Occurrence and Stabilities of Oat Trypsin and Chymotrypsin Inhibitors

Journal of Cereal Science 30 (1999) 227–235 Article No. jcrs.1999.0280, available online at http://www.idealibrary.com on

Occurrence and Stabilities of Oat Trypsin and Chymotrypsin Inhibitors M. Mikola∗ and A. Mikkonen† ∗University of Helsinki, Department of Food Technology, P.O. Box 27, FIN-00014 Finland; †Technical Centre of Finland, Biotechnology and Food Research, P.O. Box 1501, FIN-02044, Finland Received 2 June 1998

ABSTRACT Nine chymotrypsin and four trypsin inhibitors have been extracted and separated from ungerminated oat grains. The inhibitors fell into two groups, based on their heat and pH stabilities. Members of the most abundant group are labile and are inactivated at 80 °C or at pHs of 3·3 or lower. Members of the second group are stable and are resistant to boiling for 30 min. On germination, the labile inhibitors are inactivated after 2 days and the stable chymotrypsin inhibitors after 3 days. Most of the labile inhibitors from ungerminated grain are destroyed when incubated at 20 °C for 20 h but addition of PMSF, a serine protease inhibitor, prevented their inactivation. Labile inhibitors in extracts of ungerminated oats are inactivated on incubation with an extract prepared from germinated oats, but not in the presence of PMSF. Most oat chymotrypsin and trypsin inhibitors are heat labile and pH sensitive. These inhibitors are apparently inactivated by serine proteinase(s) already present in ungerminated grain.  1999 Academic Press

Keywords: proteinase inhibitors, trypsin inhibitors, chymotrypsin inhibitors.

INTRODUCTION Oats are used in foods such as biscuits, muesli and porridge, and a recent report shows that oats are suitable for use by adult coeliac disease patients1. The good overall nutritional quality of oats has been well documented2. However, oats contain proteinase inhibitors which, though considered to be antinutritional compounds, have not been studied in detail. Proteinaceous proteinase inhibitors are wide-

 : PAGE=Polyacrylamide gel electrophoresis, OTI=Oat trypsin inhibitor; OCI= Oat chymotrypsin inhibitor; PMSF=Phenylmethylsulphonyl fluoride, EDTA=Ethylenediaminetetraacetic acid; E-64=trans-epoxysuccinyl-L-lucylamid(4guanidino) butane; Pep A=pepstatin A; O-Phen=1, 10 phenanthroline. Corresponding author: Markku Mikola, Phone: 358-9-708 58638; Fax: 358-9-708 58212; e-mail: [email protected] 0733–5210/99/110227+09 $30.00/0

spread in nature. They regulate protein hydrolysis and, in plants, may serve in defence, against pest attack3. Trypsin and chymotrypsin inhibitors have been detected in all cereal grains. Several barley, wheat and rye trypsin and chymotrypsin inhibitors have been purified and characterized, but those of oats (Avena sativa L.) are less well studied4. The amounts of these inhibitors vary in different cereals. The trypsin inhibiting activity of rye is about double that of barley and 10-fold higher than that of wheat and oats5. The inhibitors have been detected in both endosperm and embryo, the embryo levels being approximately 10-fold higher than the endosperm5. Barley6,7 and rye8 amylase/trypsin inhibitors have been investigated with respect to their relationship with baker’s asthma. Barley protein Z7 (barley serpin) was shown to have a weak inhibitory effect towards chymotrypsin9. Recently Hejgaard and co-workers purified the chymotrypsin inhibiting Z type serpin from wheat10. They also  1999 Academic Press

