLC separation was approximately 100 times faster than conventional chromatofocusing and shows potential for use in rapid quantitative analysis. The a-amylases might be expected to elute in approximate order of decreasing isoe1ectric point. The early peaks (1-4 Fig. 1) are likely to correspond to a-amylase 2 and the later peaks (5-8 Fig. I) to a-amylase 1. Most protein ( > 50 %) does not bind to the 4·2
76
V. G. BATTERSHELL AND R. J. HENRY
.
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.
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FIGURE 1. Separation of wheat o:-amylases by HPLC on an anion-exchange column. A linear gradient of sodium chloride was applied starting at 2 min and increasing to 0·15 M at 12 min. Wheat germinated for seven days in sterile distilled water following surface sterilisation and treatment with antibiotics. a-Amylase activity is in arbitrary units based upon the A 620 in a standard assay.
column under these conditions and elutes at 1-2 min resulting in considerable purification of the a-amylases in one very rapid step. The purification of a-amylase possible with this method is similar to that reported for metal affinity chromatography14. The sample analysed in Fig. 1 was germinated under conditions designed to eliminate the possibility of contribution of enzymes of fungal or microbial origin. However, other samples produced without such precautions did not give significantly different profiles. Identity of HPLC peaks determined by IEF
The pI of a-amylases in the major peaks was investigated by IEF. Fractions from several HPLC separations were pooled and concentrated by ultrafiltration using Centricon 10 microconcentrations (Amicon). The 22 broad fractions analysed each contained several a-amylase isoenzymes (Table I). These results indicate that a-amylase I isoenzymes with pI values between 4'55 and 5·2 eluted between 7·4 min and 7,7 min. a-Amylase 2 isoenzymes with pI values between 5·2 and 5·85 elute mainly between 5·5 min and 6'5 min. These pI values are within the range previously reported for wheat a-amylase. Based upon this evidence the a-amylase eluting before 7·24 min (peaks 1-4 Fig. 1) was attributed to group 2 a-amylases and the later fractions (peaks 5-8 Fig. 1) to group 1 a-amylases.
77
HPLC OF WHEAT IX-AMYLASE TABLE 1. pI values of ex-amylases in fractions separated by HPLC' HPLC fraction number
Mean elution time (min)
1 2 3 4 5 6 7 8 9
5·54 5·73 5'92 6'11 6·30 6-49 6·68 6-87 7·06 7-25 7-44 7-63 7·92
10
11 12 13
pI values b
5'5, 5'85, 5'5, 5'5, 5'5, 5'5,
5'85, 5'5, 5-85, 504, 5,4, 5,4,
5,6, 5-6, 5,6, 5'3, 5'3, 5'3,
5-4 5·4 5·3 5·2 5-2 5-2
4'7,
5'0,
4,55,
4-6
5'2, 5'2,
5,0, 5'0,
4'7, 4·7
4'55 4·55
• Missing values indicate that no IX-amylase bands were detected in these fractions. b Determined by IEF (listed in order of decreasing band intensity).
•
8-0
6-0
2-0
.;/0 . 0
.----
• 2
3
4
Time (days)
FIGURE 2. Increase in, group 1 (.); group 2 (0) and total (e) lX-amylase in germinating wheat. Germinated without antibiotics.
78
V. G. BATTERSHELL AND R. J. HENRY
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4 Day
3 Day
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FIGURE 3. Changes in a-amylase content during germination as measured by HPLC; conditions as in Fig. L Germinated without antibiotics.
Changes during germination The group 2 isoenzymes are most abundant and increase from 74 % of total activity after 2 days of germination to 83 % after 4 days (Fig. 2 and Fig. 3). During this 48 h period of rapid ct-amylase production, ex-amylase levels increase more than five-fold. These results agree with the proportions of high pI a-amylase in Canadian wheat estimated by MacGregor et al,1" using chromatofocllsing; 84 % in red spring wheat and 79 % in amber durum wheat. However in the Canadian red spring wheat cultivar, Neepawa, the proportion of high pI enzyme was found to decline with time of germination. These two groups of ex-amylase are differentially controlled by gibberellic acid 6 • Genetic or environmental differences may explain slight variations in proportions of these enzymes and the pattern of change during germination.
