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Spectrofluorimetric assays for hydrolytic activity in germinating wheat
Journal of Cereal Science 2 (1984) 179-185
Spectrofluorimetric Assays for Hydrolytic Activity in Germinating Wheat FLEMMING HELTVEO
Carlsberg Research Laboratory, Department of Biotechnology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark and The Technical University of Denmark, Department of Technical Biochemistry, DK-2800 Lyngby, Denmark Received 25 January 1984 and in revised form 22 March 1984 Two rapid and sensitivespectrofluorimetric methods for detecting hydrolytic activity in flour suspensions from germinated wheat are described. The methods are based upon the enzymically-catalysed hydrolysis of non-fluorescent 4-methylumbelliferyl heptanoate (MUH) and fluorescein dibutyrate (FOB) to the highly-fluorescent compounds 4-methylumbelliferone and fluorescein respectively. MUH hydrolysis was best correlated with lipase activity (using olive oil as substrate) and with ex-amylase activity, all three assays showing an exponential development of enzyme activity with increasing germination. Hydrolytic activity, measured using the FOB assay, and esterase activity (using ethyl butyrate as substrate) displayed similar development curvesduring germination, but at a much lower rate. The fluorimetric assays are rapid, sensitive and simple to perform, and were able to detect hydrolytic activity as early as 6--12 h after germination at 15 °C of grain previously steeped for 24 hat 15°C.
Introduction In recent years the distributions in grain of barley':" wheat"-5, 7, and rice" of a number of de novo-synthesised hydrolytic enzymes such as o-amylase>", cell-wall degrading enzymes 1,2,4,5, lipases/esterases':", proteases", and ribonucleases", have been studied, A similar pattern, in which enzyme activity spreads from the scutellum into the endosperm, was demonstrated for all of the enzymes investigated. It has been demonstrated that lipase'"?", esterase'"-", and protease" activities appear after 12 h of imbibition of water in wheat and increase up to the sixth day of germination as does ex-amylase activityI4-17 ,20. Thus, it is evident that several enzyme systems have potential as indicators of germination activity. Two rapid fluorescence methods have been described for measuring lipase/esterase activity based upon the hydrolysis of 4-methylumbelliferyl heptanoate (MUH)IS-20 and fluorescein dibutyrate (FOB)IS,19 to yield the highly-fluorescent compounds, 4-methylumbelliferone and fluorescein, respectively. The FOB assay has been used in recent studies5-7 ,21 as a sensitive indicator of germination to follow the pattern of development of lipase/esterase in cereal seeds. The present study was undertaken to Abbreviations used : MUH - 4-methylumbelliferyl heptanoate ; FOB - fluorescein dibutyrate; FF A - free fatty acids. 0733-5210/84/030179 +07 $03.00/0
© 1984 Academic Press Inc. (London) Limited
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determine the relationship, in germinating wheat, between lipase and esterase activities, measured using quantitative spectrofluorimetric assays based on the enzymic hydrolysis of the FDB and MUH substrates, and activities of lipase, esterase and a-amylase, measured by conventional assay techniques. In this paper, lipase activities are defined in terms of hydrolysis of water-insoluble long-chain triacylglycerols at an oil-water interface, whereas esterase activities refer to enzymes acting on water-soluble esters with short-chain acyl groups.
Materials and Methods Material Two wheat samples, one a Danish winter wheat (cv Solid) and one a sample of U.S. hard red winter wheat (a mixture of cultivars), were steeped in water for 24 hat 15°C prior to germination in rotating steel drums at 15 "C, Samples (200 g) were withdrawn after 0,6, 12,24,48, 72,96, and 120 h of germination. The comparative study of a-amylase activity and FDB and MUH hydrolase assays (Fig. 3) was carried out using seeds that had been air-dried at 30°C (not frozen) while the other analyses were performed on samples frozen immediately in liquid nitrogen, stored at - 18 "C for four months and then air-dried at 30°C immediately before analysis (Figs. I and 2).