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successfully expressed wheat serpin (WSZ1) and barley BSZx and BSZ4 in E. coli11. These recombinant proteins all have inhibitory effects against cathepsin G; two of them, BSZx and WSZ1, also inhibit chymotrypsin and BSZx additionally inhibits trypsin11. The trypsin inhibiting activity of aqueous extracts prepared from oats is partially inactivated by boiling12. Fractionation of an oat extract by ion exchange and gel filtration chromatography yielded a single trypsin inhibitor of Mr 43 5005. Three individual oat subtilisin-inhibiting activities, with pI values between 5·5 and 6·5, were detected by isoelectric focusing and specific inhibition staining13. In barley, the endosperm trypsin inhibiting activity disappeared between the fourth and fifth days of germination14. The aim of the present study was to determine whether individual oat proteinase inhibitors are heat labile in solution, whether they disappear during germination, and whether proteinases in germinating grain inactivate the inhibitors in vitro. The study was performed using nondenaturing polyacrylamide gel electrophoresis (PAGE), combined with in situ overlay assays13,15 that are specific for chymotrypsin and trypsin inhibition. EXPERIMENTAL Plant material and extraction Oat (Avena sativa) cv. ‘Veli’ seeds from the 1994 harvest were obtained from the Agricultural Research Center of Finland, Jokioinen. For the germination experiments, seeds were dehulled by hand and only sound seeds of uniform size were used. The seeds were surface sterilised with a 1% sodium hypochlorite solution for 30 min at room temperature, followed by one wash with water, a wash with 10 m HCl, and five 5-min washes with water. The seeds were germinated in the dark at 20 °C on sterile 0·5% agar. Ten-seed samples were removed every 24 h, dried with tissue paper and frozen. Ungerminated seeds were ground in a burr mill and 20 g of the resulting fine meal was suspended in 40 mL of 20 m Tris-HCl, pH 8·0 (buffer A). Germinated seeds were homogenized with a mortar and pestle with sufficient buffer A to yield 0·5 mL of extract per 10 seeds. These extractions were completed by incubating the suspensions at room temperature, in a shaker, for 1 h, followed by centrifugation for 15 min at 10 000 g. The

supernatants were stored in aliquots at −20 °C until analysed. The extract of ungerminated seeds was called ‘crude extract’. The heat stability of the inhibitors was measured by incubating the crude extract at 60, 80 and 100 °C for either 10 or 30 min. After heat treatment the extract was cooled on an ice-water bath for 5 min and centrifuged. The supernatants were either analysed immediately by electrophoresis or stored at −20 °C for later analysis. The pH stability of the inhibitors was determined in the crude extract by adjusting to pHs 11·0, 10·0, 9·0, 4·0, 3·3 or 2·0, with either 1  HCl or 1  NaOH. After incubation for 10 min at room temperature, precipitated material was removed by centrifugation. The supernatants were neutralised with sample buffer and analysed by electrophoresis and specific staining. The stability of inhibitors in crude extracts of ungerminated grain was studied by incubating the extract for 20 h at 20 °C. Inactivation of the inhibitors by proteases from germinated seed was tested using 10 seeds germinated for 4 days (0·35 g fresh weight) extracted in 1·25 mL of buffer A which resulted in 1·5 mL of extract. This preparation was designated ‘enzyme extract’. The ‘substrate’ for these studies, was a stable inhibitor preparation obtained by incubating ‘crude extract’ at 60 °C for 30 min. Proteinases in germinated seed were examined by incubating 450 lL of substrate (corresponding to 360 mg of grain) with 50 lL of enzyme extract (corresponding to 11·7 mg fresh weight of germinated oats) at 20 °C for 20 hr, prior to electrophoretic analysis. Proteinases were classified using the following class-specific inhibitors: phenylmethylsulphonyl fluoride (PMSF, 8 m) for serine proteinases, ethylenediaminetetraacetic acid (EDTA, 5 m) for metalloproteinases, Pepstatin A (Pep A, 10 l) for aspartic proteinases and trans-epoxysuccinylL-lucylamido(4-guanidino) butane (E-64, 10 l) for cysteine proteinases. In one experiment, leupeptin (10 l), an inhibitor of cysteine proteinases and some serine proteinases, was used.