Loss of a-amylase activity during heat treatment of extracts Heat treatment of extracts at 70°C for 15 min reduced total ex-amylase activity by more than 80 % (24 % of group 1 and 18 % of group 2 activity remained). The stability of wheat ex-amylases to this heat treatment is much lower than that of barley ex-amylases (Henry, unpublished). This could be due to differences in the structure of the enzymes or may be associated with the presence of an ex-amylase inhibitor protein in barley that
HPLC OF WHEAT IX-AMYLASE
79
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Li
i ~
i!
0·8
:
I I:
I
\
0·6
0·4
,
:
i
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:
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Y \~ 80
100
Elution volume (ml)
FIGURE 4. Gel filtration of wheat IX-amylase on a 100 x 1'6 cm column of TSK-50 (S); wheat IX-amylase (--); wheat IX-amylase plus barley IX-amylase inhibitor ( ..... ).
stabilizes a-amylase undergoing heat treatment more effectively than in wheats. Heat treatment of barley a-amylases converts a-amylase 3 (a complex with the a-amylase inhibitor) to free a-amylase 27 • Complexes between wheat (X-amylase and the barley (X-amylase inhibitor
Mixture of wheat a-amylase (from a wheat sample germinated for 7 days with antibiotics) and barley a-amylase inhibitor resulted in the formation of a higher molecular weight complex as judged by gel filtration (Fig. 4). The a-amylases and aamylase/a-amylase inhibitor complexes were analysed by HPLC on a new MA7P column. This sample gave a slightly different profile with possibly better resolution than that of earlier experiments. The HPLC profile suggests that many of the a-amylase 2 peaks eluted earlier following complexing with the inhibitor (Fig. 5). The peak in activity
80
V. G. BATfERSHELL AND R. J. HENRY
0·3 ~
o
'"
~
Q>
III
o
u o 1-0 z
>-
!i 0·2 I
D
0·5
0·1
6
7 Time (min)
8
9
FIGURE 5. Interaction of barley a-amylase inhibitor with wheat a-amylase analysed by HPLC wheat a-amylase (--); wheat a-amylase plus barley a-amylase inhibitor (. - .. '). The sample and columns were different to those used in other Figs.
moved from 6·28 min to 6·10 min. The gel filtration and HPLC evidence are consistent with the formation of a higher molecular weight complex with higher pI as would be expected for a complex with the a-amylase inhibitor with a pI of 7.2 17 • However, these results can not be used to distinguish differences in the binding or inhibition of specific a-amylase components. These complexes are probably similar to the a-amylase 3 of barley. Barley a-amylase 3 has a higher pI than a-amylase 2 and results from a complex between barley a-amylase 2 and the a-amylase inhibitor 7 • Zawistowska et alY recently reported that addition of the barley a-amylase inhibitor successfully reversed the harmful effects of barley a-amylase on the breadmaking quality of wheat flour. The results of the present study suggest that the inhibitor is also likely to be effective in controlling the activity of wheat a-amylase in flour. This provides further support to the suggestion that the barley a-amylase inhibitor might have a role
HPLC OF WHEAT IX-AMYLASE
81
in the commercial utilization of wheat that has been damaged by pre-harvest sprouting 18 • These results are also consistent with the report by Mundy et a/. s indicating that the barley a-amylase inhibitor was effective in inhibiting both wheat and barley a-amylases. This work was supported by funds from the Australian Wheat Research Council. David Chadbone provided technical assistance.
