- Email: [email protected]
Purification and characterization of the 11S component of soybean proteins
ARCHIVES
OF
BIOCHEMISTRY
AND
86, 1861%
BIOPHYSICS
(1959)
Purification and Characterization of the Soybean Proteins’*’
11s
Component of
W. J. Wolf3 and D. R. Briggs From the Department of Agricultural Biochemistry, University of Minnesota, St. Paul, Minnesota Received
April
27, 1959
INTRODUCTION
Studies on the cold-insoluble fraction of soybean proteins (1) indicate that it consists principally of a component having a sedimentation constant of about 118 (2, 3) which is capable of forming disulfide polymers (3) and of dissociating into subunits (4). The interesting properties exhibited by the 118 component prompted studies directed toward the further purification and characterization of this protein. Several fractionation procedures were investigated, and while they were not uniformly successful in purifying the 11S protein, they are reported in some detail since they may be useful for future work in the purification of this and some of the other soybean protein components. EXPERIMENTAL
AND RESULTS
Materials Soybean meal was prepared as described earlier (5). Salts used were of analytical grade. Mercaptoethanol was an Eastman product. A potassium phosphate-sodium chloride buffer (0.0325 M KzHOPd ,0.0026 M KHtPO( ,0.40 M NaCl) pH 7.6,0.5 ionic strength which was made 0.01 M with respect to mercaptoethanol, was used extensively in these studies as solvent and is referred to as standard buffer. The mercaptoethanol was used to eliminate disulfide polymerization of the 75 and 11s proteins (3).
Ultracentrifugal While fractionations of the soy proteins described, it should be emphasized that
Analyses
were carried all reported
out under the conditions ultracentrifuge patterns
to be were
1 Paper No. 3961 Scientific Journal Series, Minnesota Agricultural Experiment Station. * The contents of this paper constitute a part of a thesis submitted by W. J. Wolf to the Graduate Faculty of the University of Minnesota in partial fulfillment of the requirements for the Doctor of Philosophy Degree, August 1956. 8 Procter and Gamble Predoetoral Fellow 1955-56; present address: Northern Utilization Research and Development Division, U. S. Department of Agriculture, Peoria, Ill. 186
11s
COMPONENT
OF
SOYBEAN
187
PROTEINS
obtained on these fractions while in solution in the standard buffer. Transfers from the solvent conditions used in fractionation procedures to those of the standard buffer were achieved through dialysis, at constant volumes, against large volumes of the standard buffer. By thus obtaining the patterns under uniform conditions which eliminate those aggregation, polymerization, and dissociation changes that some of these components are capable of undergoing (3, 4), a better basis for comparison is made possible than would otherwise prevail. Ultracentrifuge analyses were made at room temperature with a model E Spinco ultracentrifuge, and all photographs of the schlieren patterns were taken after the equivalent of 44 min. centrifugation at 56,100 r.p.m.
Fractionation
Studies
A. Fractionation by Cooling and, Addition of Calcium Chloride. Twenty grams of soybean meal was extracted at room temperature for 30 min. by stirring with 100 ml. water. After clarification by centrifugation (at 10000 X g) and transfer of an aliquot to standard buffer, the pattern shown in Fig. la, containing four peaks having approximate SZO,~values of 2S, 7S, llS, and 15S, was obtained. The remainder of the aqueous extract (pH 6.6) was made 0.01 M to mercaptoethanol, cooled in an ice bath for 3 hr., and centrifuged in the cold to remove the cold-insoluble fraction (1). Figures lb and lc show that cooling of the aqueous extract results in a clear-cut fractionation of the 11s component. The cold-insoluble fraction (Fig. lb) represented 26% of the total protein nitrogen and contained 63 % of the total 11s protein present in the water extract. Area measurements (corrected for radial dilution) indicated that the cold-insoluble fraction had the following composition: 2S protein, 6%; 7S protein, 7 %; 11X protein, 82%; and 15s protein, 5%. Addition of small increments of calcium ion to the aqueous extract (pH
(a) FIG. 1. Ultracentrifuge
(b)
(4
Cd)
6-4
patterns for: (a) The proteins extracted from 20 g. of soybean meal by 100 ml. water, protein concn. 2.92%; (b) the cold-insoluble fraction obtained from (a), protein concn. 0.83%; (c) solution (a) after removal of the coldinsoluble fraction, protein concn. 2.16%; (d) the protein precipitated from (c) by cooling in the presence of 0.025 N calcium chloride, protein concn. 0.36%; (e) solution (c) after removal of fraction (d), protein concn. 1.86%. Patterns were photographed at a bar angle of 65” and are directly comparable. Direction of sedimentation in Fig. 1 and subsequent figures is from left to right.