Determination of lipase activity Lipase activity (liberation of free fatty acids, FFA, from emulsified olive oil) was determined by a method (T. Galliard and J. C. Taylor, unpubl.) based on extraction of FFA22 and colorimetric estimation of the copper soaps of FFA23. Grain samples (25 g) were milled in a Udy mill fitted with a 0·5 mm sieve and portions (5 g) were defatted by extracting twice with acetone (50 ml) at -18°C and once with diethyl ether at 21°C; residual ether was removed in vacuo. A sample (0'5 g) of defatted flour was mixed with 0·04 M imidazole-HCl buffer, pH 7·0 (0'5 ml) and an emulsion (0·5 ml) containing 0·02 g of a mixture of olive oil-Tween 80 (I :0·04, vIv) in 0·04 M imidazole buffer, pH 7·0. The mixture was incubated at 21°C for 30min and the reaction was then stopped by adding 5 ml of a mixture of propan-J-ol-heptane-O'I M H 2S04 (4: I :0·2 by vol.). Distilled water (2 ml) and heptane (3 ml) were added. After mixing, the phases were allowed to separate and 2·5 ml of the upper, heptane layer were taken for analysis of FFA content by the copper soap method'" using palmitic acid as a standard. Zero-time incubation values were obtained by addition of the propan-z-ol-heptane-Hjso, mixture to the flour sample before addition of substrate and buffer solution. Absorbance values (A44 0 nm) for the coloured complex between copper soaps of FFA and diethyldithiocarbamate were compared with those given by standards containing measured amounts of palmitic acid and lipase activity was calculated as ug FFA released/min. at 21°C.
Determination of esterase activity Defatted flour (0·5 g) was extracted with 0·1 M Na borate buffer, pH 8·0 (I ml) by shaking for I h and then centrifuging for 3 min at 5000 g. The supernatant was removed and an aliquot (20 Ill) used for the assay using ethyl butyrate as substrate".
Determination of a-amylase activity, (Phadebas") Ground wheat (0,5 g) was extracted by shaking for I h with 0·2 M Na acetate buffer, pH 5·0 (I ml) containing CaCI 2 (0·2 g/l), The extract was centrifuged at 2000 g for 10 min and an aliquot (50 Ill) of the supernatant was diluted with distilled water (4'0 ml) at 37,oC. a-Amylase activity in
HYDROLYTIC ACTIVITY IN GERMINATING WHEAT
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the diluted supernatant fraction was determined using Phadebasf as substrate as described by Barnes and Blakeney's.
Spectrofluorimetric measurement ofjluorescein released in the FDB hydrolase assay All fluorescence measurements were carried out with a Jasco FP 550 Spectrofluorometer. Flour (20 mg) was transferred to a 25 ml test tube and suspended in 0·2 M Tris-HCI buffer, pH 8·0 (20 ml) and mixed carefully. An aliquot (3 ml) of the flour suspension was transferred to a quartz cuvette and a 0·1 M solution of FDB in acetone (25 Ill) was added and mixed vigorously. The change in fluorescence intensity with time at 20°C was recorded at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The enzyme activity is expressed as the change in fluorescence intensity with time (LlF/ Llt).
Spectrojluorimetric measurement of 4-methylumbelliferone released in the MUH hydrolase assay The fluorescence measurement of MUH was carried out by suspending the wheat flour (20'0 mg) in 0·2 M Tris-HCI buffer, pH 8·5 (20 ml). At zero time, 0·1 M MUH in 96% ethanol (25 Ill) was added to an aliquot (3 ml) of the flour suspension. The change in fluorescence intensity with time at 20°C was recorded at excitation and emission wavelengths of 330 and 450 nm respectively.
Comparison of enzyme activites In order to compare the development patterns of the different enzymes, enzyme activities have been expressed as a ratio of the activity of each enzyme at different stages of germination to the activity of that enzyme in ungerminated wheat (0 h). Because of the low enzyme activities in the ungerminated samples, four replicate measurements were made with these, whereas duplicate measurements were made with the germinated samples.
Results and Discussion
Incubation of suspensions of flours (prepared from different samples of germinated wheat) with FDB and MUH, gave linear increases in fluorescence intensity when recordings were made continuously up to 5 min (Fig. la and b respectively). With increasing germination time, fluorescence intensity per unit time, AF/ AT, increased substantially. In the case of FDB hydrolysis, an increase in enzyme activity was detected after 24 h of germination whilst in the parallel study of MUH hydrolysis, an increase in enzyme activity was detected as early as 12 h after germination. The results for the hydrolysis of FDB and MUH in buffered flour suspensions were almost identical to those obtained previously with soluble extracts of flour'". The previous publication" reported that solutions of FDB were less stable with respect to non-enzymic hydrolysis (i.e. with flour that had been heated dry for 30 min at 100 "C or in buffer solution alone), than were solutions of MUH. This finding has been confirmed in the present work. In Fig. 2 the development of lipase and esterase activities during germination is illustrated for the Danish wheat sample, which had been stored at - 18 "C, and these activities are compared with the FDB and MUH hydrolase assays. Lipase assays were also carried out on the same variety dried immediately after germination at 30 "C and
F. HELTVED
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FIGURE 1. Continuous recordings of the development of fluorescence intensity with time for the enzymic hydrolysis of (a) fluorescein dibutyrate (FOB) and (b) 4-methylumbelliferyl heptanoate (MUH) in samples of Danish winter wheat (cv, Solid) germinated from {}-120 h. (See text for details.)
