Resolution of pea legumin subunits by high-performance liquid chromatography

ANALYTICAL

BIOCHEMISTRY

160, 202-210

(1987)

Resolution of Pea Legumin Subunits by High-Performance Liquid Chromatography J. R. BACON, N. LAMBERT, Chemistry

M. PHALP, G. W. PLUMB,

and Biochemistry Division, Institute Colney Lane, Norwich NR4

AND D. J. WRIGHT

of Food Research, Norwich 7UA. United Kingdom

Laboratory,

Received May 7, 1986 Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fmctionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six “normal” legumin subunits (M, 60,000) were identified as well as some “large” (M, 66,000) and “small” (M, 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies, Anomalous electrophoretic behavior of the legumin subunits was also observed. 0 1987 Academic Press, Inc. KEY

WORDS:

HPLC, proteins; pea legumin; subunit structure: electrophoresis.

The storage proteins of legumes, e.g., soya, pea, and bean, are of particular importance to the food industry (1). Typically there are two major classes of storage protein, referred to as 7s and I IS, properties of which have been reviewed frequently (2-4). Legumin is the name given to the 11s class of storage proteins from pea. Each legumin molecule is composed of six subunits of approximately 60,000 M,. The subunits are not identical, but come from a pool of closely related polypeptides. They are synthesized in vivo as a single 60,000 M, polypeptide which is then proteolytically cleaved to generate a ~40,000 M, polypeptide chain with an acidic isoelectric point (pl), and a polypeptide chain of ~20,000 M, with a basic pZ, linked by a disulfide bond. There are believed to be approximately eight genes encoding the different subunits (5). In addition, the subunits undergo post-translational modification (proteolysis, deamidation) to varying degrees, further adding to their intrinsic heterogeneity (4). Preparations of the hexamer, individual subunits, and the acidic and basic polypep0003-2697187

$3.00

Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

202

tide chains have been reported using a variety of conventional purification techniques (6-8). However, the extreme heterogeneity within the legumin class, has made purification to homogeneity of such species virtually impossible, especially if significant quantities of material (> 10 mg) are required. The development of recombinant DNA techniques has greatly assisted in unraveling the complexities of storage protein structure, and many different genes have been isolated and sequenced from several legume species, revealing close homologies (9,lO). However, the genetic approach is of little use if experiments are to be performed with the proteins per se. We have commenced studies on the production and characterization of monoclonal antibodies to legumin and also on its assembly from component subunits. These experiments require significant quantities of very pure material. With this aim in mind we reevaluated the approaches taken to purify legumin subunits from pea. In this communication we describe the application of an HPLC system (namely

CHROMATOGRAPHIC

RESOLUTION

Pharmacia’s FPLC) to the separation of pea legumin subunits. The FPLC system with its specially developed support matrices has been used to great effect in the purification of many diverse proteins (I 1). This to our knowledge is the first time HPLC has been used to resolve 1 IS subunits. MATERIALS

For HPLC separations of samples, an FPLC system (Pharmacia Fine Chemicals, Uppsala, Sweden) was employed using P500 pumps and a UV-1 detection unit. The columns used were: Mono Q (lo/ lo), Mono S (5/5), and Mono P (5/20) all purchased from Pharmacia, as was the Polybuffer 74 used in eluting the Mono P column. Electrophoretic analysis was performed using the GE2/4 Pharmacia electrophoresis system, and employing specially purified (Electran) reagents obtained from BDH Chemicals (Poole, Dorset UK). A!, marker proteins (see legend to Fig. 5a) were obtained from Sigma Chemical Co. (Poole, Dorset, UK). All other chemicals used were of analar grade, and were obtained from BDH Chemicals. Peas (Pisum sativum cv. Birte) were a gift from PGRO (Thornhaugh, Cambridgeshire, UK).