188
WOLF
AND
BRIGGS
6.6), while causing no precipitation of the proteins at room temperature, increases the amount of precipitate formed at cold temperatures. The nature of this additional protein precipitated in the cold in the presence of added calcium ion was investigated. Calcium chloride was added to the supernatant from the cold-insoluble fraction at room temperature to make it 0.025 N with respect to added calcium ion, and the pH was adjusted to 6.6 with N sodium hydroxide. After standing at 4°C. overnight, the resulting precipitate was removed by centrifugation in the cold. Figures lc-e show that calcium chloride, at 4”C., precipitates an additional amount of the 118 protein but of lower purity, since the 11s peak in Fig. Id accounts for only 65% of the total area of this pattern. The protein precipitated by calcium chloride (Fig. Id) represented 10% of the protein nitrogen and contained 23% of the 11s protein originally present in the water extract. In addition to being a more heterogeneous preparation, the protein precipitated by cooling after calcium chloride addition contained 1.40 % phosphorus compared to a 0.17 % phosphorus content of the initial cold-insoluble fraction. R. Fractionation of a Salt Extract by Ammonium Sulfate Precipitation and Dialysis. Forty grams of soybean meal was stirred overnight at 4°C. with 400 ml. of 1 M sodium chloride buffered at pH 7.0 (0.02 M NaH2P04 , 0.03 M NazHP04 plus sodium hydroxide to give a pH of 7.0). The meal residue was removed by centrifugation at room temperature, and the turbid supernatant was recentrifuged at 10000 X g until clear. The extract was brought to approximately 70% saturation with ammonium sulfate (532 g./l. extract), The precipitated proteins were separated by centrifugation and dissolved in standard buffer. Figure 2a is the pattern for the total proteins precipitated from the extract at 70 % saturation with ammonium
(a)
6)
Cc)
Cd)
(4
patterns for: (a) soybean proteins precipitated with 70% saturated ammonium sulfate from a salt extract, protein concn. 2.82yc; (b) proteins precipitated on dialyzing solution of (a) against water for 40 hr., protein concn. 1.06%; (c) supernatant after removal of (b), protein concn. 1.56%; (d) proteins precipitated on dialyzing solution of (a) against water for 112 hr., protein concn. 2.34yc; (e) supernatant after removal of (d), protein concn. 0.30%. All patterns were photographed at a bar angle of 65” and are directly comparable.
FIG. 2. Ultracentrifuge
11s
COMPONENT
OF
SOYBEAN
TABLE
I
189
PROTEINS
Effect of Time and pH on the Amount of Protein Precipitated by Dialysis against Distilled Water of a Solution (in Standard Buffer) of the Soy Proteins Previously Precipitated by 70% Saturation with Ammonium Sulfate Sample No.
Conditions of dialysis
Dialysis for 40 hr. to pH 6.25 Dialysis for 112 hr. to pH 5.60 Dialysis for 112 hr. and adjusted to pH 4.50 Dialysis for 112 hr. and electrodialysis for 4 hr. to pH 5.12
Per cent of protein N pptd.‘=
Per cent of protein N insolublea,b
% 43 87 86
% 3.5 4.3 4.3
90
4.7
a Based on total protein nitrogen in original salt extract. The nonprotein content of the meal was determined by the loss of nitrogen from the salt extract on dialysis and was found to be 5.2% of the total nitrogen of the meal. b Represents protein nitrogen precipitated on dialysis but insoluble under the standard conditions used for ultracentrifugal analysis.