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FIGURE 2. Increases in lipase (e), esterase (0) and MUH (A) and FOB (,0.) hydrolase activities assays in samples of Danish winter wheat (cv. Solid) germinated for {}-120 h at 15°C. Enzyme activities are expressed relative to the activities in ungerminated wheat. Germination times were measured from the end of an initial steeping period of 24 h at 15°C.
183
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FIGURE 3. Increases in ce-amylase (.) and MUH (0) and FDB (e) hydrolase activities in samples of Danish (a) and American (b) winter wheats germinated for 0-120 h. See Fig. 2 for explanations of enzyme activities and germination times.
stored at 5 "C for three months. The blanks in this material showed a high background with regard to FFA values, (T. Galliard, pers. comm.) compared to the material stored, without drying, at -18°C. Therefore, the latter material was used in the lipase and esterase studies reported here. After 12 h of germination, a two-fold increase was observed in lipase activity and this approached a l5-fold increase after 120 h of germination. The parallel study of esterase activity, however, showed only a four-fold increase after 120 h of germination. These findings coincide well with previous reports, which have shown that lipase activity increases considerably during the germination of wheat - especially in the starchy endosperm" - whereas esterase activity, using ethyl acetate and ethyl butyrate as substrates, showed only a slight increase during the course of germination". It is also seen in Fig. 2 that the exponential increase in the MUH hydrolase activity compared well with the increase in lipase activity, whereas the FOB hydrolase and esterase activity curves showed a smaller and more linear increase with germination time. The correlation between MUH hydrolase activity and lipase activity was highly significant (r = 0·998) confirming the findings of Guilbault and co-workers" that MUH is the fluorochrome substrate best suited for the measurement of wheat lipase activity. On the other hand, FOB hydrolase activity was also correlated highly with lipase activity (r = 0·985) although the hydrolysis of FOB occurred at a much lower rate. Slightly lower
184
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correlations with esterase activity were obtained for MUH hydrolase activity (r = 0'980) and for FDB hydrolase activity (r = 0'953). In Fig. 3 the logarithmic increases in a-amylase activity and in the FDB and MUH hydrolase activities are shown for Danish and American winter wheats. It is apparent that a-amylase activity increased rapidly in the early stages of germination. After germination for 6 h, four-fold and three-fold increases in a-amylase activity were observed for the Danish and American wheats, respectively. After prolonged germination, the a-amylase activity increased exponentially in both samples (up to a 500-700 fold increase after 120 h of germination). In the fluorimetric assays, traces of hydrolysis products were detectable in the FDB and MUH assays as early as 6 h after the start of germination (Fig. 3). The values obtained for germinated wheats, analysed immediately after drying, were substantially higher than those for samples stored at -18°C and then dried, (see Figs 1 and 2). When expressed relative to the enzyme activity in ungerminated samples, activities in germinated grain, as measured using both fluorometric assays (Fig. 3a and b), were lower than the a-amylase activity at all stages of germination. After 96 h of germination (Fig. 3a) the MUH assay gave 25, the FDB assay 4 and the a-amylase assay 400 relative units of enzyme activity, respectively. The correlation coefficients with a-amylase activity were (r = 0,971) and (r = 0'796) for the MUH and FDB hydrolase activities respectively (Fig.3a). The present data demonstrate that the MUH substrate possesses several advantages in a spectrofluorimetric assay as compared with the FDB substrate. Firstly, MUH has a greater stability in solution than does FDB, thus resulting in lower blank values. Secondly, MUH hydrolase activity is correlated