OF

PEA

203

LEGUMIN

at room temperature. Purified legumin (50 mg) was dissolved in approximately 6 ml 50 mM sodium phosphate buffer, adjusted to pH 7.5 with NaOH, containing 6 M urea. (All urea-containing solutions were prepared fresh to minimize the buildup of cyanates and the resulting carbamylation of proteins.) Samples were loaded, with the aid of a 10 ml “superloop,” onto a Mono Q ( 1O/ 10 anionexchange column equilibrated in the above buffer. The flow rate throughout was 1 ml/ min. Unbound material was removed by washing with the above phosphate buffer, and bound proteins were eIuted with a stepwise NaCl gradient as shown in Fig. 1. Protein peaks PI -P6 (P7 had insignificant levels of protein) were pooled individually and the urea was removed by exhaustive dialysis against distilled water at 5°C prior to lyophilization. Further Fractionation

of Pl, P3, and P.5

Fractionation of Pl. P 1 (10 mg) was dissolved in 0.5 ml 25 mM bis-Tris adjusted to pH 7.1 with iminodiacetic acid, containing 6 M urea, and loaded onto a Mono P (5/20) chromatofocusing column equilibrated in

METHODS

Preparation of Legumin from Pea (Pisum sativum cv. Birte) Pea legumin was isolated from a pea flour extract by ammonium sulfate fractionation and purified by hydroxyapatite-ultrogel column chromatography as described by Lambert et al. (12). The final purified legumin fraction was stored as a lyophilized powder at 5°C. Isolation of Legumin Subunits by Anion-Exchange FPLC All buffers and samples applied to the FPLC columns were filtered through a 0.22 pm filter, and all separations were carried out

0

22

4s

Time

90

(min)

FIG. I. Separation of pea legumin subunits on a Mono Q anion exchange column, in the presence of 6 M urea. Pea legumin (50 mg) was dissolved in 50 mM sodium phosphate buffer pH 7.5 containing 6 M urea, and loaded onto a Mono Q (lo/IO column equilibrated in the same buffer. Fractions Pl-P7 were obtained as detailed under Methods. -, AzgO;---, NaCl concentration.

204

BACON

the same buffer. The flow rate throughout the separation was 0.5 ml/min. Unbound material was washed off with the above buffer, and the bound polypeptides were eluted with a pH gradient generated by passing through 10% Polybuffer 74 adjusted to pH 4.0 with iminodiacetic acid, containing 6 M urea. The resolved fractions Pla-Pld (see Fig. 2) were pooled individually, dialyzed against distilled water, and lyophilized. Fractionation of P3. P3 (30 mg) was dissolved in 5 ml 50 mM sodium phosphate buffer adjusted to pH 7.5 with NaOH, containing 6 M urea, and loaded onto a Mono Q (lo/ 10) anion exchange column equilibrated in the same buffer. Unbound material was washed off with the phosphate buffer, before eluting the bound protein with a shallow, linear NaCl gradient (see Fig. 3). The flow rate was 1 ml/min throughout. Fractions P3a-P3c were pooled individually, dialyzed, and lyophilized. Fractionation of PS. P5 (5- 10 mg) was dissolved in 0.5 ml 50 mM sodium acetate buffer, pH 4.1, containing 6 M urea, and loaded onto a Mono S (5/5) cation exchange column equilibrated in the same buffer.

ET

AL.

l% I

r.-0

_._____-

-

L-. 33

100

66 Time

(min)

FIG. 3. Separation of P3 on a Mono Q anion exchange column. Fraction P3 (see Fig. 1; 30 mg) was dissolved in 50 mM sodium phosphate buffer pH 7.5, containing 6 M urea, and loaded onto a Mono Q (IO/ 10) column equilibrated in the same buffer. Fractions P3a-P3c were obtamed by eluting with a shallow NaCl gradient as described under Methods. -, &a; ---, NaCl.

