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Isolation of α-amylases and other starch degrading enzymes from endosperm of germinating maize
Plant Science, 78 (1991) 143-150 Elsevier Scientific Publishers Ireland Ltd.
143
Isolation of c -amylases and other starch degrading enzymes from endosperm of germinating maize* Donn A. Warner and Clarence A. Knutson Seed Biosynthesis Research Unit. Agricultural Research Service, U.S. Department of Agriculture, National Center .['or Agricultural Utilization Research, 1815 N. University Street, Peoria. IL 61604 (U.S.A.) (Received February 28th, 1991; revision received May 6th, 1991; accepted May 13th, 1991 ) An improved purification procedure preserved high levels of a-amylase activity, and revealed and separated other starch-degrading activities from endosperm of germinating maize (Zea mays L.). Amylases were partially purified by hydroxylapatite chromatography, and separated by chromatofocusing and affinity chromatography on cycloheptaamylose. Chromatofocusing resolved eight protein peaks with amylase activity. The binding of amylase activity to cycloheptaamylose ranged from 0 to 90% for these peaks, lsoelectric focusing and analysis of substrate specificities showed two major groups of a-amylase with five subgroups, two pullulanase enzymes, and one ~3-amylasc. This procedure is a significant improvement over the use of protein precipitation and affinity chromatography which results in large, and possibly selective, losses of amylase activity. Key words." or-amylase; t3-amylase; pullulanase; maize; endosperm; starch hydrolysis
Introduction
Previous work in this laboratory has shown that multiple forms of starch degrading enzymes occur in germinating maize [1]. Because they differ in substrate specificities and reaction products, each form may have a specific role in the degradation of storage starch granules in vivo [21. Individual forms of each enzyme must be isolated to determine their biochemical characteristics, and describe their roles in the pathway of starch granule degradation. Our goal is to determine the ability of these individual enzyme forms to digest granular starch in vitro. In our previous work, amylases were fractionated by a combination of affinity chromatoCorrespondence to." Donn A. Warner, USDA/NCAUR. 1815 N. University Street, Peoria, IL 61604, U.S.A. Abbreviations: CHA, cycloheptaamylose; IEF, isoelectric focusing. *The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.
graphy, ion exchange chromatography, hydroxylapatite chromatography, and chromatofocusing. Four major fractions of c~-amylase (three affinitybound, one unbound) had distinctive action patterns. This combination procedure provided a convenient method for separation of major forms of amylases found in maize. A significant disadvantage of that isolation scheme was the loss of 50°/,, of the amylase activity in the acetone precipitation step used to concentrate the proteins in the crude extract. The possibility that the loss of activity was selective for certain enzyme forms precluded a complete characterization of the maize amylases. We could not be certain that the isolated enzymes were present in the same proportions as they are in vivo, or that all of the amylases had been identified and separated. Furthermore, we did not know whether decreases in activity were due to losses or inactivation of enzymes during processing, or were due to loss of synergistic action. Maltase and debranching activities were present in crude extracts, but were lost during purification. Another limitation of the original isolation scheme was that only
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
144 about half of the total activity bound to the affinity chromatography column, with substantial variation in the amount of activity that was bound. Therefore it was unclear whether enzymes isolated from one purification were identical to those from another. For these reasons we devised an alternate approach for separation of these enzymes which preserved more of the original activity, verified and extended our previous results, and more accurately characterized the roles of the many amylolytic enzymes in the pathway of starch degradation in germinating maize. Our goal in this study was to increase the yield of amylase activity, and to prevent the possible selective loss of particular enzyme species. The new procedure used chromatography on hydroxylapatite as the first step in purification, followed by chromatofocusing. Chromatofocusing resolved eight protein peaks. Each chromatofocusing peak had a characteristic binding profile on cycloheptaamylose (CHA). Five major c~-amylases, two pullulanase enzymes, and one 3-amylase were separated. Materials and Methods
Kernels of maize (Zea mays L.) inbred B73 were surface-sterilized with 1% (w/v) NaOCl for 10 min, imbibed overnight in 1 mM CaCI2, and germinated for 6 days at 25°C in the dark. Seedling axes were - 7 cm long. All germinated kernels were free of visible mold. Endosperms plus pericarps were stored in a freezer at -80°C for 2 days. We have observed no change in extractable activity nor in isozyme banding pattern with up to three months of storage. The frozen tissue was ground with a Polytron homogenizer in - 1.6 ml/g tissue of extraction buffer containing 20 mM Naacetate (pH 5.0) and 1 mM CaCIe. The homogenate was stirred on ice for 15 min, then strained through number 20 wire mesh and centrifuged at 20 000 x g for 10 min. The supernatant fraction was saved, and the solids resuspended in extraction buffer and centrifuged as before. The combined supernatants were filtered through two layers of Whatman number 1 filter paper and a Millipore 0.22 /~m type GS membrane, then con-
centrated with Centriprep 10 filtration concentrators (Amicon, Danvers, MA). The concentrated extract was dialyzed at 4°C overnight against a solution containing 10 mM K-phosphate (pH 6.0) and 0.1 mM CaCI2, and was then filtered through a 0.22-t~m membrane. All chromatography was done at 4°C on an FPLC liquid chromatography system (PharmaciaLKB, Piscataway, N J). Bio-Gel HT hydroxylapatite (Bio-Rad, Richmond, CA) was packed into a Pharmacia HR 10/10 column (10 cm long x 1 cm diam.) to a bed volume of 7.5 ml. A preliminary experiment using a linear gradient showed that all hydroxylapatite-bound amylase activity eluted in the range from 10 to 300 mM Kphosphate. Thereafter, amylases were eluted in one batch with 300 mM K-phosphate to prevent unnecessary dilution. Estimated capacity was 30 mg protein for the hydroxylapatite column, so the extract was divided into two portions which were chromatographed separately. The starting buffer for hydroxylapatite contained 10 mM Kphosphate pH 6.0 and 0.1 mM CaCI 2, and the flow rate was 1.0 ml/min. After unbound proteins