Characterization of Low-Molecular-Weight Protein

Cereal Chem. 63(5):414-419 A low-molecular-weight protein (S protein) fraction with a high affinity and SDS-PAGE patterns were similar for both varieties, but RP-HPLC for flour polar lipid was isolated from flours of one hard and one soft wheat patterns were quite different. Defatting the flour did not affect the yield of S variety. The fraction was characterized by lipid content and composition, protein or the PAGE and SDS-PAGE patterns. The two major acidic polyacrylamide gel electrophoresis (PAGE), gradient gel sodium components of the S protein fraction were isolated by Sephadex G-50 dodecyl sulfate PAGE (SDS-PAGE), and reversed-phase highgel-filtration chromatography and characterized by electrophoresis, amino performance liquid chromatography (RP-HPLC). The content of this acid analysis, and RP-HPLC. PAGE, SDS-PAGE, and amino acid protein in the two wheat varieties was approximately the same. Lipid analysis results did not show any significant intervarietal differences, but contents and compositions of the two preparations were similar. PAGE the S protein fractions appeared distinctly different upon RP-HPLC. Numerous reports have implicated interactions between flour yield, protein (N X 5.7), and ash contents were 72, 15.7, and 0.44% proteins and lipids with breadmaking potential of wheat flour for Neepawa and 66, 14.9, and 0.50% for Chile, respectively. (Hoseney et al 1970, Chung et al 1978, MacRitchie 1980, Pomeranz Protein and ash contents, expressed on dry bases, were determined 1980, Frazier et al 1981, Bushuk et al 1984). Generally, lipidby approved AACC methods (AACC 1983). binding ability has been attributed to glutenin or gliadin (Olcott and Mecham 1947; Ponte et al 1967; Hoseney et al 1970; Chung Defatting and Tsen 1975; Bekes et al 1983; Zawistowska et al 1984; Completely and partially defatted flours were prepared by Zawistowska et al 1985a,b). Recently it was shown (Zawistowska extraction with water-saturated butanol and n-hexane, et al 1985a) that a low-molecular-weight protein fraction respectively (Bekes et al 1983). tentatively named "S" protein, constituting about 5% of total flour protein, was associated with as much as 20% of flour polar lipid. Preparation of Glutens Because flour polar lipids may positively affect loaf volume Gluten was hand washed in a stream of distilled water from (Chung et al 1982, Bekes et al 1986), the high affinity of S protein doughs mixed in air to peak development in a farinograph; for polar flour lipids suggested that this protein may be important washing was continued until water was clear. Wet gluten was in bread dough functionality. Accordingly, S protein fractions freeze-dried, ground by mortar and pestle, and stored at 40 C. from two wheat varieties of different baking potential were compared by electrophoresis, in terms of lipid content and Preparation of Gliadin composition, and by reversed-phase high-performance liquid Undefatted Neepawa flour was extracted with 70% ethanol with chromatography (RP-HPLC). Additionally, S protein fractions agitation using a Buchler vortex-evaporator for 15 mI , were prepared from partially and totally defatted flour samples in centrifuged at 27,000 X g for 10 min, and immediately used for order to investigate the effect, if any, of flour lipid on yield and HPLC separation. composition of S protein. The two major components of S protein were also isolated by Sephadex G-50 gel-filtration chromatography Fractionation of Gluten and partially characterized. Results are described and discussed in Gluten was fractionated by ammonium sulfate precipitation of this article. the acetic acid-soluble proteins dissolved in AUC solvent (0.1M acetic acid, 3M urea, and 0.01M cetyltrimethylammonium MATERIALS AND METHODS bromide) (Wasik and Bushuk 1974). Three concentrations of Chemicals ammonium sulfate (7.8, 14.5, and 20.2%, w/v) yielded four Molecular weight reference proteins, Coomassie Brillant Blue fractions, three precipitates P1, P2, P 3, and a supernatant (5) R250, 2-mercaptoethanol, and 3-methylaminopropionitrile were fraction. Each precipitate was separated by centrifugation at obtained from Sigma (St. Louis, MO). Acrylamide, bisacrylamide, 20,000 X g for 15 min. The S fraction was concentrated on a model and sodium dodecyl sulfate (SDS) were of electrophoresis grade 8200 Amicon standard cell using YM5 membrane (5,000 mol wt and were obtained from Bio-Rad (Richmond, CA). All other cut off), dialyzed against deionized water, and freeze-dried. Gluten chemicals were of analytical reagent grade. fractionation was performed in two replications, and results were averaged. A t test was calculated at the 0.05 level of significance for Preparation of Flours dry matter distribution in ammonium sulfate fractions. The wheat varieties used in this study were Neepawa, a hard red spring wheat, and