sulfate. Except for a somewhat lower proportion of the 28 fraction, this pattern is identical with the pattern for the proteins present in the salt extract prior to precipitation with ammonium sulfate and is very similar to Fig. la, the pattern obtained for the proteins present in a water extract of soybean meal. Apparently complete precipitation of the globulin proteins occurs at 70% saturation with ammonium sulfate, while a small fraction of t,he albumins, which appear in the 28 (and 78) peaks (5), remains soluble under this condition. The solution of the 70 % ammonium sulfate-precipitated proteins in standard buffer was divided into four aliquots and dialyzed against cold (4”C.), running dist,illed water for 24 hr. Dialysis was then continued in the cold against large volumes of distilled water saturated with toluene. For these four samples, Table I shows the t’imes and conditions of dialysis, the final pH of the dialyzed sample, the percentage of the total nitrogen present in the original sodium chloride extract which was precipitated during the dialysis, and the percentage which precipitated but remained insoluble in the st,andard buffer. Since each was virtually salt-free after only 24 hr. dialysis, it’ is apparent that t,he pH attained by the protein solution on dialysis is the important factor in determining the amount of protein that is precipitated. Figures 20 and 2c show the patterns for the precipitated and supernatant fractions, respectively, of sample 1. The precipitate consisted mainly of 11’8 protein (70 %), but a considerable part of this component remained in the supernatant under these conditions. Figure 2d is the pattern for the precipitated fraction of sample 2, while
190
WOLF
AND
BRIGGS
Fig. 2e is that for its supernatant. A comparison of Figs. 2d and 2e with Fig. 2a shows that, under these conditions all of the 11X and 15s proteins were precipitated, together with most of the 7S fraction and part of the 2S fraction. Table I shows that after prolonged dialysis (112 hr.) there was no appreciable change in the total amount of protein precipitated when the system was acidified to pH 4.50 or when it was electrodialyzed. Ultracentrifuge patterns for the proteins precipitated at pH’s 4.50 and 5.12 were indistinguishable from Fig. 2d. Osborne and Campbell (12) originally proposed that the fraction which precipitates on dialysis of the proteins which are precipitated by ammonium sulfate from a sodium chloride extract be called “glycinin.” Both of the protein fractions represented by Figs. 2b and 2d could logically be called “glycinin.” It is apparent that the composition of glycinin prepared by dialysis of a buffered solution is dependent on the time allowed for dialysis and on the resultant pH attained by the dialyzing procedure. In addition to the ultracentrifugal differences noted in Figs. 2b and 2d, these fractions also differed in nitrogen content. The proteins precipitated at pH 6.25 (Fig. 2b) contained 16.80% nitrogen (dry basis), while the proteins precipitated at pH 5.60 contained 16.48 % nitrogen (dry basis). This is in confirmation of, and supplies a basis of explanation for earlier reports of variation in the nitrogen content of “glycinin” (17). C. Fractionation by Isoelectric Precipitation. A water extract was prepared by adjusting a suspension of 12.5 g. meal in 100 ml. water to a pH of 7.2, stirring for an hour, and removing the meal residue by centrifugation. The separation of the soybean globulins by adjusting the extract to pH 4.8 is illustrated in Figs. 3a-c. All of the 11s and 15s components are precipitated together with most of the 7X protein and an appreciable amount of the 2X protein. The latter two fractions appear in the supernatant (Fig. 3c) in larger amounts than was noted when the globulins (obtained as the 70% ammonium sulfate-precipitated fraction) were precipitated by dialyzing to a pH of 5.60 (compare Fig. 3c with 2e). Redissolving the acid-precipitated fraction in water by adjusting to pH 7.6 with NaOH and reprecipitating at pH 4.8 with HCl had no effect on the ultracentrifuge pattern (Fig. 36), indicating that the amount of occluded proteins present in this fraction was negligible. [Smith et al. (13) have found that washing or reprecipitation had no effect on the electrophoretic pattern of the protein fraction precipitated at pH 4.5 from an aqueous extract of soybean meal.] If the acid-precipitated protein is dissolved at pH 7.6 and reprecipitated in the presence of 0.2 M sodium chloride at pH 4.8, the 2S and 7s fractions do not precipitate completely, as shown in Figs. 3d and 3e. If the solution containing the 2S and 75 fractions at pH 4.8 in 0.2 M sodium chloride (Fig. 3e) is cooled to O”C., the 7S fraction precipitates preferentially as shown in Figs. 3f and 39.