were washed off, bound proteins were eluted with a solution containing 300 mM K-phosphate pH 6.0 and 0.1 mM CaCI2 which had been filtered to remove precipitated Ca3(PO4)> All fractions with amylase activity were pooled, concentrated with Centriprep 10 concentrators, and dialyzed at 4°C against 25 mM BisTris (pH 6.3) (Sigma, St. Louis, MO) containing 0.1 mM CaCI, for chromatofocusing. Chromatofocusing was done with a Pharmacia Mono P column. The starting buffer contained 25 mM BisTris (pH 6.3) and 0.1 mM CaCIz, and the elution buffer was Polybuffer 74 (PharmaciaLKB, Piscataway, N J) pH 3.8 containing 0.1 mM CaCI> The flow rate was 0.75 ml/min, and 0.75 ml fractions were collected. Bovine serum albumin was added to active fractions from one of two identical runs to a concentration of 1.5 mg/ml. CHA-epoxy sepharose was prepared according to Silvanovich and Hill [3], and packed into a Pharmacia-LKB HR 10/10 column to a bed volume of 7.5 ml. Centricon 10 concentrators were used to change the buffer of chromatofocusing peak fractions by repeatedly concentrating the
145
protein to - 1 0 0 /A, then restoring the original volume with extraction buffer. These samples were applied to the CHA-sepharose column at a flow rate of 1.0 ml/min. The unbound proteins were collected, and bound proteins were eluted with 10 mg/ml CHA in extraction buffer. Fraction size was 2.0 ml. Isoelectric focusing (IEF) was done on a Pharmacia Ampholine Pagplate (pH 4.0-6.5). Approximately 0.2 units total activity was loaded in each lane. Amylase activity of focused bands was detected by laying the focused gel on top of a 1-mm thick starch-containing polyacrylamide gel (containing 5.5% (w/v) acrylamide and 0.3% (w/v) soluble starch (Sigma S-2630)) for 20 min at room temperature, then staining the starch gel with a solution containing 1 mM 12 and 500 mM KI. Amylase activity was measured as increase in reducing power using the dinitrosalicylic acid assay [4]. All substrates were 2% (w/v) in a solution containing 20 mM Na-acetate (pH 5.0) and 1.0 mM CaCI2, and all except granular starch were boiled. One unit equalled 1 tzmol maltose equivalent per rain. Enzymes were designated c~amylases (EC 3.2.1.1) if they were active on soluble starch and ~3-limit dextrin, but not on pullulan. Enzymes that were active on soluble starch, but not on t3-1imit dextrin or pullulan, were designated/3amylases (EC 3.2.1.2). Enzymes that were active on pullulan were designated as pullulanases (EC 3.2.1.41). Pullulanases produced dark blue bands on starch-containirlg gels. The starch debranching activity of these enzymes produces more straightchain fragments of starch which interact with iodine, enhancing the formation of the colored complex.
Protein was measured by the Bradford [5] method using fatty acid-free bovine serum albumin (Sigma, St. Louis, MO) as a standard. Results
The crude extract could be concentrated on 10 000 MW cutoff membranes (Centriprep filtration concentrators with YMT membranes) if first filtered through a 0.22 ~zm membrane (Millipore type GS). Recovery of activity from these two steps was 75%, and amylase specific activity increased slightly (Table I). The loss of activity during concentration was assumed to be non-specific and due to mechanical losses during handling, because protein lost was equivalent to activity lost, and a small amount of amylase activity was detected in the Centriprep filtrates. Chromatofocusing of the crude extract resolved amylases poorly. Purification by hydroxylapatite chromatography was sufficient to greatly improve the resolution by chromatofocusing. Recovery of loaded activity was 80% (3300 units recovered from 4106 units loaded, Table 1), with < 1% in the void volume. The remaining activity was either inactivated or stayed bound to the column. Chromatofocusing of the hydroxylapatite fraction resolved eight protein peaks with amylase activity. These were numbered 1-8 in order of elution (Figs. 1 and 2). The peak which eluted just before peak 4 (Fig. 1) contained 30 units of activity, and had no major bands of activity on IEF, so it was not studied further. Specific activities of peaks 6 and 8 on soluble starch were 228 and 151 units/mg, respectively; other peaks ranged from about 70 to 100 units/mg (Table II). Addition of
Table I.
Partial purification by hydroxylapatite chromatography IHAP) of amylase activity from endosperm and pericarp of maize kernels after 6 days of germination (135 g fresh wt.I
Step
Volume (ml)
Protein (mg)
Total act. (units~
Spec. act. lunits/mg protein)
Recovery I'~,,)
Purification (fold)
Crude Filtrate Concentrate HAP
500 500 50 75
101 100 70 46
5550 6450 4190 3300
55 65 60 72
100 I 16 75 59
1.00 1.17 1.09 1.31
146
2 120
1 O0
5
I,,-
8O 0 oO
<
4-
60
40
20
V 10
20
I
I
1
3O
4.0
50
6O
1
I
70
80
90
Fraction Number Fig. i. Chromatofocusing of partially purified maize endosperm extract on a Pharmacia mono P column. Gradient of pH: 6.3-3.8. Flow rate: 0.75 ml/min. Sample load: 19 mg protein !1360 units of activity) in 6.0 ml. Fraction size: 0.75 ml. Horizontal bars numbered 1-8 indicate major protein peaks with amylase activity. The unbound protein peak contained 29 units of activity. V indicates start of elution buffer. The figure shows the second of two identical runs.
bovine serum albumin to purified fractions had little effect on stability or IEF banding patterns of aamylases, but did stabilize the appearance of pullulanase enzyme activities on IEF gels (data not shown). Binding of activity to CHA was very low for chromatofocusing fractions 3, 4, 5 and 5'. Only peak 8, which bound completely, exceeded 50% binding (Table III). CHA-unbound (U) and -bound (B) enzymes from peaks 1-5 were determined to be a-amylases by their ability to hydrolyze amylopectin, amylose, and B-limit dextrin, but not pullulan (Table IV). Compared with the rate on soluble starch, activi,ties on amylopectin ranged from 39% for 3B to 74% for 2B, and on amylose from 58% for 5B to
107% for 3U. Both unbound and bound forms of peaks 1-5 had very low activity on granular starch. Chromatofocusing fraction 62 (Fig. 1) was designated peak 5'. Most of the activity in this fraction did not bind to CHA (Table II1), and was determined to be B-amylase (Table IV, 5'U). The CHA-bound fraction, after being concentrated, proved to be a pullulanase (Table IV, 5'B). Peak 6, which was /~-amylase (Table IV), was not applied to the CHA column. It is our experience that maize endosperm B-amylase does not bind to CHA. Peak 7 contained three kinds of activity (Table IV). The CHA-unbound fraction (7U) was a /3amylase, as it hydrolyzed neither B-limit dextrin
147 1
2
3
4
5
6
7
8 , pH 4.0
~pH
6.5
Fig. 2. lsoelectric focusing ofchromatofocusing peaks numbered 1-8. Activitieswere detected by transfer to starch-containing polyacrylamide gel.