more highly with lipase and a-amylase activities than is FOB hydrolase activity. On the other hand , FOB seems best suited for visual identification of hydrolytic enzyme activity in germinated cereal seeds because of the intense appearance of the yellow fluorescence from fluorescein compared with the blue fluorescence due to the release of 4-methylumbelliferone, which is much more difficult to observe by eye. In the light of similar results obtained with germinated barley-- 5 , it is concluded that, in the endosperm of wheat, the pattern of development of FDB hydrolase activity approximately follows those of cell-wall degrading enzymes and of a-amylase. The FDB substrate is suitable, therefore, as an indicator of the extent of a-amylase distribution in germinated wheat endosperm': 7, whereas the MUH substrate, as used in the spectrof1uorimetric assay, is preferable as a sensitive indicator of hydrolytic activity that correlates well with that of a-amylase , at least in the two different samples of wheat that were used in the present study . Furthermore, the MUH hydrolase assay offers promise as a means of assessing the degree to which lipase activity is present in cereals and cereal products. The fluorescence methods presented here are well-suited as checks for hydrolytic enzyme activity in large numbers of samples, because of the rapid sample preparation followed by the short incubation time of 2-5 min per sample. Part of the present study was performed during a Workshop on ' The Uses of Fluorescence Analysis in Cereal Science' at Carlsberg Research Center and arranged by the International Association
HYDROLYTIC ACTIVITY IN GERMINATING WHEAT
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of Cereal Chemistry and the Danish Cereal Society in August 1983. The advice of Professor G. G. Guilbault and Dr R. Saunders during the Workshop is greatly acknowledged . I would also like to thank Dr T. Galliard and Mrs. J. C. Taylor for supplying details of the lipase assay, Mr S. Pedersen for technical assistance, Ms M. Hq,j for preparing the figures and Ms K. Kirkegaard for typing the manuscript. I am indebted to Civ. Ing. K. Jorgensen, Cando Scient. S. Aastrup, Drs. S. X. Jensen and L. Munck for stimulating discussions and for reviewing the manuscript. The work was partly supported by a grant from Statens Teknisk Videnskabelige Forskningsrad, Copenhagen, Denmark.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Gibbons, G. C. Carlsberg Res. Commun.45 (1980) 177-184. Gibbons, G. c., Aastrup, S. and Munck L. Proceedings of EBC Malt Meeting, (1981) pp 154-166 . Okamoto, K., Kitano, H . and Akazawa, T. Plant and Cell Physiol. 21 (1980) 201-204. Aastrup, S. and Erdal, K. Carlsberg Res. Commun. 45 (1980) 369-379 . Jensen, S. Aa. and Heltved , F. Carlsberg Res. Commun. 47 (1982) 297-304. Jensen, S. Aa. and Heltved, F. Carlsberg Res. Commun. 48 (\983) 1-8. Jensen, S. Aa., Munck, L. and Kruger, J. E. J. Cereal Sci. 2 (1984) 187-201. Drapron, R., Anh, N. X., Launay, B. and Builbot, A. Cereal Chem . 46 (1969) 646--655. Pancholy, S. K. and Lynd, J. Q. Phytochemistry 11 (1972) 643-645. Stauffer , C. E. and Glass , R. L. Cereal Chem . 43 (1966) 644-657 . Sullivan , B. and Howe , M. A. J. Am . Chem. Soc . 55 (1933) 320-324. Tavener, R. J. A. and Laidman, D . L. Phytochemistry 11 (1972) 989-997. Kruger, J. E. and Preston, K . Cereal Res. Commun. 4 (1976) 213-219. Barnes, W. C. and Blakeney, A. B. Die Starke 26 (1974) 193-197. Campbell, J . A. Cereal Res. Commun. 8 (1980) 107. Hagberg, S. and Perten, H . International Association for Cereal Chemistry, ICC Standard No. 107 (1967). Kruger, J. E. and Tipples , K. H. Cereal Res. Commun . 8 (1980) 97-105. Guilbault, G. G . and Sadar, M. H. Anal. Chem. 41 (1969) 366--368. Guilbault, G . G ., Sadar, M. H. and Arcenaux, D. Anal. Lett. 9 (1968) 551-536 . Jacks, T. J. and Kircher, M. W. Anal. Biochem. 21 (1967) 279-285 . Heltved, F., Aastrup, S.• Jensen, 0 ., Gibbons, G. C. and Munck , L. Carlsberg Res. Commun. 47 (1982) 291-296 . 22. Mosinger, J . Lipid Res. 6 (1965) 157-159 . 23. Shipe, J. Dairy Sci. 63 (1980) 193-198 . 24. Boehringer Mannheim Blochem. In! 2 (1975) 64-65.