Separation and specific staining of the inhibitors Nondenaturating PAGE in the absence of sodium dodecyl sulphate (SDS) and reducing agent were run on a BioRad MiniProtean II apparatus at pH 8·3, with a Tris-glycine buffer system (0·025  Tris, 0·192  glycine)16,17. The acrylamide con-

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centrations of the 0·75 mm thick gels were 10% (separation gel) and 4% (stacking gel). Four parts of sample was mixed with one part of sample buffer. Sample buffer consisted of 1 mL of 0·5  Tris-HCL pH 6·8, 0·8 mL of glycerol and 0·01 mL of 0·5% Bromophenol Blue. Typically, 16 lL of extract, corresponding to 12·8 mg of grains, was applied per lane and the gels were run at 200 V, 20 mA/gel. Specific negative staining13, with minor modifications, was used to detect the inhibitory activities. Briefly, after electrophoresis, the gels were rinsed with water and incubated in a 100 m Na-phosphate, pH 7·6 enzyme solution containing either 5 lg/mL of chymotrypsin or 50 lg/mL of trypsin for 5 or 15 min, respectively. After rinsing with water for 1 min, the gels were incubated in substrate solution until clear bands, indicating inhibitors, were visible on a pink background. The staining took up to 15 min and was stopped by immersing the gels into 5% acetic acid. The substrate solution contained: 24 mg of Fast Blue B salt dissolved in 1 mL of water and diluted to 44 mL with 100 m Na-phosphate buffer, pH 7·6, after which 12 mg of N-acetyl-D,L-phenylalanine2-naphthyl ester in 5 mL of N,N,dimethylformamide was added.

In vitro measurement of inhibition activities Trypsin inhibiting activities were measured using benzoyl-D,L-arginine-p-nitroanilide substrate (40 mg/ 100 mL buffer) according to Kakade et al.18 with modifications. The preincubation time was 20 min at 37 °C and filtration was omitted. The volumes were scaled down to give a final volume of 1·5 mL. Briefly, the sample (0·3 mL) was mixed with the same volume of enzyme solution (2 mg/100 mL), after preincubation, substrate (0·75 mL) was added. The reaction was stopped after 10 min incubation by adding 30% acetic acid (0·15 mL) and the absorbance read at 410 nm. Chymotrypsin inhibiting activity was measured using substrate, N-glutarylL-phenylalanine-p-nitroanilide (20 mg/100 mL)19. The volumes used were as found for trypsin assays and the concentration of chymotrypsin in the enzyme solution was 0·01 mg/mL. To remove the acid before the analysis of extracts treated at pH 2·0, these were filtered using disposable PD-10 gel filtration columns (Pharmacia Biotech) and washed with extraction buffer. For comparison crude, untreated extracts were also passed though gel filtration columns.

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Soluble protein Soluble protein was measured using Bio-Rad soluble protein kit according to the manufacturer’s instructions with bovine serum albumin as standard. Reagents Trypsin, (L-1-p-tosylamino-phenylethyl chloromethyl ketone-treated bovine trypsin), chymotrypsin, (tosyl lysine chloromethyl ketone-treated bovine chymotrypsin). N-acetyl-D,L-phenylalanine-2-naphthyl ester, Fast Blue B salt, N-glutaryl-L-phenylalanine-p-nitroanilide, benzoyl-D, L-arginine-p-nitroanilide, PMSF, EDTA, E-64, leupeptin and Pepstatin A were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Other reagents were of analytical grade. Milli-Q water was used throughout. RESULTS Heat stability of inhibitors Oat trypsin and chymotrypsin inhibitors were detected in extracts by nondenaturing PAGE, followed by negative staining in an overlay assay. Nine chymotrypsin and four trypsin inhibitors were found; these were numbered according to their decreasing electrophoretic mobilities [Fig. 1(a–d), first lanes, 30;0]. The chymotrypsin inhibitor bands were named OCI (oat chymotrypsin inhibitor), and the trypsin inhibitor bands OTI (oat trypsin inhibitor). The bands OCI 2 and OCI 4 were the most intensely stained and both were preceded by low intensity bands, OCI 1 and OCI 3. The two other well-separated bands, OCI 8 and 9, had the lowest electrophoretic mobilities and were more diffuse than OCI 2 and OCI 4. Three low intensity bands, OCI 5, 6 and 7, which were not always visible, were located between OCI 4 and OCI 8 [Fig. 1(a,b) lanes 30;0]. The trypsin inhibitor pattern was not as clear as that of chymotrypsin. Two trypsin inhibitors were located in adjacent bands, OTI 2 and OTI 3. Two diffuse bands, OTI 1 and OTI 4, that were not always visible, ran ahead and behind the major bands, respectively [Fig. 1(c,d) lanes 30; 0]. In some experiments additional bands were detected (data not shown). For example, when gels were run for longer times, OTI 3 separated into two distinct bands. A diffuse band with pH and