nor puilulan. Pullulanase (7BI and 7B2) and c~amylase (7B4) were separated by different retention times on CHA. Peak 8 was an c~-amylase which was active on amylopectin and amylose (76 and 88%, respective-
ly of activity on soluble starch), and ~3-1imit dextrin, but not pullulan (Table IV). Peak 8 (bound fraction) had the highest activity on granular starch of any enzyme obtained in this study. For convenience of discussion, we have ten-
148 Table II.
Chromatofocusing of peak from hydroxylapatite chromatography. Specific activities are based on soluble starch as substrate, nd, not determined Peak
pH
Recovery (units)
Specific act. (units/mg protein)
I 2 3 4 5 5' 6 7 8
5.63 5.31 5.20 4.72 4.60 nd 4.13 nd 4.05
215 460 125 162 60 10 164 12 22
106 79 88 114 71 nd 228 94 151
HAP 1 2 3 4 5 5' 7 8
% of loaded activity Unbound
Bound
336 206 444 120 155 57 9 11 21
76 38 52 87 74 68 78 45 0
24 44 46 l0 9 7 <1 17 90
Enzyme
4 U/8
Table Iii. Affinity chromatography on cycloheptaamylose of chromatofocusing peaks. Affinity chromatography of a subsample of the fraction from hydroxylapatite chromatography (HAP) is shown for comparison.
Units loaded
Activity ofcycloheptaamylose-unboundand bound fractions of chromatofocusing peaks on various substrates
1 U/B b 2 U/B 3 U/B
tatively considered the a-amylases in c h r o m a t o f o c u s i n g p e a k s 1 - 5 with isoelectric p o i n t s from a p p r o x . 5 . 1 - 5 . 7 on the I E F gel as one m a j o r g r o u p , a n d the a - a m y l a s e s in p e a k s 7 a n d 8 a r o u n d p I 4.6 as a n o t h e r (Fig. 2). T h e m a j o r high p I g r o u p was a r b i t r a r i l y d i v i d e d into four s u b g r o u p s , each o f which was enriched in c h r o m a t o f o c u s i n g p e a k s 1, 2, 3 o r 4 (Fig. 2). T h e m o s t basic s u b g r o u p , consisting o f three b a n d s n e a r p i 5 . 7 , was the most a b u n d a n t in p e a k I. Peak 2 was enriched in a s u b - g r o u p c o n t a i n i n g two b a n d s near p I 5.4. A single b a n d at p I 5.2 was p r e d o m i n a n t in p e a k 3. The m a j o r b a n d in p e a k 4 was at p l 5.1. P e a k 5 a p p e a r e d to c o n t a i n all the b a n d s that were present in the first four peaks.
Peak
Table IV.
Recovery (%) 100 82 99 97 83 75 78 62 90
5 U/B 5' U/B 6 7U 7 BI 7 B2 7 B3 7 B4 8B
% Activity on soluble starch a 0-Limit dextrin
Pullulan
Granular starch
85/81 76/103 83/68 67/55 69/67 5/101 1 0 100 100 109 85 88
0/0 0/0 0/0 0/0 0/0 0/49 0.3 0 13 10 2 0 0
0.03/0.03 0.03/0.04 0.02/0 0.02/0
0.06/0 nd/nd c nd 0 nd nd nd nd 0.17
aActivity on soluble starch (units/ml): 1 U/B, 3.9/6.7; 2 U/B, 7.2/9.8; 3 U/B, 4.2/0.77; 4 U/B, 4.2/0.76; 5 U/B, 1.6/0.43; 5' B, 4.0/0.07; 6, 124; 7 U, 0.50; 7 B1, 0.04; 7 B2, 0.03; 7 B3, 0.44; 7 B4, 0.33; 8 B, 1.7. bu, unbound; B, bound. end, not determined
Peak 7 c o n t a i n e d 13-amylase, pullulanase, and a low p I a - a m y l a s e . P e a k 8 c o n t a i n e d the low p I g r o u p o f a - a m y l a s e s (pI - 4 . 6 ) consisting o f three bands. Peak 6, f3-amylase, was free o f o t h e r activities. In the high p I g r o u p , the two higher p l s u b g r o u p s h a d m o r e t e n d e n c y to bind to C H A t h a n the two lower p l s u b g r o u p s . T h e m o r e basic o f the two b a n d s o f the 5.4 p I s u b g r o u p , a n d b a n d s o f the 5.7 p I s u b g r o u p were p r o m i n e n t in the C H A - u n b o u n d fractions from p e a k s !, 2, 3 a n d 5. T h e b o u n d fraction o f p e a k 4 was similar to the u n b o u n d fraction ( d a t a not shown). I E F c o n f i r m e d the presence o f b o t h / l - a m y l a s e a n d p u l l u l a n a s e in 5 ' , a n d their s e p a r a t i o n by CBA chromatography (data not shown). Pullulanase ( 5 ' B , T a b l e IV), which b o u n d to the column, p r o d u c e d a d a r k blue b a n d ( p l 5.2) on the s t a r c h - c o n t a i n i n g gel. H-Amylase ( 5 ' U , T a b l e IV) did n o t bind, a n d p r o d u c e d two white bands, one m a j o r a n d o n e minor. T h e three activities in p e a k 7, a n d their separation on C H A , were also seen on I E F ( d a t a not
149
shown). B-Amylase (7U, Table IV), which did not bind, was the more basic of the two white bands in peak 7. Pullulanase and the c~-amylase bound to the column, but the pullulanase (7B1, Table IV) eluted before the c~-amylase (7B4, Table IV). Pullulanase in 7BI produced three dark blue bands at pl 5.1. Discussion
This separation procedure significantly improved the yield of multiple forms of c~-amylases from germinating maize, and preserved the B-amylase and pullulanase activities. Use of membranes for filtration and concentration of the crude extract substantially increased yield and specific activity of amylases compared with precipitation by acetone [1]. Filtration on a 0.22-t~m membrane actually increased the total activity recovered, presumably due to removal of material that interfered with enzyme activity (or its detection) in the crude homogenate. Multiple forms of c~- and B-amylases and pullulanases were retained by hydroxylapatite, and recovery after hydroxylapatite was greater than recovery after acetone precipitation. Because 20% of the loaded activity was lost after hydroxylapatite chromatography (Table I), it is possible that losses were selective at this step. It is more likely, however, that the loss was non-specific, because the IEF pattern of the hydroxylapatite fraction was identical to that of the crude fraction (data not shown). Compared with the acetone precipitate, a higher proportion of the hydroxylapatite sample is in the CHA-unbound fraction, showing that the enzymes lost when using acetone were largely CHA-unbound forms. Enzymes in the crude extract could not be successfully separated by chromatofocusing (data not shown), but partial purification of the extract by hydroxylapatite chromatography was sufficient for effective chromatofocusing. IEF patterns of o~-amylases in peaks 1, 2, 3, 4 and 8 matched those produced by chromatofocusing of CHA fractions in our previous study [1]. Peaks 5, 5' and 7 were not seen before, perhaps because most of the activity in these peaks did not bind CHA. These peaks were composed of
