Agric. Biol. Chern., 47 (10), 2273-2280, 1983 2273

Properties of Winged { tetragonolobus) in Comparison with (Glycine max) and CommonBean {) 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 , 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 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 . 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 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.

£1111. vl (IB JhH IU Mi 2276 S. Ochiai Yanagi

Fig. 2. Sedimentation Diagrams of Whole Seed Extracts from Winged Bean, Soybean, and CommonBean (Kintokimame) at Various Ionic Strengths. Whole extracts in standard buffer were dialyzed against the same buffer or the same phosphate buffers with ionic strengths of 0.1, 0.3, and 0.7. The patterns shown were at 30 min after reaching 55,430 rpm at 20°C.

The sedimentation coefficients of the main that the S values of peaks were smaller at high soybean protein were about "7S" and "US," ionic strengths and larger at low ionic and minor components of about 3S and about strengths. Soybean proteins, however, showed 15S were seen at pH 7.5. But the main 7S and quite different sedimentation diagrams at vari- US peaks shifted to about 8.5S and 14S at pH ous ionic strengths. The well known soybean 4.5 and a little smaller S values at pH 9.0. The protein pattern of two clear main compo- association-dissociation phenomenon of com- nents, "7S" and "US," was seen at an ionic mon bean proteins for pH change were quite strength of 0.5 and the separation of two different from those observed above with peaks was clearer at an ionic strength of 0.7, winged bean and soybean. Most of the "7S" showing that the "7S" became smaller. At protein of common beans (Kintokimame) at ionic strengths of 0.1 and 0.3 the S values of neutral pH were shown to associate to become the two main peaks were very close to each about 18S protein at pH under 6.0. This other. Thus the main winged bean "6.5S" pro- phenomenon with French bean (Phaseolus vul- tein was shown to be different from soybean garis) was reported by Sun et al.9) At pH 10.0 "7S" protein too in physical or chemical all proteins of the three beans were dissociated character. At ionic strengths of lower than to 2 to 4S components. This dissociation was 0.1, not only soybean but also winged bean partially reversible. From these results winged and common bean protein too changed bean"6.5S" protein(s) was distinctly shown to drastically in their structure. The details of have a different physicochemical nature from these phenomena will be mentioned on most of common bean "7S" protein(s) though nother occasion. Psophocarpus and Phaseolus are thought to be a Whole winged bean extract was fractionated rom closely related families. with Sepharose 6B column chromatography. In Fig. 2 schlieren patterns of whole seed As shown in Fig. 3-a, the elution profile mainly extracts from the three beans at ionic strengths consisted of three peaks: the first peak at void of0.1to 0.7 are demonstrated. Proteins from volume, the 2nd and 3rd peaks designated as A winged beans and common beans did not have andB. The sedimentation pattern of each peak

intensivef differences with ionic change, except is shown in Fig. 3-b. The void peak contained Winged Bean Protein Compared with Soybean and CommonBean 2277

B 0.5r ^~ ' r~^q ,' 1.0' 5.0 - 0D280 A / ,/' 0D260 J ^0.4- 0.8 z Aå /' 4.0à" ;*

3'°" 1st \ o0,6à" u /' / I ^-^ §" 20.2- /1

n-i \*M i 1 1--i 1-===

0 10 20 30 40 1.0- A FRACTION NUMBER 0.8- I a

0.6- o \

O.tJå \

SEPHAROSE 6B DEAE-SEPHADEX 1ST PEAK-A MAIN PEAK 0 20 40 60 80 FRACTION NUMBER b a Fig. 4. a: DEAE Sepharose Chromatography of the 1st 1ST VOID 1ST PEAK-A 1ST PEAK-B Sepharose 6B Peak A, the "6.5S" Component(s) of Winged Bean.