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Time, min 30 10 30 10 30 10 30 Time, min 30 Temp C 0 60 60 80 80 100 100 Temp C 0 OCI# 9

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Figure 1 Photographs and schematic drawings of separation of the chymotrypsin (a and b) and trypsin (c and d) inhibitors in extracts of ungerminated oat grain by nondenaturing PAGE and their thermal stabilities. Band numbers are listed on the left side of photographs and drawings of the gels. The crude extracts were heated for either 10 or 30 min at 60, 80 or 100 °C, as indicated above the lanes. The control was held for at least 30 min at 0 °C.

heat stability characteristics similar to those of, and running behind, OTI 1 was also sometimes detected. When the heat stabilities of the inhibitors were tested, no changes were detected on incubation of the crude extract at 60 °C for up to 30 min [Fig. 1(a,b) lanes 10;60 and 30;60]. However, differences became apparent when the incubation temperature was increased to 80 °C. The main chymotrypsin inhibitors, OCI 1–4, were barely visible after 10 min incubation at 80 °C and disappeared completely after 30 min [Fig. 1(a,b)]. OTI 2 and 3 were the most heat sensitive trypsin inhibitors, behaving like the OCI 1–4 group [Fig. 1(c,d)]. For comparison, the in vitro inhibition activities of untreated and 30 min boiled samples were measured. The inhibiting activities extracted from 0·1 g of oats inhibited 1·2 lg of chymotrypsin and 5 lg of trypsin. The chymotrypsin inhibiting activity declined 38% after boiling and the trypsin inhibiting activity by 14%. Heat stable inhibitors of both chymotrypsin (OCI 5–9) and trypsin (OTI 1 and OTI 4) were detected. These heat stable

inhibitors retained part of their activities even when held at 100 °C for 30 min [Fig. 1(a–d)]. Effect of pH on the stabilities of the inhibitors Raising the pH of the crude extract from 6·2 to 11 did not affect any of the chymotrypsin inhibitors [Fig. 2(a)]. However, when the incubation pH was lowered to 3·3, OCI 1–4 disappeared, leaving bands OCI 5–9. After incubation at pH 2, only OCI 6, 8 and 9 remained. Similarly, the trypsin inhibitors were stable at alkaline pH values but under acidic conditions (pH 3·3) most were inactivated [Fig. 2(b)]. OTI 1 and 4, however, were still active after incubation at pH 2. The in vitro measurements showed that lowering the pH to 2·0 inactivated 18% of the chymotrypsin and 14% of the trypsin inhibiting activity. The amount of soluble protein declined from 2·8 mg/mL of crude extract to 2·1 mg/mL of the pH 2 treated sample. The possible inactivating effect of cysteine and aspartyl proteinases at low pH was tested by re-

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Figure 2 The effect of pH on the chymotrypsin (a) and trypsin (b) inhibitors in ungerminated oat extracts. Extracts were adjusted to the pH values indicated above the lanes and incubated at room temperature for at least 10 min prior to electrophoresis. The control (lane marked 6.2) contained unincubated, pH 6·2 crude extract.