associated enzymes which separated on CHA. The purified u-amylase in peak 7 (7B4, Table IV) appeared to be equivalent to c~-amylase in peak 8 (Fig. 2), but it eluted much earlier from the chromatofocusing column, perhaps due to its association with B-amylase and pullulanase. Peak 5 was extremely heterogeneous, and the relation of these a-amylases to those in other peaks is not clear. c~-Amylases were present in seven of the eight chromatofocusing peaks, and five peaks contained only c~-amylase. Specific activities of these enzymes agreed well with a value reported for maize c~-amylase [6]. Peak 8 c~-amylase had the highest activity on granular starch (Table IV, 8B), in agreement with our previous results [1]. We have tentatively designated two major groups of c~-amylases in endosperm of germinating maize. This classification is supported by the general appearance of the IEF banding pattern (Fig. 2), and the later time of appearance of the low pI group during the course of germination (unpublished data). Action patterns on soluble starch, however, indicate that the isoform at pl 5.1 has biochemical properties different from the other high pl o~-amylases [1], and thus may not belong in the same major group. A detailed genetic analysis of maize c~-amylases will be required so that individual isoforms can be assigned to specific genes or gene families. IEF banding patterns of CHA-unbound and bound c~-amylases were similar. However, since the CHA columns were not overloaded, differential binding indicated some biochemical difference between unbound and bound forms. In our previous study, all the bands in the high pl group of oe amylases, as well as the low pl or-amylase, appeared to bind [1]. In that study, CHA-unbound and -bound forms of the high pI c~-amylases had different action patterns on both soluble and granular starch. Action pattern studies will help to elucidate the biochemical differences of the unbound and bound enzymes obtained in this study, and to compare them with those enzymes obtained previously by chromatofocusing of CHA fractions. B-Amylases were present in peaks 5', 6 and 7. The form in peak 6, which consisted of two bands,
150
was free of contaminating activities, and corresponded to the H-amylase in our previous study. ~3-Amylases in 5' and peak 7 were separated from other acti¥ities by chromatography on CHA, which binds low pl o~-amylases and pullulanases, but not 13-amylases. The form in peak 5U was the same as that in peak 6, while the form in peak 7U corresponded to the more acidic of the two bands. The use of chromatofocusing to isolate u-amylases eliminated the need for heat treatment or other methods to inactivate ¢3-amylase, since the latter is not present in any of the major u-amylasecontaining chromatofocusing fractions. Pullulanases in peaks 5' and 7 were purified by virtue of binding to CHA. IEF showed that for peak 7, the first fraction eluting from CHA contained only pullulanase, the next two fractions were mixtures of pullulanase and o~-amylase, and fraction 7-B4 was pure a-amylase (data not shown). This observation was confirmed by the data in Table IV. The pullulanase enzymes in the two peaks were determined to be different forms based on the following observations. Pullulanase in fraction 5' produced one or two dark blue bands on the starch gel at pI - 5.2. Enzymes in fraction 781 produced three dark blue bands with pI - 5.1. The two forms in fractions 5 ' B and 7B have different relative reaction rates on pullulan. Activity rates in unfractionated samples reflect the synergism which results from the combination of the different starch-degrading activities that are present in vivo. Loss of activity during purification, though doubtless due partly to loss of enzyme, is apparently also due partly to loss of synergism. This loss of synergism is seen clearly in Table IV. Recovery in affinity-bound and unbound fractions from the hydroxylapatite peak was 100%, indicating that various starchdegrading activities were still present and acting
synergistically in both fractions. Recovery of activity after affinity chromatography of chromatofocusing fractions that contained only a-amylase was >82%, indicating little or no synergism between bound and unbound forms. However, total recovery for peak 7 was only 62% when /3-amylase was separated from c~-amylase and pullulanase. Comparison of the protein recovery in the affinity fractions with the recovery of activity would help to determine whether enzyme or synergism was lost when peak 7 was fractionated. In summary, this purification scheme has enabled us to isolate five forms of c~-amylase, one /3amylase, and two forms of pullulanase activity. Four of the c~-amylase forms contained both CHA-binding and non-binding activities. This procedure forms the basis for studies of the synergism of starch-degrading activities during digestion of granular starch in vitro, as well as studies of the expression and regulation of starchdegrading enzymes in germinating maize. References I
2
3 4 5
6
D.A. Warner, M.J. Grove and C.A. Knutson, Isolation and characterization of alpha-amylases from endosperm of germinating maize. Cereal Chem., (1991) in press. E. Beck and P. Ziegler, Biosynthesis and degradation of starch in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40 (1989) 95-117. M.P. Silvanovich and R.D. Hill, Affinity chromatography of cereal u-amylases. Anal. Biochem., 73 (1976) 430-433. P. Bernfeld, Enzymes of starch degradation and synthesis. Adv. Enzymol., 12 (1951) 379-428. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. D. Lecommandeur, Y. Sirou and C. Lauriere, Glycan research on barley, maize, oats, and sorghum grain c~amylases: Comparison with rice u-amylase. Arch. Biochem. Biophys., 278 (1990) 245-250.