References 1. Gale, M. D, in 'Third International Symposium on Pre-harvest Sprouting in Cereals' (J. E. Kruger and D. E. LaBerge, eds,) Westview Press, Boulder (1983) pp 105-110. 2. Nishikawa, K. and Nobuhara, M, Japan J. Genetics 46 (1971) 345-353. 3. Marchylo, B. A., Lacroix, L. J. and Kruger, J. E. Can. J. Plant Sci. 60 (1980) 433-443. 4. Marchylo, B., Kruger, J, E. and Irvine, G, N. Cereal Chemistry 53 (1976) 157-173. 5. Marchylo, B. A. and Kruger, J. E. in 'Third International Symposium on Pre-harvest Sprouting in Cereals' (J. E. Kruger and D. E. LaBerge, eds.), Westview Press, Boulder (1983) pp 96-104, 6. Henry, R. J. J. Chrornatogr. 481 (1989) 397-402. 7. Weselake, R. J., MacGregor, A. W. and Hill, R. D. Plant Physiol. 72 (1983) 809-812. 8. Mundy, J., Hejgaard, J. and Svendsen,!. FEBS letters 167 (1984) 210-214. 9. Zawistowska, D. in 'Wheat is unique' (Y. Pomeranz, ed.), American Association of Cereal Chemists, St Paul (1989) pp 117-129. 10. Meredith, P. and Pomeranz, Y, Adv. Cereal Sci. Technol. 7 (1985) 239-320. II. Corder, A. M. and Henry, R. J. Cereal Chern. 66 (1989) 435-439. 12. Hoy, J, T" Macauley, B. J. and Fincher, G, B. J. Inst. Brew. 87 (1981) 77-80. 13. Henry, R. J. J. Sci. Food Agric. 49 (1989) 15-24. 14. Zawistowska, D., Sangster, K., Zawistowski, J., Langstaff, J. and Frieser, A. D. Cereal Chern. 65 (1988) 413-416. 15. MacGregor, A. W., Marchylo, B. A. and Kruger, J. E. Cereal Chem. 65 (1988) 326-333. 16. Marchylo, B. A., Kruger, J. E. and MacGregor, A. W. Cereal Chern. 61 (1984) 305-310. 17. Mundy, J., Svendsen, 1. and Hejgaard, J. Carlsberg Res. Cornmun. 48 (1983) 81-90. 18. Zawistowska, D., Langstaff, J. and Bushuk, W. J. Cereal Sci. 8 (1988) 207-209.
High-performance Liquid Chromatography of IXAmylases from Germinating Wheat and Complexes with the IX-Amylase Inhibitor from Barley V. G. BATTERSHELL and R. J. HENRY
Queensland Wheat Research Institute PO Box 2282, Toowoomba, Qld 4350, Australia Received 11 December 1989
Wheat (X-amylases were separated into about eight major components in less than 10 min by high-performance liquid chromatography (HPLC) on non-porous ion exchange columns. This allowed rapid quantitative estimation of (X-amylase isoenzyme groups. The proportions of group 1 (low pI) and group 2 (high pI) isoenzymes changed very little from 2 days (74 % group 2) to 4 days (83 % group 2) of germination at 20°C despite a five-fold increase in total ex-amylase activity. Addition of the (X-amylase inhibitor from barley resulted in an apparent increase in the molecular weight of wheat (X-amylase on gel filtration. Wheat ex-amylases eluted from the HPLC earlier in the presence of the barley inhibitor probably because of the formation of enzyme/inhibitor complexes with higher pI. This suggests that the barley (X-amylase inhibitor may be useful in the utilization of wheat damaged by preharvest sprouting. This technique has potential for use in rapid quantitative analysis of genetic variations in (X-amylases and (X-amylase/inhibitor complexes.
Introduction ex-Amylase (EC 3.2.1.1) is produced in germinating grain and is associated with a serious loss of quality in wheat subjected to pre-harvest sprouting. The lX-amylase I group (pI 4·7 to 5'2) is associated with the ex-amy 2 genes on chromosomes 7A, 7B and 7D and the iX-amylase 2 group (pI 6·0 to 6'2) with the ex-amy I genes on the chromosomes 6A, 6B and 6D 1 ,2. The different forms of ex-amylase found in germinating wheat are usually separated by isoelectric focusing 3 • These isoenzymes have also been separated by liquid chromato~ graphy on ion-exchange columns 4 and more recently by chromatofocusing 5 • ex-Amylases from barley have been separated by HPLC on non-porous anion-exchange columns 6 • In this investigation HPLC has been applied to the rapid separation of ex-amylases from wheat and to quantitative determination of changes in specific isoenzyme groups during germination. Barley contains an endogenous ex-amylase inhibitor protein 7 that has a less effective analogue in wheatS. Zawistowska 9 suggested that the barley ex-amylase inhibitor may be Abbreviations used: HPLC
= high-performance liquid chromatography; IEF = isoe1ectricfocusing.