13
CER
2
Spectrofluorimetric Assays for Hydrolytic Activity in Germinating Wheat FLEMMING HELTVEO
Carlsberg Research Laboratory, Department of Biotechnology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark and The Technical University of Denmark, Department of Technical Biochemistry, DK-2800 Lyngby, Denmark Received 25 January 1984 and in revised form 22 March 1984 Two rapid and sensitivespectrofluorimetric methods for detecting hydrolytic activity in flour suspensions from germinated wheat are described. The methods are based upon the enzymically-catalysed hydrolysis of non-fluorescent 4-methylumbelliferyl heptanoate (MUH) and fluorescein dibutyrate (FOB) to the highly-fluorescent compounds 4-methylumbelliferone and fluorescein respectively. MUH hydrolysis was best correlated with lipase activity (using olive oil as substrate) and with ex-amylase activity, all three assays showing an exponential development of enzyme activity with increasing germination. Hydrolytic activity, measured using the FOB assay, and esterase activity (using ethyl butyrate as substrate) displayed similar development curvesduring germination, but at a much lower rate. The fluorimetric assays are rapid, sensitive and simple to perform, and were able to detect hydrolytic activity as early as 6--12 h after germination at 15 °C of grain previously steeped for 24 hat 15°C.
Introduction In recent years the distributions in grain of barley':" wheat"-5, 7, and rice" of a number of de novo-synthesised hydrolytic enzymes such as o-amylase>", cell-wall degrading enzymes 1,2,4,5, lipases/esterases':", proteases", and ribonucleases", have been studied, A similar pattern, in which enzyme activity spreads from the scutellum into the endosperm, was demonstrated for all of the enzymes investigated. It has been demonstrated that lipase'"?", esterase'"-", and protease" activities appear after 12 h of imbibition of water in wheat and increase up to the sixth day of germination as does ex-amylase activityI4-17 ,20. Thus, it is evident that several enzyme systems have potential as indicators of germination activity. Two rapid fluorescence methods have been described for measuring lipase/esterase activity based upon the hydrolysis of 4-methylumbelliferyl heptanoate (MUH)IS-20 and fluorescein dibutyrate (FOB)IS,19 to yield the highly-fluorescent compounds, 4-methylumbelliferone and fluorescein, respectively. The FOB assay has been used in recent studies5-7 ,21 as a sensitive indicator of germination to follow the pattern of development of lipase/esterase in cereal seeds. The present study was undertaken to Abbreviations used : MUH - 4-methylumbelliferyl heptanoate ; FOB - fluorescein dibutyrate; FF A - free fatty acids. 0733-5210/84/030179 +07 $03.00/0
© 1984 Academic Press Inc. (London) Limited
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determine the relationship, in germinating wheat, between lipase and esterase activities, measured using quantitative spectrofluorimetric assays based on the enzymic hydrolysis of the FDB and MUH substrates, and activities of lipase, esterase and a-amylase, measured by conventional assay techniques. In this paper, lipase activities are defined in terms of hydrolysis of water-insoluble long-chain triacylglycerols at an oil-water interface, whereas esterase activities refer to enzymes acting on water-soluble esters with short-chain acyl groups.
Materials and Methods Material Two wheat samples, one a Danish winter wheat (cv Solid) and one a sample of U.S. hard red winter wheat (a mixture of cultivars), were steeped in water for 24 hat 15°C prior to germination in rotating steel drums at 15 "C, Samples (200 g) were withdrawn after 0,6, 12,24,48, 72,96, and 120 h of germination. The comparative study of a-amylase activity and FDB and MUH hydrolase assays (Fig. 3) was carried out using seeds that had been air-dried at 30°C (not frozen) while the other analyses were performed on samples frozen immediately in liquid nitrogen, stored at - 18 "C for four months and then air-dried at 30°C immediately before analysis (Figs. I and 2).