Chile, a soft white spring wheat. Wheats were Fractionation of S Fraction grown in 1983 under identical conditions in western Canada. A Gel-filtration chromatography of S protein fractions was Buhler experimental mill was used to mill grain into flour. Flour performed on Sephadex G-50 in AUC solvent. Column size was 2.6 >< 90 cm, flow rate was 12 ml/ hr, and 3-ml fractions were collected. •Presented at the AACC 70th Annual Meeting, Orlando, FL, September 1985. Prticoenofheeuewamntrdat20mbynLK Contribution no. 739 of the Department of Plant Science, University of Manitoba, Uvicord SII ultraviolet absorbance monitor. Appropriate Winnipeg, Canada R3T 2N2, with financial assistance from the Natural Sciences and fractions were pooled, dialyzed against distilled water, and freezeEngineering Research Council of Canada. dried. Ovalbumin (mol wt 45,000) and trypsinogen (mol wt 24,000) 2 Present address: ABI Biotechnology Inc., 1150 Waverley, Winnipeg, Manitoba, weeudtoclbaeheoum tofiiaeesmtonf Canada R3T 0P4.weeuetoalbaeteclm tofclttesitonf 3 Northern Regional Research Center, Agricultural Research Service, U.S. molecular weights of S protein peaks. Percentages of eluted Department of Agriculture, 1815 N. University St., Peoria, IL 61604. fractions were estimated from peak areas of the fractions collected. ________________________________________________ Areas from three separations were averaged; the experimental © 1986 American Association of Cereal Chemists, Inc. error was ±+3%. 414 CEREAL CHEMISTRY Lipid Analysis RESULTS AND DISCUSSION For lipid determination, 60 mg of S fraction was extracted at 400 C by shaking with three portions of water-saturated butanol Characterization of S Protein (ratio of solvent to sample in each extraction was 12.5:1, v/w). The Distribution of fractions from ammonium sulfate precipitation first extraction was performed overnight, and subsequent is shown in Table I. Defatting had little effect on the dry matter extractions for 1 hr. Extracts were clarified by filtration, by distribution among the four fractions. Only for Chile it was found centrifugation at 12,000 X g for 20 min, or both. Filtrates were that defatting with n-hexane affected the amount of P2 as combined and evaporated with a stream of nitrogen at 400 C. The compared with control flour, and there was a significant difference dry residues were extracted with chloroform, filtered, and in amount of P 3 in n-hexane defatted and totally defatted sample. chloroform was evaporated with a stream of nitrogen at 400 C. All other differences in dry material distribution among Lipid content was determined gravimetrically from the residue ammonium sulfate fractions were not significant. The S fraction remaining after solvent evaporation. Total lipid was fractionated constituted approximately 12% of the acetic acid-soluble gluten by silicic acid column chromatography into neutral lipid (eluted protein. Similar yields were obtained previously (Zawistowska et with chloroform) and polar lipid (eluted with acetone followed by al 1985a) with the cultivar Neepawa (from different crop year) and methanol) (Kates 1972). Lipid analyses were performed in by Frazier et al (1981) for an unspecified U.S. hard red spring triplicate and averages are reported. wheat variety. Lipid content determination of the S fraction of nondefatted Polyacrylamide Gel Eletrophoresis flours showed the S fraction of Neepawa to contain 22.6% (± 1.0) Polyacrylamide gel eletrophoresis (PAGE) was performed on lipid, and that of Chile, 24.6% (± 0.9). Most of the lipid was polar 6.3% gels in aluminum lactate buffer at pH 3.1 (Bushuk and lipid. The S fraction of Neepawa contained 3.8% (± 0.1) neutral Zillman 1978), except that run time was decreased from 5 to 2 hr. lipid and 18.8% (± 1.0) polar lipid, whereas that of Chile contained Sodium dodecyl sulfate (SDS)-PAGE was done on an LKB 2001 3.9% (± 0.1) neutral lipid and 20.7% (± 1.1) polar lipid. The total vertical electrophoresis unit (Zawistowska and Bushuk 1986) using lipid content for Neepawa was similar to the result obtained for 10-18% gradient gels and the discontinuous buffer system Neepawa from a different crop year (Zawistowska et al 1985a). (Laemmli 1970). Proteins were dissolved in 62.5 mMTris-HC1, pH However, the proportion of neutral to polar lipid obtained for the 6.8, containing 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol. two Neepawa samples was different. The reason for this is Samples were heated for 1.5 min in a boiling water bath to ensure unknown, but it could be due to environment. An effect of complete unfolding of proteins and were cooled before application to the gel. Molecular weights were estimated from migration distances of bovine serum albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), betaNeepawa Chile lactoglobulin (18,400), and lysozyme (14,300). IN P T Ir N P TI MWx Amino Acid Analysis 10-3 Amino acid compositions were determined on an LKB 4151 Alpha Plus automatic amino acid analyzer. Samples were hydrolyzed in 6N HC1 for 16 hr at 1210C under vacuum, evaporated to dryness, dissolved in 0.2M sodium citrate buffer, pH 2.2, and filtered before analysis in duplicate. Cysteine and methionine were determined as cysteic acid and methionine 66 sulfone following performic acid oxidation (Hirs 1967). 