Properties of Winged Bean (Psophocarpus Tetragonolobus
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Agric. Biol. Chern., 47 (10), 2273-2280, 1983 2273 Properties of Winged Bean {Psophocarpus tetragonolobus) Protein in Comparison with Soybean (Glycine max) and CommonBean {Phaseolus vulgaris) Protein Sonoe Ochiai Yanagi National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Kannondai, Yatabe-machi, Tsukuba-gun, Ibaraki 305, Japan Received February 28, 1983 Conditions were defined which extract more than 90% of winged bean {Psophocarpus tetragonolobus) seed proteins. Sedimentation profiles of whole seed extract from winged bean, soybean, and commonbean (variety "Kintokimame") at various pHs and ionic strengths were compared, because winged bean and soybean are resemble each other closely in their protein- and lipid-rich nature, and winged bean and commonbean {Phaseolus vulgaris) are thought to be of nearly related families. However, a clear dissimilarity of their "6 to 7S" component(s), one of the main storage proteins in the three beans, was represented. Two main peaks of winged bean protein by Sepharose 6B chromatography were shown to correspond to the "6.5S" and "2.5S" com- ponents.Extrapolateds20tV, or ^o.w of the "6.5S" component seemed to have no practical meaning because the actual structure of the "6.5S" protein distilled water or very low ionic concentrations were altered discontinuously from the usual patterns. Further purification of the "6.5S" component(s) could be carried out by rechromatography on Sepharose 6B or DEAE Sepharose, eliminating minor components. However, the electrophoretic or ultracentrifugal patterns showed the occurrence of small amounts of aggregation simultaneously. The structure of the "6.5S" component was preserved for several months by freezing. A few reports on basic properties of winged (Phaseolus vulgaris) by plant classification. bean (Psophocarpus tetragonolobus) seed pro- Therefore, winged bean protein was compared tein have been published in the recent studies with soybean and commonbean protein, using on various aspects of winged beans. Gillespie he same method of extraction, in this paper. and Blagrove reported the isolation and prop- In another presentation3* "6.5S" component erties of winged bean seed protein extracted was confirmed to be the general storage pro- with an acidic buffer (acetate chloride pH 4.5 teint in many selections of winged bean seeds. buffer) and they mentioned that the major The component(s) of "6.5S," therefore, were proteins had sedimentation coefficients of 2S fractionated and their properties were investi- and 6S.1} Sathe and Salunkhe studied subunits' gated in this paper. molecular weights of winged bean protein extracted with water.2) MATERIALS AND METHODS One of the reasons why winged bean is considered to be noteworthy is the abundance Winged bean lines were Nigeria Tpt 2 and Indonesia of lipid and protein content which, as has 909, harvested at the Okinawa Branch of the Tropical become well known recently, is almost equal to Agriculture Research Center, Ministry of Agriculture, Forestry and Fisheries. The soybean variety was Norin that of soybeans. From another point of view, o. 2, and the commonbean was Taisho Kintokimame. winged bean is regarded as being in a family The methods cited by Gomori4) was mainly followed in closely related to that of common beans N preparing buffers. Thirty him phosphate buffer, pH 7.5, Abbreviations: DTT, dithiothreitol, SDS, sodium dodecyl sulfate, PAGE, polyacrylamide gel electrophoresis. 2274 S. Ochiai Yanagi containing 0.4m NaCl and 0.5 him dithiothreitol (DTT) 6ml. Sample fractions from the Sepharose 6B column was was called the standard buffer. Nitrogen content was dialyzed against 30 mM Tris-HCl buffer, pH 7.5, contain- determined by the micro-Kjeldahl method.5) Protein was ing 0.1 m NaCl and 0.5mM DTT and loaded onto a DEAE measured by the method of Lowry6) and by ultraviolet Sepharose column (0.7 x 14cm) previously equilibrated absorption7) or calculated by multiplying the nitrogen withthe same buffer. The column was eluted with the same value by 6.25. Whole seed extract was prepared as men- buffer containing 0.1 m NaCl to 0.5 m NaCl, stepwise or as ioned in another of our papers.3) a