Determination of lipase activity Lipase activity (liberation of free fatty acids, FFA, from emulsified olive oil) was determined by a method (T. Galliard and J. C. Taylor, unpubl.) based on extraction of FFA22 and colorimetric estimation of the copper soaps of FFA23. Grain samples (25 g) were milled in a Udy mill fitted with a 0·5 mm sieve and portions (5 g) were defatted by extracting twice with acetone (50 ml) at -18°C and once with diethyl ether at 21°C; residual ether was removed in vacuo. A sample (0'5 g) of defatted flour was mixed with 0·04 M imidazole-HCl buffer, pH 7·0 (0'5 ml) and an emulsion (0·5 ml) containing 0·02 g of a mixture of olive oil-Tween 80 (I :0·04, vIv) in 0·04 M imidazole buffer, pH 7·0. The mixture was incubated at 21°C for 30min and the reaction was then stopped by adding 5 ml of a mixture of propan-J-ol-heptane-O'I M H 2S04 (4: I :0·2 by vol.). Distilled water (2 ml) and heptane (3 ml) were added. After mixing, the phases were allowed to separate and 2·5 ml of the upper, heptane layer were taken for analysis of FFA content by the copper soap method'" using palmitic acid as a standard. Zero-time incubation values were obtained by addition of the propan-z-ol-heptane-Hjso, mixture to the flour sample before addition of substrate and buffer solution. Absorbance values (A44 0 nm) for the coloured complex between copper soaps of FFA and diethyldithiocarbamate were compared with those given by standards containing measured amounts of palmitic acid and lipase activity was calculated as ug FFA released/min. at 21°C.
Determination of esterase activity Defatted flour (0·5 g) was extracted with 0·1 M Na borate buffer, pH 8·0 (I ml) by shaking for I h and then centrifuging for 3 min at 5000 g. The supernatant was removed and an aliquot (20 Ill) used for the assay using ethyl butyrate as substrate".
Determination of a-amylase activity, (Phadebas") Ground wheat (0,5 g) was extracted by shaking for I h with 0·2 M Na acetate buffer, pH 5·0 (I ml) containing CaCI 2 (0·2 g/l), The extract was centrifuged at 2000 g for 10 min and an aliquot (50 Ill) of the supernatant was diluted with distilled water (4'0 ml) at 37,oC. a-Amylase activity in
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the diluted supernatant fraction was determined using Phadebasf as substrate as described by Barnes and Blakeney's.
Spectrofluorimetric measurement ofjluorescein released in the FDB hydrolase assay All fluorescence measurements were carried out with a Jasco FP 550 Spectrofluorometer. Flour (20 mg) was transferred to a 25 ml test tube and suspended in 0·2 M Tris-HCI buffer, pH 8·0 (20 ml) and mixed carefully. An aliquot (3 ml) of the flour suspension was transferred to a quartz cuvette and a 0·1 M solution of FDB in acetone (25 Ill) was added and mixed vigorously. The change in fluorescence intensity with time at 20°C was recorded at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. The enzyme activity is expressed as the change in fluorescence intensity with time (LlF/ Llt).
Spectrojluorimetric measurement of 4-methylumbelliferone released in the MUH hydrolase assay The fluorescence measurement of MUH was carried out by suspending the wheat flour (20'0 mg) in 0·2 M Tris-HCI buffer, pH 8·5 (20 ml). At zero time, 0·1 M MUH in 96% ethanol (25 Ill) was added to an aliquot (3 ml) of the flour suspension. The change in fluorescence intensity with time at 20°C was recorded at excitation and emission wavelengths of 330 and 450 nm respectively.
Comparison of enzyme activites In order to compare the development patterns of the different enzymes, enzyme activities have been expressed as a ratio of the activity of each enzyme at different stages of germination to the activity of that enzyme in ungerminated wheat (0 h). Because of the low enzyme activities in the ungerminated samples, four replicate measurements were made with these, whereas duplicate measurements were made with the germinated samples.
Results and Discussion
Incubation of suspensions of flours (prepared from different samples of germinated wheat) with FDB and MUH, gave linear increases in fluorescence intensity when recordings were made continuously up to 5 min (Fig. la and b respectively). With increasing germination time, fluorescence intensity per unit time, AF/ AT, increased substantially. In the case of FDB hydrolysis, an increase in enzyme activity was detected after 24 h of germination whilst in the parallel study of MUH hydrolysis, an increase in enzyme activity was detected as early as 12 h after germination. The results for the hydrolysis of FDB and MUH in buffered flour suspensions were almost identical to those obtained previously with soluble extracts of flour'". The previous publication" reported that solutions of FDB were less stable with respect to non-enzymic hydrolysis (i.e. with flour that had been heated dry for 30 min at 100 "C or in buffer solution alone), than were solutions of MUH. This finding has been confirmed in the present work. In Fig. 2 the development of lipase and esterase activities during germination is illustrated for the Danish wheat sample, which had been stored at - 18 "C, and these activities are compared with the FDB and MUH hydrolase assays. Lipase assays were also carried out on the same variety dried immediately after germination at 30 "C and
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FIGURE 1. Continuous recordings of the development of fluorescence intensity with time for the enzymic hydrolysis of (a) fluorescein dibutyrate (FOB) and (b) 4-methylumbelliferyl heptanoate (MUH) in samples of Danish winter wheat (cv, Solid) germinated from {}-120 h. (See text for details.)