After removing unbound material with the same buffer, bound proteins were eluted with a linear NaCl gradient as shown in Fig. 4. The flow rate was 1 ml/min throughout. Fractions P5a and P5b were pooled individually, dialyzed, and lyophilized. SDS’-Polyacrylamide-Gel (SDS-PAGE)

Electrophoresis

Electrophoresis was performed in 1.5 mm 14 X 18 cm 14% acrylamide slab gels using the Laemmli ( 13) buffering system. Samples prepared under reducing conditions were boiled for 15 min in 2% (w/v) SDS and 5% (v/v) /I-mercaptoethanol. Samples prepared under nonreducing conditions were denatured with 2% (w/v) SDS in the presence of 1 mM iodoacetamide to prevent thiol-disulfide interchange reactions. These samples were not boiled, as this has been shown to cleave the intermolecular disulfide bond linking acidic and basic chains (14). X

Time

(mid

FIG. 2. Separation of PI on a Mono P chromatofocusing column. Fraction PI (see Fig. I ; 20 mg) was dissolved in 25 mM bis-Tris buffer adjusted to pH 7.1 with iminodiacetic acid, containing 6 M urea, and loaded onto a Mono P (5/20) column equilibrated in the same buffer. Fractions Pla-Pld were obtained as described under Methods, The arrow indicates the commencement of the pH gradient using Polybuffer 74.

RESULTS AND DISCUSSION

Pea legumin was purified by a method developed in our laboratory to ensure maxi’ Abbreviations used: SDS, sodium dodecyl PAGE, polyacrylamide gel electrophoresis.

sulfate;

CHROMATOGRAPHIC

2 5 I’ 1’ 1’ ‘;:i---_-2 0 25

,’ I’

I’

/’

,

2’

50 Time

/’ ,/ ‘Sb , /’ -

75

RESOLUTION

/’

1oc

hin)

FIG. 4. Separation of P5 on a Mono S cation exchange column. Fraction PS (see Fig. I; 5 mg) was dissolved in 50 mM sodium acetate buffer pH 4.1 containing 6 M urea, and loaded onto a Mono S column equilibrated in the same buffer. Fractions P5a and P5b were obtained as described under Methods. -, A280; ---, NaCl gradient.

mum integrity of the final product (12). SDS-PAGE analysis of pea legumin under reducing (Fig. 5a, lane 2), and nonreducing conditions (Fig. 5b, lane 2) confirm that its polypeptide composition is similar to those reported by other groups (6,15) and in addition reveal some of the complex heterogeneity typically observed within the 11s fraction, not only from pea but also from other legume species (2). The reduced gel shows many bands between 21,000 and 24,000 M, which are generally referred to as the basic polypeptide chains. The broad band around 40,000 M, corresponds to the acidic chains. On nonreducing gels, the major band of about 60,000 M, corresponds to the various combinations of acidic and basic polypeptide chains, disulfide bonded to give legumin subunits. Also visible is a minor band at 40,000 M, which has been termed “small legumin” and comprises basic and acidic moieties of approximately 20,000 M, each (15). A faint band seen at aproximately 64,000 M, has been attributed to a large legumin subunit (15). Another minor band at 30,000 M, is of unknown origin. Subfractionation of Pea Legumin The uncertain composition of the legumin fraction severely restricts its usefulness in

OF PEA LEGUMIN

205

further experiments. To date, it has not proved possible to purify specific hexameric legumin species using conventional techniques ( 16,17); this is due to the close similarities between legumin hexamers, some of which will share common subunits (3). The advent of HPLC systems for protein purification has afforded dramatic improvements in the resolution and speed of separation (18). Consequently, we applied this technique, specifically the Pharmacia FPLC system, to the problem of further purifying the legumin fraction from pea. Unfortunately, even with FPLC we could not resolve the legumin into discrete 11s species using anion or cation exchange columns or by chromatofocusing; the legumin eluted as a very broad heterogeneous peak (data not shown) with a significant proportion precipitating on the columns, as evidenced by a substantial increase in column back pressure. These results clearly emphasize how closely homologous the legumins are. Another problem with this approach was that the legumin 11s fraction aggregated with time (as adjudged by gel filtration on a Superose 6 column), the mechanism of which is unclear. It may be that the composition of this fraction is dynamic, with various states of aggregation existing at any one time. Consequently, it was decided to purify the component subunits of the legumin fraction, after dissociation with 6 M urea. The 60,000 M, subunit, being the gene product, is the fundamental building block of the legumin molecule and it has been proved possible to reassemble the 1 IS complex in vitro from the subunits (19,20). Methods have been reported for the isolation of subunits from several legume 11s proteins using DEAE-Sephadex chromatography in the presence of 6 M urea (14,20), but the resolution attained was only moderate. In comparison, Fig. 1 shows the resolution of pea legumin subunits on a Mono Q anion exchange column attached to an FPLC system. The stepwise NaCl gradient was optimized to give maximum resolution. Each step was main-