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Figure 3 The disappearance of chymotrypsin (a) and trypsin (b) inhibitors during the germination of oats. Extracts of grains, germinated at 20 °C for the number of days indicated above the lanes, were separated using nondenaturing PAGE and stained for inhibitor activity.

peating the experiment (between pH 6·2 and pH 2·0) in the presence of pepstatin A and E-64. Before electrophoretic analysis the samples were filtered on PD-10 columns to remove the inhibitors. No differences in activities were observed. In a similar manner the effect of gel filtration was examined by running the samples in parallel with the original samples diluted by the same factor (results not shown). The inhibitor patterns in both cases were exactly the same. Changes in inhibitors during seed germination During the germination of oat grains at 20 °C on agar gel, the activity of chymotrypsin inhibitors OCI 1–4 and the trypsin inhibitors OTI 2 and 3 began to decrease after two days and had totally disappeared by the third day (Fig. 3). The chymotrypsin inhibitor bands OCI 8 and 9 were still

visible after 3 days of germination but disappeared by the fourth day. Inactivation of the inhibitors present in crude extracts Inactivation of most of the heat- and pH-labile chymotrypsin inhibitors and essentially all the corresponding trypsin inhibitors occurred when crude extracts of ungerminated grains were incubated at 20 °C for 20 h [Fig. 4(a,b) lanes C] at pH 6·2. Under these conditions OCI 1 and 2 activities had also decreased but were not totally inactivated. The inactivation of OCI 1–4 was totally prevented by the addition of PMSF, a serine proteinase inhibitor, to the incubation mixture [Fig. 4(a)]. A similar, partial, effect of PMSF was observed with the trypsin inhibitors [Fig. 4(b)]. Neither EDTA, (a metalloproteinase inhibitor), Pepstatin A (an

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Figure 4 In vitro inactivation of chymotrypsin (a) and trypsin (b) inhibitors by extracts of ungerminated oats. The crude extract was incubated at room temperature for 20 h in the presence or absence of class-specific proteinase inhibitors (as indicated above the lanes). U=unincubated crude extract and C=extract incubated in the absence of added class specific inhibitors. In (a) lanes U′ and C′ are photographs of lanes U and C in (a) stained first for chymotrypsin inhibition and immediately afterwards with Coomassie Brilliant Blue to locate protein bands.

aspartic proteinase inhibitor) nor E-64 (a cysteine proteinase inhibitor) retarded the inactivation of the inhibitors. When PMSF and EDTA were added together, OTI 2 and 3 were unaffected. Thus, it appears that ungerminated oats contain serine proteinase(s) that inactivate some of endogenous trypsin and chymotrypsin inhibitors. On one of the gels stained for chymotrypsin inhibitors the positions of the OCI 1–4 inhibitors was marked and the gel immediately stained with Coomassie Brilliant Blue [Fig. 4(a) U′, C′]. The protein band corresponding to OCI 4 in untreated sample (lane U′) had disappeared during the incubation (lane C′). The stained gel section shows that there are several distinct protein bands in the area of the labile inhibitors (OCI 1–4) whereas the stable inhibitors do not have corresponding bands.

Inactivation of the inhibitors by proteinases from germinating seeds Germinated oats also contain proteinases that inactivated the heat and pH labile trypsin and chymotrypsin inhibitors [Fig. 5(a,b) lanes I]. Reaction mixtures containing inhibitors with and without enzyme extracts from 4-day germinated seeds were prepared and incubated as described in Materials and Methods. When analysed by electrophoresis, the OCI 1–4 and OTI 2–3 inhibitor bands had disappeared only from reaction mixtures containing extracts from germinated seed [Fig. 5(a,b) lane I]. When various class-specific proteinase inhibitors, including leupeptin, were added to the incubation mixtures, only PMSF clearly prevented inactivation. Thus both germinated and un-

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Figure 5 In vitro inactivation of chymotrypsin (a) and trypsin (b) inhibitors in ungerminated oats by proteases from germinated oats. An ungerminated oat extract, heated at 60 °C for 30 min was used as ‘substrate’ for enzymes from 4-day germinated grain. The substrate and enzymes were incubated at 20 °C for 20 h. Class-specific protease inhibitors were added to incubation mixtures as indicated. Lane C=control, the ‘substrate’ solution incubated in the absence of ‘enzyme extract’; I=a mixture of substrate and enzyme extracts incubated in the absence of class specific inhibitors, LP=Leupeptin.

