The three globulins of the seeds of L. angustifolius cv. Uniwhite may be satisfactorily resolved in 10 min by electrophoresis on cellulose acetate strips. These globulins, conglutins α, β and γ, vary markedly in their amino acid compositions, with conglutin Ω differing from conglutins α and β and most other legume storage proteins in its relatively high content of cystine and methionine and lower content of arginine and glutamic acid. When examined on sodium dodecyl sulphate-polyacrylamide gels, both in the absence and presence of β-mercaptoethanol, the three globulins were found to differ completely in the type of subunit proteins they contain and in the significance of intrachain disulphide bonding. Conglutin α was found to contain three or four types of non-covalently linked subunits with apparent molecular weights in the range 55 000-80 000, each of which may contain a disulphide-bonded moiety with a molecular weight near 20 000. Conglutin γ was found to contain disulphide-bonded chains of molecular weights 17 000 and 30 000, whereas the four major subunits of conglutin β, whose molecular weights lie in the range 20 000-60 000, were not covalently linked together. The latter globulin does not appear to be homogeneous, for it can be separated by fractional precipitation with ammonium sulphate into a series of fractions which differ markedly in the proportion of subunit types they contain.

Seeds from L. angustifolius grown at three levels of applied sulphur contain similar amounts of protein whereas the ratio of total nitrogen to total sulphur in the whole seed and in the extracted protein is greatly increased under sulphur deficiency. This large change in nitrogen to sulphur ratio is accompanied by suppression of the synthesis of conglutins α and γ which usually contain most of the sulphur-containing amino acids found in these seeds. This is balanced by synthesis of an increased amount of conglutin β which normally contains no methionine and a lower proportion of cystine. Studies of the protein subunit composition show that the overall molecular weight distribution of the polypeptides is independent of the level of sulphur, but under sulphur deficiency the higher molecular weight subunits do not contain the disulphide linkages normally present. It is not known whether these higher-molecular-weight conglutin β subunits are normally absent or present only in trace amounts in the seeds.

Physicochemical studies are reported for conglutin �, the minor globulin isolated from seeds of L. angustifolius cv. Uniwhite. Isoelectric focusing of the native protein in polyacrylamide gel slabs resolved major and minor broad bands near pH 8.0 and 7.8 respectively. Following reduction of disulfide bonds with β-mercaptoethanol in 8 M urea, the smaller polypeptide chain of known sequence focused near pH 6.9 while the larger chain focused near pH 8.0. Sedimentation equilibrium studies showed that the major component in aqueous buffers at neutral pH is a hexamer of molecular weight 280 000 which dissociates to the monomer of molecular weight 47 000 at pH 4.8. The sequence molecular weight of the small subunit polypeptide is 16 517 [Elleman, T.C. (1977). Aust. J. Biol. Sci. 30, 33-45]. The molecular weights determined for the larger chain by sedimentation equilibrium or column chromatography in 6 M guanidine hydrochloride, and by dodecyl sulfate-polyacrylamide gel electrophoresis, were in the range 28 000-30 000. Optical rotatory dispersion and circular dichroism measurements have been used to establish the approximate proportions of α-helix (15%), β-structure (35%), β-turns (18%) and unordered regions (32%) in the native protein. The denaturation curve for guanidine hydrochloride and the proportions of α-helix (50%), β-turns (18%) and unordered regions (32%) in 80 % trifluoroethanol have been determined.

Wool and many mammalian keratins consist of two classes of proteins, one which is higher in sulphur content than the parent keratin and is of basic character in the unmodified keratin and one which is lower in sulphur content and is acidic in character (Alexander and Earland 1950;Corfield, Robson, and Skinner 1958;Gillespie and Simmonds 1960). In the intact fibre the low-sulphur proteins are thought to occur in the microfibrils and the high-sulphur proteins in the matrix, and the two are probably linked together by disulphide bonds (Birbeck and Mercer 1957; Rogers 1959).After the disulphide bonds have been broken, by reduction followed by alkylation with iodoacetate, or by oxidation, the two classes of protein can be separated in two ways. The high-sulphur component may be preferentially extracted in the presence of salt (Corfield, Robson, and Skinner 1958;Gillespie 1962) or of certain specific ions (e.g. zinc or cetyltrimethylammonium ion) which suppress the solubility of the low-sulphur proteins (Gillespie 1962). Alternatively a mixture of the proteins can be extracted at low ionic strength and the lowsulphur proteins separated from the others by precipitation at acid pH values. It. has recently been observed that the latter separation when applied to the reduced and S-carboxymethylated proteins can be complicated by co-precipitation of the two classes of proteins. This paper presents an account of this phenomenon and a summary of conditions suitable for separating mixtures of high-and low-sulphur proteins. Results and Discussion(i) Reduced and Oarboxymethylated Proteins.-Erratic and variable yields of high-sulphur protein were obtained when the low-sulphur proteins were removed from mixtures by precipitation at pH 4·1 at an ionic strength of 0 ,1. It. has been reported that a number of reprecipitations were necessary to remove the highsulphur proteins completely from the low-sulphur protein precipitated under these conditions (Gillespie 1960). A study of artificially prepared mixtures of high-and low-sulphur proteins showed that, as the ratio of low-sulphur protein to high in the mixture increases (Fig. I), so the recovery of high-sulphur protein after precipitation falls. Treatment of the supernatant with Zn2+ at pH 6 (Gillespie 1957) showed that the low-sulphur proteins were always quantitatively precipitated.Further data on this co-precipitation of high-and low-sulphur proteins are shown in Table 1. The mixed high-and low-sulphur proteins used in this experiment were prepared from Merino wool, either by extraction with 0 ·IM potassium * Manuscript