206

BACON ET AL. 1

2345678

FIG. 5. SDS-PAGE analysis of pea legumin and fractions Pl-P6 obtained as detailed in Fig. 1. (a) Samples analyzed in the presence offl-mercaptoethanol. Lane 1, M, standard proteins (10 /.rgeach of rabbit muscle phosphorylase b 92,000 M,, bovine serum albumin 67,000 M,, hen ovalbumin 45,000 M,, rabbit muscle lactate dehydrogenase 35,000 M,, bovine pancreas chymotrypsinogen 25,000 M,, soyabean tryp sin inhibitor 2 1,000 M,, bovine milk @-lactoglobulin 18,000 M,, and horse heart cytochrome c 12,500 M,); lane 2, pea legumin (30 pg); lane 3, Pl (20 pg); lane 4, P2 (20 fig); lane 5, P3 (20 wg); lane 6, P4 (2Oeg); lane 7, PS (30 rg); lane 8, P6 (30 pg). (b) Samples analyzed in the absence of &mercaptoethanol. Lane 1, M, standard proteins (as in a); lane 2, pea legumin (15 pg); lane 3, PI (10 pg); lane 4, P2 (20 pg); lane 5, P3 (20 pg); lane 6, P4 (15 pg); lane 7, P5 (20 pg); lane 8, P6 (20 pg).

tained for at least one column volume to ensure that the fractions were discrete; six such fractions were resolved, although heterogeneity within some peaks was clearly visible. Fraction 7 was found to be essentially nonproteinaceous. Using this procedure up to 100 mg of legumin could be processed in 90 min. The speed and resolution was substantially superior to previous, conventional, anion exchange fractionations reported. Minimizing the time the proteins were in 6 M urea also reduced the possibility of articially increasing the heterogeneity still further through side-chain modification. The elution profile was highly reproducible, even between different preparations of pea legumin. SDS-PAGE analysis of PI-P6 (Figs. 5a, b) shows that although distinct, most were still not homogeneous. The unbound material Pl, consisted of species between

38,000 and 43,000 M, under nonreducing conditions, which in the presence of /3-mercaptoethanol dissociated to give several bands between 20,000 and 24,000 M,. This fraction therefore corresponds to the small legumin population of subunits. P2, a relatively minor component, migrated as a lO,OOO-12,000 it& polypeptide in the presence or absence of reducing agent. The origin of this protein is unknown; sequence analysis may show whether it is derived from one of the subunits by proteolysis as was demonstrated for soya (3). The largest fraction, P3, comprised a major band at 60,000 AI, (the typical 40,000 + 20,000 I%& subunit composition) and a lesser component at 64,000 AI, which is probably the large legumin subunit described by Matta et al. (15). Under reducing conditions, in addition to the 40,000 and 20,000 M, bands, a minor band of 49,000 M,