Pooled peak A fractions were dialyzed against 30 niM Tris- I if 1H l HC1 buffer, pH 7.5, containing 0.1m NaCl and 0.5niM DTT and charged to the column equilibrated with the same buffer. Elution was carried out with the same buffer containing 0.1 to 0.5m of NaCl in a gradient. 2ND PEAK-A b: Sedimentation Profile of the "6.5S" Component(s) of Winged Bean before and after DEAE Chromatography. b Pictures were at 30 min after reaching 55,430 rpm. Fig. 3. a: Fractionationinof Whole Winged Bean Proteins through Sepharose 6B Column Chromatography (Top) and Rechromatography of Peak A Fraction from the 1st Chromatography (Bottom). Therefore peak A, the "6.5S" component(s), The column was equilibrated and eluted with standard was assumed in this paper to be the main buffer,and the fraction volume was 6ml. winged bean storage protein. As seen by the b: Sedimentation Patterns of Fractionated Winged Bean elution pattern of rechromatography and the Proteins through Sepharose 6B Chromatography. sedimentation pattern, peak A, the "6.5S" Photographs were taken 30 min after reaching 55,430 rpm. component(s), was almost isolated as a chro- matographically and centrifugally single peak only very large molecular weight substance(s) by one passage through a Sepharose 6B gel which could not be observed under these cen- column. Rather larger amounts of high molec- trifugal conditions. Peak A and peak B cor- ular weight substances could be observed in responded to the "6.5S" and "2.5S" com- the sedimentation profile after the 2nd chro- ponents, respectively. As mentioned in our matography (bottom-side tailing). These high other report,3) peak B was a mixture of many molecular component(s) might have been proteins which were different for various enerated after chromatography elution. strains of winged beans. The main component The Sepharose 6B fraction A was chroma-

of peak A seemed to be commonto all wing- tographedg with DEAE Sephadex or DEAE ed beans in the results of chromatography Sepharose (Fig. 4-a). Both results were fun- profiles, PAGE, and sedimentation analyses.3) damentally the same. At 0.1m NaCI con- 2278 S. Ochiai Yanagi

centration, almost all the protein components The acturally measured s20 value came in a were bound to the ion exchange gel. A main wide range in proportion to the ionic strength peak began to be eluted at 0.25 m NaCl. Before of the solution, and the measured s20 w in the and after the main peak, small peaks were solution of OD280 = 3.0 with and without DTT separated according to the NaCl gradient. In were6.7 and 7.0, but the 520w extrapolated spite of this purification procedure, the sedi- from the measured values of\i=0.05 to fi=0.5 mentation profile of the DEAE Sepharose was8.1 (Fig. 6-b). Besides the actual s20,w in main peak was not as sharp as the 1st chro- concentrated solutions higher than OD280 = 3 matography of Sepharose 6B peak A, and were much less than 6.7 (data not shown). The the high molecular side tailing was seen, just structure of the protein seemed to be changed as after Sepharose 6B rechromatography (Fig. 3-b, Fig. 4-b). The alteration of ionic strength of the solution, an ion exchange reaction or some other unknown factor might have cause BSA° .JLJBl aggregation. 6B peak-A As seen in Fig. 5-a, DEAE Sepharose frac- DEAE #27 ZZ^M tions, like the Sepharose 6B fractions, did not DEAE #30 present a sharp PAGE band. Electrophoresis DEAE #32^H in high ionic strength, adding 0.4m NaCl, could not prevent the band from becoming broad (Fig. 5-b). The "6.5S" components of 0 winged bean might have some variations of BSA HHIII molecular size or amino acid components. 6B peak-AiHili

The sedimentation coefficient, s209 of DEAE #27^H»å :å :å :å :å :å :å :à":à":å :;:>:::¥::>:å :å :å :::å : Sepharose 6B peak A of UPS-99 was analyzed DEAE #30(^1 at several concentrations (Fig. 6-a) and in DEAE #32iilll solutions of various ionic strengths (Fig. 6-b). When the protein concentration became lower, Fig.5. Electrophoresis Pattern of the Winged Bean "6.5S" Peak before and after DEAE Chromatography. the value of s20 went up slightly at the ionic strength of 0.5. Calculated ^20 w were about a, by the ordinary PAGE method of Davis9); b, in high ionicconcentration buffer. O, origin; F, front; #27 to 32, 0.5S larger than the measured s20 of this correspond to the fraction numbers in Fig. 4-a; BSA, condition, so the extrapolated s% w was 6.9. bovineserum albumin.

a b 8.0å N

å .: Xn å

S ' <> '

...* t? iy. 0 4 8 12 0.2 0.4 0D280 IONIC STRENGTH

Fig. 6. Sedimentation Coefficient of the Sepharose 6B Peak A of Winged Bean Seed Proteins (UPS-99). a, with change of protein concentration. Ionic strength of the solution was 0.5. b, with change of ionic strength of the solution. OD280 of the solution was 3.0. O and A, measured in distilled water adjusted to pH 7.5 with and without 0.5 him DTT, respectively. Winged Bean Protein Compared with Soybean and CommonBean 2279