143
Isolation of c -amylases and other starch degrading enzymes from endosperm of germinating maize* Donn A. Warner and Clarence A. Knutson Seed Biosynthesis Research Unit. Agricultural Research Service, U.S. Department of Agriculture, National Center .['or Agricultural Utilization Research, 1815 N. University Street, Peoria. IL 61604 (U.S.A.) (Received February 28th, 1991; revision received May 6th, 1991; accepted May 13th, 1991 ) An improved purification procedure preserved high levels of a-amylase activity, and revealed and separated other starch-degrading activities from endosperm of germinating maize (Zea mays L.). Amylases were partially purified by hydroxylapatite chromatography, and separated by chromatofocusing and affinity chromatography on cycloheptaamylose. Chromatofocusing resolved eight protein peaks with amylase activity. The binding of amylase activity to cycloheptaamylose ranged from 0 to 90% for these peaks, lsoelectric focusing and analysis of substrate specificities showed two major groups of a-amylase with five subgroups, two pullulanase enzymes, and one ~3-amylasc. This procedure is a significant improvement over the use of protein precipitation and affinity chromatography which results in large, and possibly selective, losses of amylase activity. Key words." or-amylase; t3-amylase; pullulanase; maize; endosperm; starch hydrolysis
Introduction
Previous work in this laboratory has shown that multiple forms of starch degrading enzymes occur in germinating maize [1]. Because they differ in substrate specificities and reaction products, each form may have a specific role in the degradation of storage starch granules in vivo [21. Individual forms of each enzyme must be isolated to determine their biochemical characteristics, and describe their roles in the pathway of starch granule degradation. Our goal is to determine the ability of these individual enzyme forms to digest granular starch in vitro. In our previous work, amylases were fractionated by a combination of affinity chromatoCorrespondence to." Donn A. Warner, USDA/NCAUR. 1815 N. University Street, Peoria, IL 61604, U.S.A. Abbreviations: CHA, cycloheptaamylose; IEF, isoelectric focusing. *The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.
graphy, ion exchange chromatography, hydroxylapatite chromatography, and chromatofocusing. Four major fractions of c~-amylase (three affinitybound, one unbound) had distinctive action patterns. This combination procedure provided a convenient method for separation of major forms of amylases found in maize. A significant disadvantage of that isolation scheme was the loss of 50°/,, of the amylase activity in the acetone precipitation step used to concentrate the proteins in the crude extract. The possibility that the loss of activity was selective for certain enzyme forms precluded a complete characterization of the maize amylases. We could not be certain that the isolated enzymes were present in the same proportions as they are in vivo, or that all of the amylases had been identified and separated. Furthermore, we did not know whether decreases in activity were due to losses or inactivation of enzymes during processing, or were due to loss of synergistic action. Maltase and debranching activities were present in crude extracts, but were lost during purification. Another limitation of the original isolation scheme was that only
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
144 about half of the total activity bound to the affinity chromatography column, with substantial variation in the amount of activity that was bound. Therefore it was unclear whether enzymes isolated from one purification were identical to those from another. For these reasons we devised an alternate approach for separation of these enzymes which preserved more of the original activity, verified and extended our previous results, and more accurately characterized the roles of the many amylolytic enzymes in the pathway of starch degradation in germinating maize. Our goal in this study was to increase the yield of amylase activity, and to prevent the possible selective loss of particular enzyme species. The new procedure used chromatography on hydroxylapatite as the first step in purification, followed by chromatofocusing. Chromatofocusing resolved eight protein peaks. Each chromatofocusing peak had a characteristic binding profile on cycloheptaamylose (CHA). Five major c~-amylases, two pullulanase enzymes, and one 3-amylase were separated. Materials and Methods
Kernels of maize (Zea mays L.) inbred B73 were surface-sterilized with 1% (w/v) NaOCl for 10 min, imbibed overnight in 1 mM CaCI2, and germinated for 6 days at 25°C in the dark. Seedling axes were - 7 cm long. All germinated kernels were free of visible mold. Endosperms plus pericarps were stored in a freezer at -80°C for 2 days. We have observed no change in extractable activity nor in isozyme banding pattern with up to three months of storage. The frozen tissue was ground with a Polytron homogenizer in - 1.6 ml/g tissue of extraction buffer containing 20 mM Naacetate (pH 5.0) and 1 mM CaCIe. The homogenate was stirred on ice for 15 min, then strained through number 20 wire mesh and centrifuged at 20 000 x g for 10 min. The supernatant fraction was saved, and the solids resuspended in extraction buffer and centrifuged as before. The combined supernatants were filtered through two layers of Whatman number 1 filter paper and a Millipore 0.22 /~m type GS membrane, then con-
centrated with Centriprep 10 filtration concentrators (Amicon, Danvers, MA). The concentrated extract was dialyzed at 4°C overnight against a solution containing 10 mM K-phosphate (pH 6.0) and 0.1 mM CaCI2, and was then filtered through a 0.22-t~m membrane. All chromatography was done at 4°C on an FPLC liquid chromatography system (PharmaciaLKB, Piscataway, N J). Bio-Gel HT hydroxylapatite (Bio-Rad, Richmond, CA) was packed into a Pharmacia HR 10/10 column (10 cm long x 1 cm diam.) to a bed volume of 7.5 ml. A preliminary experiment using a linear gradient showed that all hydroxylapatite-bound amylase activity eluted in the range from 10 to 300 mM Kphosphate. Thereafter, amylases were eluted in one batch with 300 mM K-phosphate to prevent unnecessary dilution. Estimated capacity was 30 mg protein for the hydroxylapatite column, so the extract was divided into two portions which were chromatographed separately. The starting buffer for hydroxylapatite contained 10 mM Kphosphate pH 6.0 and 0.1 mM CaCI 2, and the flow rate was 1.0 ml/min. After unbound proteins were washed off, bound proteins were eluted with a solution containing 300 mM K-phosphate pH 6.0 and 0.1 mM CaCI2 which had been filtered to remove precipitated Ca3(PO4)> All fractions with amylase activity were pooled, concentrated with Centriprep 10 concentrators, and dialyzed at 4°C against 25 mM BisTris (pH 6.3) (Sigma, St. Louis, MO) containing 0.1 mM CaCI, for chromatofocusing. Chromatofocusing was done with a Pharmacia Mono P column. The starting buffer contained 25 mM BisTris (pH 6.3) and 0.1 mM CaCIz, and the elution buffer was Polybuffer 74 (PharmaciaLKB, Piscataway, N J) pH 3.8 containing 0.1 mM CaCI> The flow rate was 0.75 ml/min, and 0.75 ml fractions were collected. Bovine serum albumin was added to active fractions from one of two identical runs to a concentration of 1.5 mg/ml. CHA-epoxy sepharose was prepared according to Silvanovich and Hill [3], and packed into a Pharmacia-LKB HR 10/10 column to a bed volume of 7.5 ml. Centricon 10 concentrators were used to change the buffer of chromatofocusing peak fractions by repeatedly concentrating the