CHROMATOGRAPHIC

RESOLUTION

can be seen, the acidic polypeptide of the large legumin subunit. Whether the two different acidic polypeptides share a common 20,000 M, basic polypeptide, or whether there is more than one basic species unresolvable by SDS-PAGE is not known. P4 is a homogeneous 60,000 1M, subunit producing normal acidic and basic polypeptides under reducing conditions. P5 is very similar in composition to P3 having a large subunit in addition to the more typical type, although slight migrational differences can be observed and the band intensities are different. The fact that P3 and P5 eluted quite separately on the Mono Q column further demonstrates that these components although similar as adjudged by SDS-PAGE are quite distinct entities. Interestingly, a combination of the large acidic polypeptide (49,000 MJ and the basic polypeptide (22,000 A&) resulted in a species migrating at approximately 65,000 M,, demonstrating that at least one of the polypeptides was migrating anomalously. Such behavior is discussed below. Finally the major component of P6 was a species of 32,000 M, both under reducing and nonreducing conditions; the nature of this is unclear. Typical 40,000 and 20,000 M, polypeptides can be seen but these are less abundant components. The above results aptly demonstrate how complex the legumin fraction is, being composed of many similar polypeptides, some of which are not resolved by SDS-PAGE. Further PuriJication

of Legurnin

Subunits

Pl and P3, the major fractions (comprising ~60% of the recoverable protein from the first separation) were purified further by FPLC. Figure 2 shows the separation of Pl into four major and distinct fractions, P 1a-P 1d, using a Mono P chromatofocusing column. SDS-PAGE analysis revealed the complex composition of the four fractions (Figs. 6a, b). Pla comprised bands between 21,000 and 24,000 M, under both reducing

OF

PEA

LEGUMIN

207

and nonreducing conditions. It could be that they represent detached small acidic and/or basic polypeptides. Plb is a small legumin subunit of 40,000 M, composed of 19,000 and 23,000 M, polypeptides. In addition a band at 32,000 M, is prominent in both reducing and nonreducing gels. The nature of this is unknown, although it was only a minor component of the original Pl (see Fig. 5b, lane 3). P lc and Pld were composed of different types of small legumin subunits. P3 was further resolved on a Mono Q column, using a very shallow NaCl gradient (Fig. 3), into two major fractions, P3a and P3b, and a minor broad fraction, P3c (the sharp peak eluting at 1 M NaCl, was nonproteinaceous, and like P7 in Fig. 1 may be a “refractive” artifact). SDS-PAGE analysis (Figs. 6a, b) revealed P3a and P3b to be typical 60,000 M, subunits comprising 40,000 and 20,000 M, polypeptides, although slight migrational differences could be seen. P3c contained the large legumin acidic polypeptide observed in the original P3 (Fig. 5a, lane 5) as well as a more typical 40,000 M, species and basic polypeptides around 22,000 M,. There was insufficient material in P5 and P6 to fractionate further at a meaningful level. In any case, as mentioned previously, P6 comprised mainly a polypeptide of unknown source. Sufficient amounts of P5 were obtained however, by pooling several runs, and fractionation on a Mono S cation exchange column produced two distinct components (P5a and P5b) using a linear salt gradient (Fig. 4). Analysis by SDS-PAGE under reducing conditions (Fig. 6a, lanes 9, 10) showed that P5a was made up of typical 40,000 and 20,000 M, polypeptides, and P5b comprised in addition a large acidic polypeptide of 44,000 M,, although only the large subunit was observed on nonreducing gels (Fig. 6b, lane 9). Anomalous Behavior of Pea Legumin Fractions on Electrophoretic Analysis It is clear from our data that SDS-PAGE analysis can give misleading information on

208

BACON ET AL.

b

FIG. 6. SDS-PAGE analysis of fractions Pla-PSb obtained as described in Figs. 2-4. (a) Samples analyzed in the presence of &mercaptoethanol. Lane 1, M, standard proteins (see legend to Fig. 5a); lane 2, Pla (10 fig); lane 3, Plb (20 rep); lane 4, Plc (20 pg); lane 5, Pld (20 pg); lane 6, P3a (20 rep);lane 7, P3b (20 fig); lane 8, P3c (20 pg); lane 9, P5a (20 pg); lane 10, P5b (15 pg). (b) Samples analyzed in the absence of ~-mercaptoethanol.Lane1,Pla(lO~g);lane2,Plb(15~g);1ane3,Plc(lO~g);lane4,P1d(lO~g);1ane5, P3a (20 pg); lane 6, P3b (15 pg); lane 7, P3c (IO pg); lane 8, P5a (20 pg); lane 9, P5b (15 pg); lane 10, M, standard proteins (see legend to Fig. 5a).