germinated oats contain serine proteinases that can inactivate some oat trypsin and chymotrypsin inhibitors. DISCUSSION We have studied oat chymotrypsin and trypsin inhibitors to clarify their physical properties and to determine their sensitivities to proteinases present in ungerminated and germinated oat grain. Using nondenaturing PAGE and specific staining, we demonstrated the existence of nine chymotrypsin and four trypsin inhibitors. This is a first step in studying these poorly understood oat inhibitors. The partial inactivation of the trypsin inhibition activity of an oat extract by boiling, reported earlier12, was apparently due to the presence of at least two different types of trypsin inhibitors. As shown here, both heat stable and heat labile types are present. The situation with chymotrypsin inhibitors seems to be quite similar. The pattern of chymotrypsin inhibition seen in oats is similar to that in barley20, from which two different chymotrypsin inhibitors have been purified. One of these is resistant to boiling, whereas the other is inactivated at temperatures above 60 °C20. Our studies on the stabilities of the inhibitors at different pHs also indicated that at least two different types of both trypsin and chymotrypsin inhibitors are present in ungerminated oats. On the other hand the results obtained using in vitro inhibition measurements were quite different. The in vitro measurements contrary to the electrophoretic results

indicate that most of the inhibitors are stable. The in vitro measurements may be affected by some interfering factors. Oat extracts cause high backgrounds in the blank samples of the analysis. Also in the untreated extracts there is present activity that does hydrolyse benzoyl-D,L-arginine-p-nitroanilide, the substrate used in trypsin inhibition experiments. This activity is not detected in heat, or low pH treated extracts, and this difference may affect the in vitro measurements. The results may also be affected by inhibition of other substances than proteins such as phytate21. Oat trypsin and chymotrypsin inhibitors are inactivated by proteinase(s) that are inhibited by PMSF. These proteinase(s) are present in both ungerminated and germinated oats. A cysteine proteinase has been reported to initiate the hydrolysis of soybean trypsin inhibitor during germination22. This proteinase was not present in ungerminated beans but was purified from the cotyledons of four-day-germinated beans. In addition to this ‘initiating’ cysteine proteinase, other proteinases were detected that inactivated the proteinase inhibitors22. In mung beans, a serine proteinase was present in ungerminated seeds that initiates the degradation of one trypsin inhibitor23. This mung bean proteinase had a molecular size of 65 kD and a pH optimum of about 523. In our study, the experiments were conducted at pH 6·2, which was the pH of the crude extract. In barley, at least one serine proteinase has been detected in ungerminated seeds and seven electrophoretically separable serine proteinases were present in germinated seed24,25. The major barley serine pro-

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teinase, which was also present in small amounts in ungerminated seeds, had an apparent pH optimum between pH 5·3–6·5. Four cysteine proteinases purified from barley grains have been shown to be inhibited by E-64 and/or leupeptin whereas only one was slightly (28%) inhibited by PMSF26–29. Our results show that the inactivation of some of the oat trypsin and chymotrypsin inhibitors was prevented by PMSF but not by E-64 or leupeptin (Fig. 5) indicating that serine proteinase(s) are responsible for the inactivation. At least two types of both trypsin and chymotrypsin inhibitors are present in oats. The groups differ in their pH and thermal stabilities. The inhibitors of pH and heat labile groups were shown to be inactivated in vitro by serine proteinase(s) present in both ungerminated and germinated oat seeds. For human use oats are usually heat treated to prevent spoiling, but for animal feed they are often used in the farm without processing. The low inhibitor activity of oats5 together with our results of their inactivation at low pH (pH of human stomach) and high temperatures show that oat chymotrypsin and trypsin inhibitors should not cause nutritional problems.

7.

8.

9. 10. 11.

12.

13.

Acknowledgments The Wilhuri Foundation, Raisio Foundation and Foundation for Biotechnical and Industrial Fermentation Research are gratefully acknowledged for partial funding of this study. We thank Dr B.L. Jones for reviewing the manuscript. We also thank Dr M. Saastamoinen for providing the seed sample and Mr J. Mattila for technical assistance.

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