The seeds of 99 genotypes of L. angustifolius were examined by electrophoresis on cellulose acetate and found to contain two major salt-extracted globulins, conglutins α and β, and a third minor globulin, conglutin γ. Dissociation with sodium dodecyl sulfate and electrophoresis on polyacrylamide gel showed variation in the number, proportion and molecular size of the subunits which constitute these proteins. This was confirmed by electrophoresis in polyacrylamide gel containing urea at acid pH. The globulins in the salt extracts of some of the seeds were subject to partial enzymic degradation unless conditions which inactivate proteolytic enzymes were used during extraction. Crude protein level of the seed meal, determined for many of the genotypes from their nitrogen content, varied from about 30 to 50%. Only 5-10% of this total protein was water-extracted albumins. Amino acid analysis showed that the albumin fraction contains higher levels of many of the essential amino acids including cystine and particularly methionine.

The amino acid composition of winged bean seed meal is similar to that of soybean but their storage globulins are quite different. Winged bean proteins are soluble to the extent of 60% at the pH of a meal-water slurry (pH 6.6), 80% at pH 11 but only 12% at pH 5. However, the proteins are soluble to the extent of 80% from pH 5 to 9 in 10% NaCl rising to 90% at pH 11. There are no satisfactory ways of recovering all the proteins from solution by simple changes in pH or ionic strength. Winged bean seed contains major proteins with sedimentation coefficients of 2 S and 6 S. Electrophoresis on cellulose acetate resolves three globulin fractions which we have named psophocarpins A, B, and C. The proteins from these electrophoretic regions have been isolated and partially purified. Psophocarpin A is essentially a single protein comparatively rich in sulfur-containing amino acids while the other fractions are composed of a number of related components which have not been separated. When examined by SDS-polyacrylamide gel electrophoresis, the globulin fractions differed in the kind of subunit proteins they contain and in the extent of disulfide bonding. The 40 000 mol. wt subunit of psophocarpin A contains disulfide bonded chains of mol. wt 16 000 and 24 000. The proteins corresponding to the other electrophoretic regions are more complex.

SummaryA study has been made on the extraction of reduced protein from wool by two methods. In one the aim has been to solubilize as much protein as possible and in the other to use the mildest conditions possible. In the first method extraction was made with potassium thioglycollate in the presence of urea and the variables temperature, pH, urea concentration, and thioglycollate concentration were studied. It was found that between 80 and 85% of wool can be solubilized at room temperature by 0-2M potassium thioglycollate at pH 10-5 in the presence of 6M urea. In the second method a total of more than 60% of the wool was solubilized; the highsulphur protein was first preferentially extracted by O· 8M potassium thioglycollate at pH 10'5 and O°C, and low-sulphur protein then released from the residue by plasmolysis by immersing the residue in ice-cold distilled water. The advantage of this method is that at no time does the temperature rise above O°C or the pH above 10-5.Although the low-sulphur proteins (i.e. proteins precipitable at pH 4·4 after alkylation with iodoacetate) prepared by both these procedures gave essentially single-boundaried patterns on electrophoresis at pH 11-0, amino acid analyses indicated that, in addition to the SCMKAI and SCMKA2 proteins previously reported, these proteins also contain variable amounts of constituents rich in glycine and S-carboxymethyl cysteine.

The protein level in seeds of six species of lupin, grown either under sulfur deficiency or with adequate sulfur fertilization, is marginally affected by sulfur supply. However, the ratio of total sulfur to total nitrogen in the seed is greatly decreased under sulfur deficiency. This large change in sulfur-to-nitrogen ratio is accompanied by suppression of the synthesis of conglutins α and γ, which contain a significant amount of cyst(e)ine and methionine. The level of protein is maintained by increased synthesis of conglutin β, which normally contains no methionine and a low proportion of cyst(e)ine. These changes in the proportions of the proteins are reflected in the amino acid analyses for the globulin extracts. The changes in protein subunit composition which accompany the differences in the proportions of the proteins have been studied using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results emphasize the differences in subunit composition between lupin species in terms of the number of components, their molecular weights and the importance of disulfide bonding. Two-dimensional electrophoresis, using cellulose acetate and SDS-polyacrylamide gradient gels, has been used to compare the subunit composition of the individual globulins for Lupinus angustifolius and L. elegans at both sulfur levels.