0733-5210/90/040073 +09 $03.00/0 4
© 1990 Academic Press Limited CER 12
74
V. G. BATIERSHELL AND R. J. HENRY
highly suited to the control of excess a-amylase in flour from sprouted wheat because it meets the requirement for a non-toxic a-amylase inhibitor that will not alter the essential breadmaking properties of the gluten 1o . The a-amylase inhibitor from barley has been shown to be an inhibitor of a-amylase from wheat 3 • 8 • The interaction between wheat aamylase and the barley a-amylase inhibitor has now been investigated by applying the HPLC technique to the analysis of complexes between wheat a-amylase and the barley a-amylase inhibitor.
Experimental Germination of wheat Wheat (cv. Hartog) was germinated at 20°C for up to 7 days in the germination cabinet of a Seeger micro-malting plantl l• In some experiments microbial growth was eliminated by surface sterilization and germination in the presence of antibiotics 12 . In these experiments the grain was treated with 0·1 % silver nitrate for 20 min, and the silver nitrate then removed by washing with sterile 0'5 M sodium chloride followed by sterile distilled water. The grain was then steeped in sterile distilled water containing kanamycin (100 Ilgjml) chloramphenicol (l00 ).lg/ml) penicillin (100 units/ml) and nystatin (100 units/ml); (all antibiotics were from Sigma, St Louis, U.S.A.). Germination was continued on filter paper in covered germination trays with addition of sterile distilled water when necessary. The germinated grain was frozen by immersion in liquid nitrogen and freeze-dried.
Extraction of (X-amylase The freeze-dried grain was ground to pass a 0·8 mm sieve using a laboratory hammer mill (Falling Number 3100). a-Amylase was extracted by suspending ground grain (0'5 g) in 50 mM sodium hydrogen malate buffer pH 5·2 (5 ml containing 2 mM CaCI 2 and 50 mM NaCI). The mixture was agitated by occasional vortex mixing over a period of 10 min and then centrifuged for 10 min at 2000 g.
Preparation for HPLC The supernatant following centrifugation was filtered through a 0'22 ).lm filter (Millipore). The extract was desalted on a 16 mm x 50 mm column of Bio-Gel P-6 (Bio-Rad) equilibrated with 5 mM Tris-HCI pH 8·6 containing 1 mM calcium chloride.
HPLC The HPLC procedure was similar to that previously described 6 • The desalted sample (1 ml) was loaded onto a 50 x 7·8 mm MA7P (Bio-Rad) anion exchange column. rx-Amylases were eluted by applying a gradient of sodium chloride in the 5 mM Tris-HCI buffer pH 8·6 containing 1 mM calcium chloride. Protein was monitored at 280 nm and ex-amylase was determined on fractions collected.
(X-Amylase assay a-Amylase was determined by dilution of an aliquot (0'05 ml) of each fraction to 1 ml with the original extraction (50 mM sodium malate, pH 5'2,50 mM sodium chloride, 2 mM calcium chloride) and assayed using dye-labelled substrate tablets (phadebas Pharmacia, Uppsala, Sweden)13.
HPLC OF WHEAT ex-AMYLASE
75
Isoelectric fact/sing (/EF) This was performed on Ampholine PAG (polyacrylamide gel) plate gels pH 3·5 to 9'5 (LKB). Gels were run for 1'25 h at 30 W. The pH gradient was determined using pI markers (Pharmacia) and staining with Coomassie Blue. a-Amylase was detected by incubating the IEF gels in a solution of I % soluble starch (American Society of Brewing Chemists, St Paul, Minnesota, U.S.A.) in 50 mM NaCI, 2 mM CaCI 2 , 50 mM sodium hydrogen malate buffer pH 5·2 at 40°C for 15 min. The starch was dissolved by bringing the solution to 95 °C and then cooling slowly to 40°C. Amylase bands were revealed as colourless bands on a blue background by staining with a solution containing 12 (0,04 %) and KI (0,4 %) in 0'5 MHCI. In some experiments pinkish bands were detected, probably due to the presence of ~-amylase.