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FIGURE 2. Increases in lipase (e), esterase (0) and MUH (A) and FOB (,0.) hydrolase activities assays in samples of Danish winter wheat (cv. Solid) germinated for {}-120 h at 15°C. Enzyme activities are expressed relative to the activities in ungerminated wheat. Germination times were measured from the end of an initial steeping period of 24 h at 15°C.
183
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96
120
FIGURE 3. Increases in ce-amylase (.) and MUH (0) and FDB (e) hydrolase activities in samples of Danish (a) and American (b) winter wheats germinated for 0-120 h. See Fig. 2 for explanations of enzyme activities and germination times.
stored at 5 "C for three months. The blanks in this material showed a high background with regard to FFA values, (T. Galliard, pers. comm.) compared to the material stored, without drying, at -18°C. Therefore, the latter material was used in the lipase and esterase studies reported here. After 12 h of germination, a two-fold increase was observed in lipase activity and this approached a l5-fold increase after 120 h of germination. The parallel study of esterase activity, however, showed only a four-fold increase after 120 h of germination. These findings coincide well with previous reports, which have shown that lipase activity increases considerably during the germination of wheat - especially in the starchy endosperm" - whereas esterase activity, using ethyl acetate and ethyl butyrate as substrates, showed only a slight increase during the course of germination". It is also seen in Fig. 2 that the exponential increase in the MUH hydrolase activity compared well with the increase in lipase activity, whereas the FOB hydrolase and esterase activity curves showed a smaller and more linear increase with germination time. The correlation between MUH hydrolase activity and lipase activity was highly significant (r = 0·998) confirming the findings of Guilbault and co-workers" that MUH is the fluorochrome substrate best suited for the measurement of wheat lipase activity. On the other hand, FOB hydrolase activity was also correlated highly with lipase activity (r = 0·985) although the hydrolysis of FOB occurred at a much lower rate. Slightly lower
184
F.HELTVED
correlations with esterase activity were obtained for MUH hydrolase activity (r = 0'980) and for FDB hydrolase activity (r = 0'953). In Fig. 3 the logarithmic increases in a-amylase activity and in the FDB and MUH hydrolase activities are shown for Danish and American winter wheats. It is apparent that a-amylase activity increased rapidly in the early stages of germination. After germination for 6 h, four-fold and three-fold increases in a-amylase activity were observed for the Danish and American wheats, respectively. After prolonged germination, the a-amylase activity increased exponentially in both samples (up to a 500-700 fold increase after 120 h of germination). In the fluorimetric assays, traces of hydrolysis products were detectable in the FDB and MUH assays as early as 6 h after the start of germination (Fig. 3). The values obtained for germinated wheats, analysed immediately after drying, were substantially higher than those for samples stored at -18°C and then dried, (see Figs 1 and 2). When expressed relative to the enzyme activity in ungerminated samples, activities in germinated grain, as measured using both fluorometric assays (Fig. 3a and b), were lower than the a-amylase activity at all stages of germination. After 96 h of germination (Fig. 3a) the MUH assay gave 25, the FDB assay 4 and the a-amylase assay 400 relative units of enzyme activity, respectively. The correlation coefficients with a-amylase activity were (r = 0,971) and (r = 0'796) for the MUH and FDB hydrolase activities respectively (Fig.3a). The present data demonstrate that the MUH substrate possesses several advantages in a spectrofluorimetric assay as compared with the FDB substrate. Firstly, MUH has a greater stability in solution than does FDB, thus resulting in lower blank values. Secondly, MUH hydrolase activity is correlated more highly with lipase