45Tryptophan was not determined. Values for aspartic and glutamic acids include asparagine and glutamine, respectively. 36 RP-HPLC RP-HPLC was performed on a large-pore (300A) SynChropak RP-P (C18) column (4.1 X 250 mm) (Bietz 1983, Bietz and Cobb 1985). The system consisted of a Waters M6000A and M45 solvent1 8.4delivery system controlled by a model 660 solvent programmer, a WISP 710A automatic sample injector, and a model 450 variable14.3wavelength detector. For analysis, 0.5 mg of sample was dissolved in 1 ml of AUC solvent. Samples (50-100 /.1) were analyzed using a 0 linear gradient of 25-50% acetonitrile containing 0.1% trifluoroacetic acid over 50 min (total run time 60 min) at 1.0 Fig. 1. Gradient (10-18%) sodium dodecyl sulfate-polyacrylamide gel ml/min and 700 C. Detection was at 210 nm using detector settings electrophoretic patterns of S fractions from Neepawa and Chile flours. N, of 0.2-0.4 absorbance units full-scale. Data were recorded on a nondefatted; P, partially defatted; and T, totally defatted. Pattern on left is Houston Instruments Omniscribe recorder (10 mV full-scale). of molecular weight reference proteins. TABLE I Dry Matter Distribution in Fractions of Acetic Acid-Soluble Gluten Prepared by Ammonium Sulfate Precipitation' Neepawa Chile Partially Totally Partially Totally Nondefatted Defatted Defatted Nondefatted Defatted Defatted Fraction (%) (%) (%) (%) (%) (%) Precipitate P1 50.6 (±2.7) 51.1 (±1.3) 51.6 (±2.1) 56.9 (±2.8) 47.1 (±2.6) 54.1 (±1.7) P2 34.5 (±1.4) 34.0 (±1.7) 33.9 (±1.1) 28.9 (±1.8) 38.0 (±2.1) 32.2 (±0.8) P3 2.4 (±0.6) 2.2 (±0.1) 3.1 (±0.3) 2.0 (±0.4) 3.0 (±0.3) 1.5 (±0.3) Supernatant S 12.5 (±0.6) 12.7 (±0.7) 11.4 (±0.6) 12.2 (±0.7) 11.9 (±0.6) 12.2 (±0.7) a Means from two replications are given in the table; standard deviations are in parentheses. Vol. 63, No. 5,1986 415 environment on lipid content and composition has been reported patterns for the two varieties were similar, and defatting of the (Fisher et al 1964, 1966; Bekes et al 1986). flours did not change electrophoregrams. SDS-PAGE showed that all six S protein preparations were RP-HPLC of S fractions from nondefatted Neepawa and Chile quite heterogeneous (Fig. 1). Each pattern was dominated by two flours, however, showed substantive qualitative and quantitative major bands characterized by molecular weights of approximately differences between the two varieties (Fig. 3). For example, peaks 14,000 and 16,000. Additional numerous minor bands were a, b, h, 1, p, r, and s are present in the chromatogram of Neepawa observed throughout the fractionation range for all samples of but absent in that of Chile, and Chile has additional peaks g and o. both varieties. Visual evaluation of relative band intensities Major quantitative differences are also evident, for example, peak showed no major quantitative changes caused by defatting flours e is much larger in Neepawa, whereas peaks m and n are much with the exception of S protein of the partially defatted Chile flour, larger in Chile. For this sample, intensity of bands that correspond to gel-filtration Comparison of chromatograms of nondefatted, partially fraction II SDS-PAGE subunits (see below) is much lower than defatted, and totally defatted flours of each variety (results not that of the same band of the other S protein fractions. It might be shown) revealed quantitative differences in some peaks, possibly that the protein of this fraction was partially extracted by n-hexane indicating selective interactions of specific proteins with flour during defatting of flour. No similar observation, however, was lipids. made for S protein from partially defatted Neepawa flour. This RP-HPLC comparison of S fraction and 70% aqueous, ethanolfact could be explained by different protein-lipid interactions in soluble proteins (mainly gliadin) from nondefatted Neepawa flour these two wheat varieties. (Fig. 4) showed that most S fraction peaks elute between 11 and 30 Acidic PAGE of all S protein preprations (Fig. 2) revealed four min, whereas major gliadin peaks elute from 25 to 45 min. Thus, major and at least three minor bands of medium mobility, and most S protein components are less hydrophobic than most minor bands throughout the high-mobility range. Analogous gliadins. In this respect, S proteins resemble many wheat albumins and globulins (Bietz 1983, Bietz et al 1984). This observation is consistent with results that showed the low-molecular-weight S Neepawa Chile