Isolation of barley a-amylase inhibitor Barley (Hordeum l'ulgare cv. Grimmett) was pearled in a Strong-Scott barley pearIer to remove approximately 5 % of the grain weight. The pearled barley was ground to pass a 0·8 mm sieve using a Falling Number 3100 hammer mill. The ground barley (l kg) was extracted with 20 mM MOPS (morpholino propane sulphonic acid) buffer pH 7·0 (3 I containing 20 mM NaCI) with stirring for 20 min at 4°C. The extract was centrifuged for 5 min at 3000 g and filtered through Whatman No. I filter paper. The pH was adjusted to 7'0 with dilute NaOH. The extract was then passed through beds of DEAE-cellulose (DE52 Whatman) and CM-cellulose (CM52 Whatman) equilibrated with the extraction buffer. The ion-exchange chromatography was performed with the media in sintered glass funnels allowing acceleration of flow by application of vacuum. The solution was made 100 % saturated with ammonium sulphate (2280 g). The solids were collected following flocculation and resuspended in 5 mM Tris HCI pH 8·6 (500 ml containing 1 mM CaCI 2) and dialysed against 5 I of the same buffer with three changes of buffer. The dialysed solution was stored frozen as a crude inhibitor preparation.
Gel filtration Wheat a-amylase and a-amylase/inhibitor complexes were analysed by gel filtration on a TSK-50 (S) gel filtration column (100 x 1'6 cm) in 5 mM Tris pH 8'6 containing I mM CaCI 2 • Equal volumes of the wheat IX-amylase and crude barley IX-amylase inhibitor preparations were mixed and incubated at room temperature for 30 min to allow complex formation. In all experiments 0·5 ml of sample was loaded and 1'3 ml fractions were collected. a-Amylase was determined following the addition of 0·5 ml of the a-amylase assay buffer to 0·5 ml of each fraction.
Results and Discussion Separation of wheat a-amylases by HPLC Ion-exchange chromatography at a flow rate of 1·5 ml/min allowed the separation shown in Fig. 1 in less than 10 min. The profile is similar to that reported by Marchylo and Kruger 5 for the separation of a-amylases from hard red spring wheat by chromatofocusing. The HPLC separation was approximately 100 times faster than conventional chromatofocusing and shows potential for use in rapid quantitative analysis. The a-amylases might be expected to elute in approximate order of decreasing isoe1ectric point. The early peaks (1-4 Fig. 1) are likely to correspond to a-amylase 2 and the later peaks (5-8 Fig. I) to a-amylase 1. Most protein ( > 50 %) does not bind to the 4·2
76
V. G. BATTERSHELL AND R. J. HENRY
.
3 2
4
0·4
0-15
.
.-..- ..- ..- .-..- ..-.
l
..-' 0·1 ••••
,j
i;./\'-''-''-·'-·'-·
t j
, ..-
\".
". 5
7
z:
0·05
"1.•\
6
~ 0'10 ~
;'
8
Time (min)
6
7
8
.,'..•:'• ..... ..... 9
FIGURE 1. Separation of wheat o:-amylases by HPLC on an anion-exchange column. A linear gradient of sodium chloride was applied starting at 2 min and increasing to 0·15 M at 12 min. Wheat germinated for seven days in sterile distilled water following surface sterilisation and treatment with antibiotics. a-Amylase activity is in arbitrary units based upon the A 620 in a standard assay.
column under these conditions and elutes at 1-2 min resulting in considerable purification of the a-amylases in one very rapid step. The purification of a-amylase possible with this method is similar to that reported for metal affinity chromatography14. The sample analysed in Fig. 1 was germinated under conditions designed to eliminate the possibility of contribution of enzymes of fungal or microbial origin. However, other samples produced without such precautions did not give significantly different profiles. Identity of HPLC peaks determined by IEF
The pI of a-amylases in the major peaks was investigated by IEF. Fractions from several HPLC separations were pooled and concentrated by ultrafiltration using Centricon 10 microconcentrations (Amicon). The 22 broad fractions analysed each contained several a-amylase isoenzymes (Table I). These results indicate that a-amylase I isoenzymes with pI values between 4'55 and 5·2 eluted between 7·4 min and 7,7 min. a-Amylase 2 isoenzymes with pI values between 5·2 and 5·85 elute mainly between 5·5 min and 6'5 min. These pI values are within the range previously reported for wheat a-amylase. Based upon this evidence the a-amylase eluting before 7·24 min (peaks 1-4 Fig. 1) was attributed to group 2 a-amylases and the later fractions (peaks 5-8 Fig. 1) to group 1 a-amylases.