gradient. Ultracentrifugal analyses were performed with a Hitachi Polyacrylamide gel electrophoresis (PAGE) was per- 282 using a schlieren optical system and a UV absorption formed essentially by the pH 8.2 system of Davis8) ap- ystem at 20°C. plied to a slab gel. Sepharose 6B chromatography was carried out with a t 1.6s x 100cm or 2.6 x 100cm column equilibrated with the standard buffer. Two ml or 5ml of dialyzed sample, RESULTS OD280=30 to 60 for the 1st chromatography and OD280 =10 for the 2nd chromatography, was charged and The effects of pH and ionic strength on eluted with the standard buffer and fractionated to 2 to extracting winged bean protein are summa- ableI. Effect of pH and Ionic Strength on Extraction T of Winged Bean Protein S o lu b ilize d Solubilized p ro te in N -su b sta n c e /T o ta l m g /g sa m p le ' B u f erO P H if Cru de _ ., , N-su bsta ncefc . Di alyze d , 0/,e xtract J (%) A b B d A c eta te 4.5 0. 1 3 9 1 6 0.3 4 4 1 9 0.5 5 0 2 3 5.2 0. 1 4 0 1 8 0.3 4 2 2 0 72 2 3 4 2 2 6 0.5 4 7 2 4 7 5 2 38 2 5 9 P h o sp h a te 6.0 0. 1 5 5 2 0 0.3 5 7 2 1 0.5 5 6 20 7.0 0. 1 6 2 22 0.3 6 1 2 1 0.5 5 9 2 3 7.5 0. 1 6 4 2 9 0.3 6 5 30 9 3 3 2 2 3 11 0.5 6 7 2 9 T ris-H C l 7.0 0. 1 5 0 1 8 0.3 5 9 2 3 0.5 6 3 2 7 8.0 0. 1 7 6 30 9 0 3 1 1 3 0 2 0.3 7 3 30 9 3 3 2 3 3 14 0.5 7 5 3 1 9 .0 0. 1 32 1 3 0.3 34 1 3 0.5 3 1 1 5 B o ra x -N a O H 10.0 0 .1 5 3 2 3 0.3 5 0 2 5 0.5 5 3 2 7 uffer concentration was 30 mM,and ionic strength (jum) was adjusted with NaCl. Measured by the Kjeldahl method.5) Milligrams protein for dry weight sample. Measured by the Kjeldahl method.5) B Measured by the Lowry et al.'s method.6) Winged Bean Protein Compared with Soybean and CommonBean 2275 rized in Table I. Tris-HCl buffer, pH 8.0, was sistent with the percentages of solubilized-N/ the best for extracting OD280 absorbing sub- total-N. An ionic strength of 0.1 was not as stances. Phosphate buffer, pH 7.5, had the effective in extracting OD280 absorbing sub- same effect in extracting undialyzable high stances for both crude dialyzed extracts as molecular weight OD280 absorbing substances ionic strengths of0.3 and 0.5 through all of the as Tris-HCl buffer, pH 8.0. Extracted nitro- pH range. Considering these results and com- genous substances to total N-substances mea- paring them with other legume protein reports, sured by the Kjeldahl method were more than 30mM phosphate buffer, pH 7.5, including 90% for Tris-HCl (pH 8.0) and phosphate (pH 0.4m NaCl and 0.5mM DTT was chosen for 7.5) buffers. With acetate buffer, less than 75% the standard buffer for extracting winged bean of total nitrogen could be taken out even if the rotein in our experiments. constituents of the buffer were the same as Sedimentation diagrams of whole seed ex- Gillespie and Blagrove's.1} Not only pH but tracts from winged bean, soybean, and com- also the kind of buffer was significant, because mon bean at various pHs are exhibited in Fig. a large amount of precipitation was observed 1.p The sedimentation profiles of winged bean with citrate buffer extraction in a wide acidic proteins were fundamentally similar from a range of pH (pH 3.0 to 6.0, data not shown). pH of4.0 (data not shown) to pH 9.0. The two The amount of solubilized protein was mea- main peaks were about "6.5S" and about sured for dialyzed whole seed extract by the "2.5S" at pH 7.5 (patterns of 8 different Kjeldahl and Lowry methods. Results of the winged bean strains were shown in another two methods generally agreed for the most of paper3^ with larger S values at acidic pHs and the pH range and ionic strengths examined. As smaller S values at basic pHs. Soybean pro- the total protein content of this winged bean teins showed almost the same phenomena as was 34.8%,3) these results were also very con- winged bean proteins with alteration of pH. WINGED BEAN SOYBEAN COMMON BEAN P^ ^-5 Fig. 1. Schlieren Patterns of Whole Seed Extracts from Winged Bean, Soybean, and Common Bean (Kintokimame), at Various pHs. Whole extracts in standard buffer were dialyzed against the same buffer or acetate pH 4.5 or 5.2, of Tris-HCl pH 9.0 or borate pH 10.0 buffers with concentrations of 30mM and containing 0.4m NaCl and 0.5 him DTT. The figures shown were at 30 min after reaching 55,430 rpm at 20°C.