145
protein to - 1 0 0 /A, then restoring the original volume with extraction buffer. These samples were applied to the CHA-sepharose column at a flow rate of 1.0 ml/min. The unbound proteins were collected, and bound proteins were eluted with 10 mg/ml CHA in extraction buffer. Fraction size was 2.0 ml. Isoelectric focusing (IEF) was done on a Pharmacia Ampholine Pagplate (pH 4.0-6.5). Approximately 0.2 units total activity was loaded in each lane. Amylase activity of focused bands was detected by laying the focused gel on top of a 1-mm thick starch-containing polyacrylamide gel (containing 5.5% (w/v) acrylamide and 0.3% (w/v) soluble starch (Sigma S-2630)) for 20 min at room temperature, then staining the starch gel with a solution containing 1 mM 12 and 500 mM KI. Amylase activity was measured as increase in reducing power using the dinitrosalicylic acid assay [4]. All substrates were 2% (w/v) in a solution containing 20 mM Na-acetate (pH 5.0) and 1.0 mM CaCI2, and all except granular starch were boiled. One unit equalled 1 tzmol maltose equivalent per rain. Enzymes were designated c~amylases (EC 3.2.1.1) if they were active on soluble starch and ~3-limit dextrin, but not on pullulan. Enzymes that were active on soluble starch, but not on t3-1imit dextrin or pullulan, were designated/3amylases (EC 3.2.1.2). Enzymes that were active on pullulan were designated as pullulanases (EC 3.2.1.41). Pullulanases produced dark blue bands on starch-containirlg gels. The starch debranching activity of these enzymes produces more straightchain fragments of starch which interact with iodine, enhancing the formation of the colored complex.
Protein was measured by the Bradford [5] method using fatty acid-free bovine serum albumin (Sigma, St. Louis, MO) as a standard. Results
The crude extract could be concentrated on 10 000 MW cutoff membranes (Centriprep filtration concentrators with YMT membranes) if first filtered through a 0.22 ~zm membrane (Millipore type GS). Recovery of activity from these two steps was 75%, and amylase specific activity increased slightly (Table I). The loss of activity during concentration was assumed to be non-specific and due to mechanical losses during handling, because protein lost was equivalent to activity lost, and a small amount of amylase activity was detected in the Centriprep filtrates. Chromatofocusing of the crude extract resolved amylases poorly. Purification by hydroxylapatite chromatography was sufficient to greatly improve the resolution by chromatofocusing. Recovery of loaded activity was 80% (3300 units recovered from 4106 units loaded, Table 1), with < 1% in the void volume. The remaining activity was either inactivated or stayed bound to the column. Chromatofocusing of the hydroxylapatite fraction resolved eight protein peaks with amylase activity. These were numbered 1-8 in order of elution (Figs. 1 and 2). The peak which eluted just before peak 4 (Fig. 1) contained 30 units of activity, and had no major bands of activity on IEF, so it was not studied further. Specific activities of peaks 6 and 8 on soluble starch were 228 and 151 units/mg, respectively; other peaks ranged from about 70 to 100 units/mg (Table II). Addition of
Table I.
Partial purification by hydroxylapatite chromatography IHAP) of amylase activity from endosperm and pericarp of maize kernels after 6 days of germination (135 g fresh wt.I
Step
Volume (ml)
Protein (mg)
Total act. (units~
Spec. act. lunits/mg protein)
Recovery I'~,,)
Purification (fold)
Crude Filtrate Concentrate HAP
500 500 50 75
101 100 70 46
5550 6450 4190 3300
55 65 60 72
100 I 16 75 59
1.00 1.17 1.09 1.31
146
2 120
1 O0
5
I,,-
8O 0 oO
<
4-
60
40
20
V 10
20
I
I
1
3O
4.0
50
6O
1
I
70
80
90
Fraction Number Fig. i. Chromatofocusing of partially purified maize endosperm extract on a Pharmacia mono P column. Gradient of pH: 6.3-3.8. Flow rate: 0.75 ml/min. Sample load: 19 mg protein !1360 units of activity) in 6.0 ml. Fraction size: 0.75 ml. Horizontal bars numbered 1-8 indicate major protein peaks with amylase activity. The unbound protein peak contained 29 units of activity. V indicates start of elution buffer. The figure shows the second of two identical runs.
bovine serum albumin to purified fractions had little effect on stability or IEF banding patterns of aamylases, but did stabilize the appearance of pullulanase enzyme activities on IEF gels (data not shown). Binding of activity to CHA was very low for chromatofocusing fractions 3, 4, 5 and 5'. Only peak 8, which bound completely, exceeded 50% binding (Table III). CHA-unbound (U) and -bound (B) enzymes from peaks 1-5 were determined to be a-amylases by their ability to hydrolyze amylopectin, amylose, and B-limit dextrin, but not pullulan (Table IV). Compared with the rate on soluble starch, activi,ties on amylopectin ranged from 39% for 3B to 74% for 2B, and on amylose from 58% for 5B to
107% for 3U. Both unbound and bound forms of peaks 1-5 had very low activity on granular starch. Chromatofocusing fraction 62 (Fig. 1) was designated peak 5'. Most of the activity in this fraction did not bind to CHA (Table II1), and was determined to be B-amylase (Table IV, 5'U). The CHA-bound fraction, after being concentrated, proved to be a pullulanase (Table IV, 5'B). Peak 6, which was /~-amylase (Table IV), was not applied to the CHA column. It is our experience that maize endosperm B-amylase does not bind to CHA. Peak 7 contained three kinds of activity (Table IV). The CHA-unbound fraction (7U) was a /3amylase, as it hydrolyzed neither B-limit dextrin
147 1
2
3
4
5
6
7
8 , pH 4.0
~pH
6.5
Fig. 2. lsoelectric focusing ofchromatofocusing peaks numbered 1-8. Activitieswere detected by transfer to starch-containing polyacrylamide gel.