the composition of pea legumin. Anomalies can arise from one of three possible sources. First, we have identified five distinct 60,000 &I, subunits by FPLC presumably exploiting minor charge differences present. These components could not be separated by SDSPAGE analysis under nonreducing conditions, and even showed similar migrational properties on reducing gels. Therefore SDSPAGE analysis of legumin only gives a limited indication of the heterogeneity present, although the minor differences observed could be accentuated by varying electrophoretie conditions, e.g., sample loading or acrylamide concentration (data not shown). Attempts were made to resolve the legumin fractions by two-dimensional gel electrophoresis according to the method of O’Farrell (21). However, because of problems of

artifactual heterogeneity when performing isoelectric focusing in the presence of urea (22), especially when applied to legume storage proteins (23), such analyses were curtailed. Second, as was noted earlier, some legumin polypeptides migrated in a nonlinear manner. For example, the large subunit component present in P5 (II~~ 65,000) consisted of protomers of M, 49,000 and 22,000. The reason for this is not known, although similar behavior has been reported for some soya 11s subunits (3). To assist in ascribing which polypeptides formed which disulfidelinked complexes, a two-dimensional electrophoretic approach was employed (15) in which species were separated by SDS-PAGE under nonreducing conditions in the first dimension, and then under reducing condi-

CHROMATOGRAPHIC

RESOLUTION

209

OF PEA LEGUMIN

tions in the second dimension (results not ities and uncertainties that have hampered workers in this field in the past. shown). Third, it was observed that some of the basic polypeptides stained very poorly, a CONCLUSIONS phenomenon that is probably associated Table 1 summarizes the above complex with their hydrophobic nature (4). As a result, one cannot readily quantify the species data. Pea legumin dissociated by 6 M urea was separated into twelve distinct fractions present simply on staining intensities. by FPLC using a range of columns. SDSTo summarize, an assessment of homogenity of 1 IS subunits is very difficult to PAGE analysis in the absence of P-mercapachieve by electrophoretic techniques. We toethanol revealed the existence of almost believe that the utilization of HPLC for such twenty different components, most of which, assessments should be encouraged. The with the exception of a few of unknown orispeed of analysis ensures that one is less gin, were consistent with the proposed model likely to obtain artifactual data, while a very for legumin subunits. This number is consishigh degree of resolution is still attained. As tent with the conclusions of Matta et al. (15). the use of HPLC for 1 IS proteins expands, In addition to the classic 60,000 M, subunits, and the variety of columns increases, we small subunits of 40,000 J4, and large subunits of 65,000 M, were observed. The FPLC would hope that the speed and resolution elution profiles presented in this paper are a demonstrated in this study will similarly imculmination of many experiments in which prove, thereby solving many of the complex-

TABLE 1 SUMMARY OF FRACTIONATION OF PEA LEGUMIN SUBUNKS BY PPLC

Fraction Pl

% Composition of legumin fraction’

Pla

25 2

Plb

S

Plc Pld P2 P3 P3a P3b P3c

10 8 I 35 15 15 1

P4 P5 P5a P5b P6

I 10 4 3 9

M, of species in the absence of fi-mercaptoethanol (X 10-9 21 21.5 32 40 40 40 10 60 60 60 65 60 60 65 22 32 60

1 1

1

1

’ % Composition based on integration of peaks detected at 280 nm.

Comments Free subunits? Free subunits? Unknown Small legumin subunit Small legumin subunit Small legumin subunit Proteolytic fragment? Normal subunit Normal subunit Normal subunit Large subunit Normal subunit Normal subunit Large subunit Unknown Unknown Normal subunit

210

BACON ET AL.