77
HPLC OF WHEAT IX-AMYLASE TABLE 1. pI values of ex-amylases in fractions separated by HPLC' HPLC fraction number
Mean elution time (min)
1 2 3 4 5 6 7 8 9
5·54 5·73 5'92 6'11 6·30 6-49 6·68 6-87 7·06 7-25 7-44 7-63 7·92
10
11 12 13
pI values b
5'5, 5'85, 5'5, 5'5, 5'5, 5'5,
5'85, 5'5, 5-85, 504, 5,4, 5,4,
5,6, 5-6, 5,6, 5'3, 5'3, 5'3,
5-4 5·4 5·3 5·2 5-2 5-2
4'7,
5'0,
4,55,
4-6
5'2, 5'2,
5,0, 5'0,
4'7, 4·7
4'55 4·55
• Missing values indicate that no IX-amylase bands were detected in these fractions. b Determined by IEF (listed in order of decreasing band intensity).
•
8-0
6-0
2-0
.;/0 . 0
.----
• 2
3
4
Time (days)
FIGURE 2. Increase in, group 1 (.); group 2 (0) and total (e) lX-amylase in germinating wheat. Germinated without antibiotics.
78
V. G. BATTERSHELL AND R. J. HENRY
2 Day
4 Day
3 Day
0·6 0·5
f·
~
o
.£'"
'" >. "
0·4
u>
E
<:(
0·3
I
o
0·2
o· I
~ ~.~.~.~.~.~.~.~/~.~
r"" )
.........
. .....\/'1\ , ...... 1"'••__ 6
7
l.
...........1
8
9
..'
'
I
6
~.~
\
I··~h.,.."••·~\ .. .j.,
7 8 Time (min)
f-\/
r\ //
A
///'~/-./---/-
9
'i
~
\
~\.J\./\\ .t.f\,
I
I
I ' "i'V
6
7
8
0·15
~ z
0·10
0·05
9
FIGURE 3. Changes in a-amylase content during germination as measured by HPLC; conditions as in Fig. L Germinated without antibiotics.
Changes during germination The group 2 isoenzymes are most abundant and increase from 74 % of total activity after 2 days of germination to 83 % after 4 days (Fig. 2 and Fig. 3). During this 48 h period of rapid ct-amylase production, ex-amylase levels increase more than five-fold. These results agree with the proportions of high pI a-amylase in Canadian wheat estimated by MacGregor et al,1" using chromatofocllsing; 84 % in red spring wheat and 79 % in amber durum wheat. However in the Canadian red spring wheat cultivar, Neepawa, the proportion of high pI enzyme was found to decline with time of germination. These two groups of ex-amylase are differentially controlled by gibberellic acid 6 • Genetic or environmental differences may explain slight variations in proportions of these enzymes and the pattern of change during germination.
Loss of a-amylase activity during heat treatment of extracts Heat treatment of extracts at 70°C for 15 min reduced total ex-amylase activity by more than 80 % (24 % of group 1 and 18 % of group 2 activity remained). The stability of wheat ex-amylases to this heat treatment is much lower than that of barley ex-amylases (Henry, unpublished). This could be due to differences in the structure of the enzymes or may be associated with the presence of an ex-amylase inhibitor protein in barley that
HPLC OF WHEAT IX-AMYLASE
79
f\
Li
i ~
i!
0·8
:
I I:
I
\
0·6
0·4
,
:
i
~.:
:
.1
0·2
Y \~ 80
100
Elution volume (ml)
FIGURE 4. Gel filtration of wheat IX-amylase on a 100 x 1'6 cm column of TSK-50 (S); wheat IX-amylase (--); wheat IX-amylase plus barley IX-amylase inhibitor ( ..... ).
stabilizes a-amylase undergoing heat treatment more effectively than in wheats. Heat treatment of barley a-amylases converts a-amylase 3 (a complex with the a-amylase inhibitor) to free a-amylase 27 • Complexes between wheat (X-amylase and the barley (X-amylase inhibitor
Mixture of wheat a-amylase (from a wheat sample germinated for 7 days with antibiotics) and barley a-amylase inhibitor resulted in the formation of a higher molecular weight complex as judged by gel filtration (Fig. 4). The a-amylases and aamylase/a-amylase inhibitor complexes were analysed by HPLC on a new MA7P column. This sample gave a slightly different profile with possibly better resolution than that of earlier experiments. The HPLC profile suggests that many of the a-amylase 2 peaks eluted earlier following complexing with the inhibitor (Fig. 5). The peak in activity
80
V. G. BATfERSHELL AND R. J. HENRY
0·3 ~
o
'"
~
Q>
III
o
u o 1-0 z
>-
!i 0·2 I
D
0·5
0·1
6
7 Time (min)
8
9
FIGURE 5. Interaction of barley a-amylase inhibitor with wheat a-amylase analysed by HPLC wheat a-amylase (--); wheat a-amylase plus barley a-amylase inhibitor (. - .. '). The sample and columns were different to those used in other Figs.