nor puilulan. Pullulanase (7BI and 7B2) and c~amylase (7B4) were separated by different retention times on CHA. Peak 8 was an c~-amylase which was active on amylopectin and amylose (76 and 88%, respective-
ly of activity on soluble starch), and ~3-1imit dextrin, but not pullulan (Table IV). Peak 8 (bound fraction) had the highest activity on granular starch of any enzyme obtained in this study. For convenience of discussion, we have ten-
148 Table II.
Chromatofocusing of peak from hydroxylapatite chromatography. Specific activities are based on soluble starch as substrate, nd, not determined Peak
pH
Recovery (units)
Specific act. (units/mg protein)
I 2 3 4 5 5' 6 7 8
5.63 5.31 5.20 4.72 4.60 nd 4.13 nd 4.05
215 460 125 162 60 10 164 12 22
106 79 88 114 71 nd 228 94 151
HAP 1 2 3 4 5 5' 7 8
% of loaded activity Unbound
Bound
336 206 444 120 155 57 9 11 21
76 38 52 87 74 68 78 45 0
24 44 46 l0 9 7 <1 17 90
Enzyme
4 U/8
Table Iii. Affinity chromatography on cycloheptaamylose of chromatofocusing peaks. Affinity chromatography of a subsample of the fraction from hydroxylapatite chromatography (HAP) is shown for comparison.
Units loaded
Activity ofcycloheptaamylose-unboundand bound fractions of chromatofocusing peaks on various substrates
1 U/B b 2 U/B 3 U/B
tatively considered the a-amylases in c h r o m a t o f o c u s i n g p e a k s 1 - 5 with isoelectric p o i n t s from a p p r o x . 5 . 1 - 5 . 7 on the I E F gel as one m a j o r g r o u p , a n d the a - a m y l a s e s in p e a k s 7 a n d 8 a r o u n d p I 4.6 as a n o t h e r (Fig. 2). T h e m a j o r high p I g r o u p was a r b i t r a r i l y d i v i d e d into four s u b g r o u p s , each o f which was enriched in c h r o m a t o f o c u s i n g p e a k s 1, 2, 3 o r 4 (Fig. 2). T h e m o s t basic s u b g r o u p , consisting o f three b a n d s n e a r p i 5 . 7 , was the most a b u n d a n t in p e a k I. Peak 2 was enriched in a s u b - g r o u p c o n t a i n i n g two b a n d s near p I 5.4. A single b a n d at p I 5.2 was p r e d o m i n a n t in p e a k 3. The m a j o r b a n d in p e a k 4 was at p l 5.1. P e a k 5 a p p e a r e d to c o n t a i n all the b a n d s that were present in the first four peaks.
Peak
Table IV.
Recovery (%) 100 82 99 97 83 75 78 62 90
5 U/B 5' U/B 6 7U 7 BI 7 B2 7 B3 7 B4 8B
% Activity on soluble starch a 0-Limit dextrin
Pullulan
Granular starch
85/81 76/103 83/68 67/55 69/67 5/101 1 0 100 100 109 85 88
0/0 0/0 0/0 0/0 0/0 0/49 0.3 0 13 10 2 0 0
0.03/0.03 0.03/0.04 0.02/0 0.02/0
0.06/0 nd/nd c nd 0 nd nd nd nd 0.17
aActivity on soluble starch (units/ml): 1 U/B, 3.9/6.7; 2 U/B, 7.2/9.8; 3 U/B, 4.2/0.77; 4 U/B, 4.2/0.76; 5 U/B, 1.6/0.43; 5' B, 4.0/0.07; 6, 124; 7 U, 0.50; 7 B1, 0.04; 7 B2, 0.03; 7 B3, 0.44; 7 B4, 0.33; 8 B, 1.7. bu, unbound; B, bound. end, not determined
Peak 7 c o n t a i n e d 13-amylase, pullulanase, and a low p I a - a m y l a s e . P e a k 8 c o n t a i n e d the low p I g r o u p o f a - a m y l a s e s (pI - 4 . 6 ) consisting o f three bands. Peak 6, f3-amylase, was free o f o t h e r activities. In the high p I g r o u p , the two higher p l s u b g r o u p s h a d m o r e t e n d e n c y to bind to C H A t h a n the two lower p l s u b g r o u p s . T h e m o r e basic o f the two b a n d s o f the 5.4 p I s u b g r o u p , a n d b a n d s o f the 5.7 p I s u b g r o u p were p r o m i n e n t in the C H A - u n b o u n d fractions from p e a k s !, 2, 3 a n d 5. T h e b o u n d fraction o f p e a k 4 was similar to the u n b o u n d fraction ( d a t a not shown). I E F c o n f i r m e d the presence o f b o t h / l - a m y l a s e a n d p u l l u l a n a s e in 5 ' , a n d their s e p a r a t i o n by CBA chromatography (data not shown). Pullulanase ( 5 ' B , T a b l e IV), which b o u n d to the column, p r o d u c e d a d a r k blue b a n d ( p l 5.2) on the s t a r c h - c o n t a i n i n g gel. H-Amylase ( 5 ' U , T a b l e IV) did n o t bind, a n d p r o d u c e d two white bands, one m a j o r a n d o n e minor. T h e three activities in p e a k 7, a n d their separation on C H A , were also seen on I E F ( d a t a not
149
shown). B-Amylase (7U, Table IV), which did not bind, was the more basic of the two white bands in peak 7. Pullulanase and the c~-amylase bound to the column, but the pullulanase (7B1, Table IV) eluted before the c~-amylase (7B4, Table IV). Pullulanase in 7BI produced three dark blue bands at pl 5.1. Discussion
This separation procedure significantly improved the yield of multiple forms of c~-amylases from germinating maize, and preserved the B-amylase and pullulanase activities. Use of membranes for filtration and concentration of the crude extract substantially increased yield and specific activity of amylases compared with precipitation by acetone [1]. Filtration on a 0.22-t~m membrane actually increased the total activity recovered, presumably due to removal of material that interfered with enzyme activity (or its detection) in the crude homogenate. Multiple forms of c~- and B-amylases and pullulanases were retained by hydroxylapatite, and recovery after hydroxylapatite was greater than recovery after acetone precipitation. Because 20% of the loaded activity was lost after hydroxylapatite chromatography (Table I), it is possible that losses were selective at this step. It is more likely, however, that the loss was non-specific, because the IEF pattern of the hydroxylapatite fraction was identical to that of the crude fraction (data not shown). Compared with the acetone precipitate, a higher proportion of the hydroxylapatite sample is in the CHA-unbound fraction, showing that the enzymes lost when using acetone were largely CHA-unbound forms. Enzymes in the crude extract could not be successfully separated by chromatofocusing (data not shown), but partial purification of the extract by hydroxylapatite chromatography was sufficient for effective chromatofocusing. IEF patterns of o~-amylases in peaks 1, 2, 3, 4 and 8 matched those produced by chromatofocusing of CHA fractions in our previous study [1]. Peaks 5, 5' and 7 were not seen before, perhaps because most of the activity in these peaks did not bind CHA. These peaks were composed of