2. Derbyshire, E., Wright, D. J., and Boulter, D. (1976) other buffering systems, eluting salts, and Phytochemistry 15, 3-24. gradients were tested. Using conventional 3. Nielson, N. C. (1984) Philos. Trans. R. Sot. Lonchromatographic procedures, such optimidon, Ser. B 304, 287-296. zation would not have been so readily 4. Gatehouse, J. A., Croy, R. R. D., and Boulter, D. achieved. (1984) CRC Crit. Rev. Plant Sci 1, 287-314. 5. Domoney, C., and Casey, R. (1985) Nucl. Acids Res. The nature of the differences between the 13,687-699. subunits is probably severalfold. There are 6. Casey, R. (1979) Biochem. J. 177,509-520. believed to be eight genes encoding different 7. Croy, R. R. D., Derbyshire, E., Krishna, T. G., and legumin subunits in pea (5). Some of these Boulter, D. (1979) New Phytol. 83, 29-35. genes have been isolated and sequenced, re8. Casey, R., March, J. F., Sharman, J. E., and Short, M. N. (1981) Biochim. Biophys. Acta. 670, vealing close homologies, as well as notice428-432. able differences (9). There also appear to be 9. Croy, R. R. D., and Gatehouse, J. A. (1985) in Plant close similarities with other plant 11 S storage Genetic Engineering (Dodds, J. H., Ed.), pp. protein subunits (10). In addition to belong143-268, Cambridge Univ. Press, London/New ing to a multigene family, the 1 IS storage York. 10. Argos, P., Narayana, S. V. L., and Nielsen, N. C. proteins are thought to undergo post-trans(1985)EMBOJ.4, 1111-1117. lational processing, e.g., deamidation and FPLC Reference List, Pharmacia Fine proteolysis within the seed, further increas- 11. (1986) Chemicals, Uppsala, Sweden. ing heterogeneity. It is also possible that arti- 12. Lambert, N., Chambers, S. J., Phalp, M., and factual processing may occur during purifiWright, D. J. (1986) Biochem. Sot. Trans. 14, 1186-I 188. cation of the legumin fraction, although our 13. Laemmli, U. K. (1970) Nature (London) 227, isolation procedure ( 12) was devised to min680-685. imize such possibilities. 14. Staswick, P. E., Hermodson, M. A., and Nielsen, Four of the fractions isolated, Plc, P3a, N. C. (1981) J. Biol. Chem. 256,8752-8755. P3b, and P4, were demonstrated to be homo1.5. Matta, N. K., Gatehouse, J. A., and Boulter, D. (1981)5. Exp. Bot. 32, 1295-1307. geneous legumin subunits as shown by 16. Utsumi, S., and Mori, T. (1980) Biochim. Biophys. SDS-PAGE and FPLC, and were prepared Acta 621, 179-l 89. in sufficient quantities to allow further stud- 17. Utsumi, S., Inaba, H., and Mori, T. (1981) Phytoies. Currently, these fractions are being used chemistry 20, 585-589. to study the subunit assembly of 1 IS legu- 18. Regmier, F. E., and Gooding, K. M. (1980) Anal. Biochem. 103, l-25. min, and to produce and characterize monoclonal antibodies. We are also presently ap- 19. Utsumi, S., and Mori, T. (1983) J. Biochem. 94, 200 I-2008. plying the methods described in this commu20. Nakamura, T., Utsumi, S., and Mori, T. (1985) nication to the separation of subunits from Agric. Biol. Chem. 49, 2733-2740. other plant 11 S storage proteins. 21. O’Farrell, P. H. (1975) J. Biol. Chem. 250,

REFERENCES 1. Wright, D. J., and Bumstead, M. R. (1984) Phz’los. Trans. R. Sot. London, Ser. B 304,38 l-393.

4007-402 1. 22. Goamazza, E., and Righetti, P. G. (1980) in Electrophoresis ‘79 (Radola, B., Ed.), pp. 124- 140, de Gruyter, Berlin. 23. Gatehouse, J. A., Croy, R. R. D., and Boulter, D. (1980) Biochem. J. 185,497-503.