and a-amylase activities than is FOB hydrolase activity. On the other hand , FOB seems best suited for visual identification of hydrolytic enzyme activity in germinated cereal seeds because of the intense appearance of the yellow fluorescence from fluorescein compared with the blue fluorescence due to the release of 4-methylumbelliferone, which is much more difficult to observe by eye. In the light of similar results obtained with germinated barley-- 5 , it is concluded that, in the endosperm of wheat, the pattern of development of FDB hydrolase activity approximately follows those of cell-wall degrading enzymes and of a-amylase. The FDB substrate is suitable, therefore, as an indicator of the extent of a-amylase distribution in germinated wheat endosperm': 7, whereas the MUH substrate, as used in the spectrof1uorimetric assay, is preferable as a sensitive indicator of hydrolytic activity that correlates well with that of a-amylase , at least in the two different samples of wheat that were used in the present study . Furthermore, the MUH hydrolase assay offers promise as a means of assessing the degree to which lipase activity is present in cereals and cereal products. The fluorescence methods presented here are well-suited as checks for hydrolytic enzyme activity in large numbers of samples, because of the rapid sample preparation followed by the short incubation time of 2-5 min per sample. Part of the present study was performed during a Workshop on ' The Uses of Fluorescence Analysis in Cereal Science' at Carlsberg Research Center and arranged by the International Association
HYDROLYTIC ACTIVITY IN GERMINATING WHEAT
185
of Cereal Chemistry and the Danish Cereal Society in August 1983. The advice of Professor G. G. Guilbault and Dr R. Saunders during the Workshop is greatly acknowledged . I would also like to thank Dr T. Galliard and Mrs. J. C. Taylor for supplying details of the lipase assay, Mr S. Pedersen for technical assistance, Ms M. Hq,j for preparing the figures and Ms K. Kirkegaard for typing the manuscript. I am indebted to Civ. Ing. K. Jorgensen, Cando Scient. S. Aastrup, Drs. S. X. Jensen and L. Munck for stimulating discussions and for reviewing the manuscript. The work was partly supported by a grant from Statens Teknisk Videnskabelige Forskningsrad, Copenhagen, Denmark.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Gibbons, G. C. Carlsberg Res. Commun.45 (1980) 177-184. Gibbons, G. c., Aastrup, S. and Munck L. Proceedings of EBC Malt Meeting, (1981) pp 154-166 . Okamoto, K., Kitano, H . and Akazawa, T. Plant and Cell Physiol. 21 (1980) 201-204. Aastrup, S. and Erdal, K. Carlsberg Res. Commun. 45 (1980) 369-379 . Jensen, S. Aa. and Heltved , F. Carlsberg Res. Commun. 47 (1982) 297-304. Jensen, S. Aa. and Heltved, F. Carlsberg Res. Commun. 48 (\983) 1-8. Jensen, S. Aa., Munck, L. and Kruger, J. E. J. Cereal Sci. 2 (1984) 187-201. Drapron, R., Anh, N. X., Launay, B. and Builbot, A. Cereal Chem . 46 (1969) 646--655. Pancholy, S. K. and Lynd, J. Q. Phytochemistry 11 (1972) 643-645. Stauffer , C. E. and Glass , R. L. Cereal Chem . 43 (1966) 644-657 . Sullivan , B. and Howe , M. A. J. Am . Chem. Soc . 55 (1933) 320-324. Tavener, R. J. A. and Laidman, D . L. Phytochemistry 11 (1972) 989-997. Kruger, J. E. and Preston, K . Cereal Res. Commun. 4 (1976) 213-219. Barnes, W. C. and Blakeney, A. B. Die Starke 26 (1974) 193-197. Campbell, J . A. Cereal Res. Commun. 8 (1980) 107. Hagberg, S. and Perten, H . International Association for Cereal Chemistry, ICC Standard No. 107 (1967). Kruger, J. E. and Tipples , K. H. Cereal Res. Commun . 8 (1980) 97-105. Guilbault, G. G . and Sadar, M. H. Anal. Chem. 41 (1969) 366--368. Guilbault, G . G ., Sadar, M. H. and Arcenaux, D. Anal. Lett. 9 (1968) 551-536 . Jacks, T. J. and Kircher, M. W. Anal. Biochem. 21 (1967) 279-285 . Heltved, F., Aastrup, S.• Jensen, 0 ., Gibbons, G. C. and Munck , L. Carlsberg Res. Commun. 47 (1982) 291-296 . 22. Mosinger, J . Lipid Res. 6 (1965) 157-159 . 23. Shipe, J. Dairy Sci. 63 (1980) 193-198 . 24. Boehringer Mannheim Blochem. In! 2 (1975) 64-65.
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