associated enzymes which separated on CHA. The purified u-amylase in peak 7 (7B4, Table IV) appeared to be equivalent to c~-amylase in peak 8 (Fig. 2), but it eluted much earlier from the chromatofocusing column, perhaps due to its association with B-amylase and pullulanase. Peak 5 was extremely heterogeneous, and the relation of these a-amylases to those in other peaks is not clear. c~-Amylases were present in seven of the eight chromatofocusing peaks, and five peaks contained only c~-amylase. Specific activities of these enzymes agreed well with a value reported for maize c~-amylase [6]. Peak 8 c~-amylase had the highest activity on granular starch (Table IV, 8B), in agreement with our previous results [1]. We have tentatively designated two major groups of c~-amylases in endosperm of germinating maize. This classification is supported by the general appearance of the IEF banding pattern (Fig. 2), and the later time of appearance of the low pI group during the course of germination (unpublished data). Action patterns on soluble starch, however, indicate that the isoform at pl 5.1 has biochemical properties different from the other high pl o~-amylases [1], and thus may not belong in the same major group. A detailed genetic analysis of maize c~-amylases will be required so that individual isoforms can be assigned to specific genes or gene families. IEF banding patterns of CHA-unbound and bound c~-amylases were similar. However, since the CHA columns were not overloaded, differential binding indicated some biochemical difference between unbound and bound forms. In our previous study, all the bands in the high pl group of oe amylases, as well as the low pl or-amylase, appeared to bind [1]. In that study, CHA-unbound and -bound forms of the high pI c~-amylases had different action patterns on both soluble and granular starch. Action pattern studies will help to elucidate the biochemical differences of the unbound and bound enzymes obtained in this study, and to compare them with those enzymes obtained previously by chromatofocusing of CHA fractions. B-Amylases were present in peaks 5', 6 and 7. The form in peak 6, which consisted of two bands,
150
was free of contaminating activities, and corresponded to the H-amylase in our previous study. ~3-Amylases in 5' and peak 7 were separated from other acti¥ities by chromatography on CHA, which binds low pl o~-amylases and pullulanases, but not 13-amylases. The form in peak 5U was the same as that in peak 6, while the form in peak 7U corresponded to the more acidic of the two bands. The use of chromatofocusing to isolate u-amylases eliminated the need for heat treatment or other methods to inactivate ¢3-amylase, since the latter is not present in any of the major u-amylasecontaining chromatofocusing fractions. Pullulanases in peaks 5' and 7 were purified by virtue of binding to CHA. IEF showed that for peak 7, the first fraction eluting from CHA contained only pullulanase, the next two fractions were mixtures of pullulanase and o~-amylase, and fraction 7-B4 was pure a-amylase (data not shown). This observation was confirmed by the data in Table IV. The pullulanase enzymes in the two peaks were determined to be different forms based on the following observations. Pullulanase in fraction 5' produced one or two dark blue bands on the starch gel at pI - 5.2. Enzymes in fraction 781 produced three dark blue bands with pI - 5.1. The two forms in fractions 5 ' B and 7B have different relative reaction rates on pullulan. Activity rates in unfractionated samples reflect the synergism which results from the combination of the different starch-degrading activities that are present in vivo. Loss of activity during purification, though doubtless due partly to loss of enzyme, is apparently also due partly to loss of synergism. This loss of synergism is seen clearly in Table IV. Recovery in affinity-bound and unbound fractions from the hydroxylapatite peak was 100%, indicating that various starchdegrading activities were still present and acting
synergistically in both fractions. Recovery of activity after affinity chromatography of chromatofocusing fractions that contained only a-amylase was >82%, indicating little or no synergism between bound and unbound forms. However, total recovery for peak 7 was only 62% when /3-amylase was separated from c~-amylase and pullulanase. Comparison of the protein recovery in the affinity fractions with the recovery of activity would help to determine whether enzyme or synergism was lost when peak 7 was fractionated. In summary, this purification scheme has enabled us to isolate five forms of c~-amylase, one /3amylase, and two forms of pullulanase activity. Four of the c~-amylase forms contained both CHA-binding and non-binding activities. This procedure forms the basis for studies of the synergism of starch-degrading activities during digestion of granular starch in vitro, as well as studies of the expression and regulation of starchdegrading enzymes in germinating maize. References I
2
3 4 5
6
D.A. Warner, M.J. Grove and C.A. Knutson, Isolation and characterization of alpha-amylases from endosperm of germinating maize. Cereal Chem., (1991) in press. E. Beck and P. Ziegler, Biosynthesis and degradation of starch in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 40 (1989) 95-117. M.P. Silvanovich and R.D. Hill, Affinity chromatography of cereal u-amylases. Anal. Biochem., 73 (1976) 430-433. P. Bernfeld, Enzymes of starch degradation and synthesis. Adv. Enzymol., 12 (1951) 379-428. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. D. Lecommandeur, Y. Sirou and C. Lauriere, Glycan research on barley, maize, oats, and sorghum grain c~amylases: Comparison with rice u-amylase. Arch. Biochem. Biophys., 278 (1990) 245-250.