proteins than the "6.5S" component were not BSA lljlj observed, unlike with soybeans, though both FRESH |jp|j beans were so prominent for high protein Jiiiill contents (more than 30%). The protein content Ac.D,-80 Ipi of commonbeans was much less than winged beans (around 25%) and the ratio of the -80 ||p| "6.5S" component(s) to "2 to 3S" components -20 pIBIl was distinctly higher than that of winged bean. So the storage protein content of beans and the content of larger molecular protein was shown to have no relationship. The "18S" com- Fig.7. Effects of Storage Conditions on the "6.5S" Protein of Winged Bean Seed. ponent, which was the major peak of the common proteins in acidic conditions, was not Fresh, fresh fraction of peak A from Sepharose 6B chromatography; Ac.D. - 80, frozen with acetone dry ice observed at all with winged beans including and stored at -80°C; -80, stored directly at -80°C; the various stages of bean development.10) -20, stored directly at -20°C; 5, stored at 5°C, for 5 Though the ultracentrifugal pattern of the months. O, origin of PAGE; F, front of PAGE; BSA, "6.5S" component of winged beans after only bovine serum albumin. one passage of Sepharose 6B chromatography considerably according to the ionic strength of was an almost symmetrical single peak and the solution and the concentration of the only traces of tailing components before and protein. Therefore, calculation and extrapo- after the peak were seen, the main PAGE band lation seemed not have practical meaning and wasbroad, besides the existence of a few minor accurate S values must be mentioned with the bands. The minor components could not be onditions of measurement. eliminated completely by DEAE Sepharose chromatography (Fig. 5) nor by CM Sepha- c Freezing was a very good method for pre- serving the structure of the "6.5S" protein. rose chromatography (data not shown). High Samples kept in the refrigerator for a long time molecular weight aggregates of the "6.5S" were degraded and the original proteins were components seemed to be present in rather not seen at all by PAGE (Fig. 7). Keeping the larger amounts after rechromatography on sample in a -80°C freezer after acetone dry Sepharose 6B or DEAE Sepharose. There- ice freezing, or keeping in a -80°C freezer fore, to know the fundamental nature of the directly, or keeping in a - 20°C freezer directly "6.5S" component, analyses of sedimenta- for five months gave electrophoretically and tion coefficient were performed using the peak ultracentrifugally the same protein profile as A fraction from the 1st Sepharose 6B chro- the freshly prepared fraction. matography. Seeing that the results of Fig. 6 and the sedimentation pattern of whole seed extract in DISCUSSION higher protein concentrations and in very low The profiles of winged bean proteins de- ionic strengths was quite different from the scribed in this paper could be thought patterns shown in Figs. 1 and 2 (data not to reflect almost the total aspect of storage shown),the actual ^2o,w or ^cw °f this com- protein in seeds, because the standard buffer ponent was difficult to determine simply, and use here could extract more than 90% of the to decide those values by extrapolation with total N-substances in the seeds, as seen in measured S values seemed to have no practical Table I. meaning. The majority of the seed storage The main winged bean proteins were sepa- protein may have a very small molecular rated into two components at various pHs and weight distribution and variation of consti- ionic strengths, and higher molecular weight tuent amino acids, seeing the results of Fig. 5- 2280 S. Ochiai Yanagi aand -b. The details of these heterogenieties Chem., 47, 2267 (1983). have to be studied in relationship to varieties G. Gomori, "Method in Enzymology," Vol. I, ed. by andmaturities, and have been left to be sub- S. P. Colowick and N. O. Kaplan, Academic Press nc., New York, 1955, p. 138. jects of a future paper. R. H. Burris and P. W. Wilson, "Methods in Enzymology," Vol. IV, ed. by S. P. Colowick and N. Acknowledgments. Author wishes to give cordial

I O. Kaplan, Academic Press Inc., New York, 1957, p. thanks to Mr. H. Takada and Mr. J. Abe, of the 356. Okinawa branch of the Tropical Agriculture Research O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Center, and Dr. S. Uemoto, Kyushu University, for sup- andall, /. Biol. Chem., 193, 265 (1951). plying the winged bean seeds. E. Layne, "Method in Enzymology," Vol. Ill, ed. by S. P. Colowick and N. O. Kaplan, Academic Press REFERENCES Inc., New York, 1957, p. 447. B.J.Davis,Ann. N. Y. Acad. Set, 121, 404 (1964). 1) J. M. Gillespie and R. J. Blagrove, Aust. J. Plant S.M.Sun,R. C. McLeester, F. A. Bliss and T. C. PhysioL, 5, 357 (1978). Hall,/. Biol. Chem., 249, 2118 (1974). 2) S. K. Sathe and D. K. Salunkhe, /. Food ScL, 46, S. O. Yanagi, M. Kato and S. Uemoto, Agric. Biol. 1389 (1981).

Chem.,R 47, 2387 (1983). 3) S. O. Yanagi, N. Yoshida and K. Saio, Agric. Biol.