moved from 6·28 min to 6·10 min. The gel filtration and HPLC evidence are consistent with the formation of a higher molecular weight complex with higher pI as would be expected for a complex with the a-amylase inhibitor with a pI of 7.2 17 • However, these results can not be used to distinguish differences in the binding or inhibition of specific a-amylase components. These complexes are probably similar to the a-amylase 3 of barley. Barley a-amylase 3 has a higher pI than a-amylase 2 and results from a complex between barley a-amylase 2 and the a-amylase inhibitor 7 • Zawistowska et alY recently reported that addition of the barley a-amylase inhibitor successfully reversed the harmful effects of barley a-amylase on the breadmaking quality of wheat flour. The results of the present study suggest that the inhibitor is also likely to be effective in controlling the activity of wheat a-amylase in flour. This provides further support to the suggestion that the barley a-amylase inhibitor might have a role
HPLC OF WHEAT IX-AMYLASE
81
in the commercial utilization of wheat that has been damaged by pre-harvest sprouting 18 • These results are also consistent with the report by Mundy et a/. s indicating that the barley a-amylase inhibitor was effective in inhibiting both wheat and barley a-amylases. This work was supported by funds from the Australian Wheat Research Council. David Chadbone provided technical assistance.
References 1. Gale, M. D, in 'Third International Symposium on Pre-harvest Sprouting in Cereals' (J. E. Kruger and D. E. LaBerge, eds,) Westview Press, Boulder (1983) pp 105-110. 2. Nishikawa, K. and Nobuhara, M, Japan J. Genetics 46 (1971) 345-353. 3. Marchylo, B. A., Lacroix, L. J. and Kruger, J. E. Can. J. Plant Sci. 60 (1980) 433-443. 4. Marchylo, B., Kruger, J, E. and Irvine, G, N. Cereal Chemistry 53 (1976) 157-173. 5. Marchylo, B. A. and Kruger, J. E. in 'Third International Symposium on Pre-harvest Sprouting in Cereals' (J. E. Kruger and D. E. LaBerge, eds.), Westview Press, Boulder (1983) pp 96-104, 6. Henry, R. J. J. Chrornatogr. 481 (1989) 397-402. 7. Weselake, R. J., MacGregor, A. W. and Hill, R. D. Plant Physiol. 72 (1983) 809-812. 8. Mundy, J., Hejgaard, J. and Svendsen,!. FEBS letters 167 (1984) 210-214. 9. Zawistowska, D. in 'Wheat is unique' (Y. Pomeranz, ed.), American Association of Cereal Chemists, St Paul (1989) pp 117-129. 10. Meredith, P. and Pomeranz, Y, Adv. Cereal Sci. Technol. 7 (1985) 239-320. II. Corder, A. M. and Henry, R. J. Cereal Chern. 66 (1989) 435-439. 12. Hoy, J, T" Macauley, B. J. and Fincher, G, B. J. Inst. Brew. 87 (1981) 77-80. 13. Henry, R. J. J. Sci. Food Agric. 49 (1989) 15-24. 14. Zawistowska, D., Sangster, K., Zawistowski, J., Langstaff, J. and Frieser, A. D. Cereal Chern. 65 (1988) 413-416. 15. MacGregor, A. W., Marchylo, B. A. and Kruger, J. E. Cereal Chem. 65 (1988) 326-333. 16. Marchylo, B. A., Kruger, J. E. and MacGregor, A. W. Cereal Chern. 61 (1984) 305-310. 17. Mundy, J., Svendsen, 1. and Hejgaard, J. Carlsberg Res. Cornmun. 48 (1983) 81-90. 18. Zawistowska, D., Langstaff, J. and Bushuk, W. J. Cereal Sci. 8 (1988) 207-209.