1153 COMPONENT
64
OF
SOYBEAN
191
PROTEINS
6)
(4
(.f)
67)
6-l)
04
FIG. 3. Ultracentrifuge
patterns for: (a) soybean proteins extracted from 12.5 g. meal by 100 ml. water adjusted to pH 7.2, protein concn. 1.94%; (b) proteins precipitated from (a) at pH 4.8, protein concn. 1.70%; (c) supernatant after removal of (b), protein concn. 0.26%; (d) precipitate obtained at pH 4.8, 0.2 ionic strength after dissolving (b) in water at pH 7.6, protein concn. 1.41%; (e) supernatant after removal of (d), protein concn. 0.28%; (f) proteins in (e) soluble in the cold, protein concn. 0.15%; (g) protein precipitating from fraction (e) on cooling at pH 4.8, 0.2 ionic strength, protein concn. 0.24%; (h) 11s protein isolated from fraction (d) by precipitation at pH 4.8 between 0.4 and 0.2 ionic strength, protein concn. 1.11%. All patterns photographed at a bar angle of 55” except (g) which was at 40”. Patterns (a)-(j) are directly comparable.
Repeated precipitation and washing of the acid-precipitated protein fraction (Fig. 36) at pH 4.8 in the presence of 0.2 M sodium chloride and 0.01 M mercaptoethanol yielded an 11s preparation quite similar to the cold-insoluble fraction (Fig. lb); it still contained appreciable amounts of the 2S, 78, and 15s fractions. Further purification of the 11s component was effected by variation of the ionic strength at pH 4.8. For example, a 1% solution of partially purified 11s fraction (obtained by reprecipitation and washing the acid-precipitated protein fraction at pH 4.8 and 0.2 ionic strength) was dissolved in the standard buffer, then adjusted to pH 4.8 with hydrochloric acid and diluted to 0.4 ionic strength. A precipitate formed which consisted primarily of 11s protein plus a high proportion of 15s protein and a small amount of a faster sedimenting material. The protein remaining soluble at 0.4 ionic
192
WOLF
AND
BRIGGS
strength was then recovered as a precipitate by diluting the solution to 0.2 ionic strength. This precipitate was dissolved at pH 7.6, 0.5 ionic strength in a volume of buffer slightly smaller than the original solution, and the entire process was repeated twice. The protein obtained finally, as a precipitate at 0.2 ionic strength, consisted of 11X component contaminated with what appeared to be only traces of 7X and 15X fractions (Fig. 3h). However, when a reference base line was fitted to the enlarged pattern, the following area distribution (corrected for radial dilution) was obtained: 2s (5 %), 7X (5 %), 11s (82 %), and 15X (8 %). While this area distribution is not appreciably different from that for the cold-insoluble fraction described above, the protein obtained by this procedure formed a clear solution in standard buffer as contrasted with the somewhat turbid, yellow solution obtained with the cold-insoluble fraction. D. Fractional Extraction of Acid-Precipitated Protein. Three hundred grams of meal was suspended in 2400 ml. water, and the pH was adjusted to 7.2 with 1 N sodium hydroxide. After stirring for 30 min., the meal residue was removed by centrifugation. Volume of supernatant was 1465 ml. The pH was adjusted to 4.8, which caused 80 % of the total nitrogen in the extract to precipitate. The precipitate (pattern same as Fig. 3b) was then extracted, as indicated in Table II, by stirring with sodium chloride solutions of pH 4.8, made 0.01 M with respect to mercaptoethanol, for lTABLE Practional Extraction 1 2 3 4 5 6 7 8 9 lob lib 126
Extraction
II
of Acid-Precipitated
Soybean Protein
NaCl concn.
N/ml. in extract
Per cent of N extracted’”
moles/l. 0.20 0.20 0.20 0.20 0.35 0.35 0.35 0.35 0.35 -
w. 0.70 0.22 0.11 0.07 0.63 0.39 0.29 0.21 0.16 3.93 0.48 0.14
% 13.2 4.1 2.0 1.3 11.9 7.3 5.4 3.9 3.0 24.2 3.1 0.8
Total a Based on total N in the protein precipitate. b Extraction made with 500 ml. of phosphate-N&l strength. For extraction number 10 the protein-buffer pH 7.6 prior to extraction.
80.2
buffer, pH 7.6, 0.5 ionic suspension was adjusted to
11s
(a)
COMPONENT
(b)
OF
SOYBEAN
PROTEIKS
(4
193
Cd)
FIG. 4. Ultracentrifuge
patterns for 11s protein preparations obtained in (a) extraction5, protein concn. 1.4270; (b) extraction 6, protein concn. 1.15%; (c) extraction 7, protein concn. 1.16%; (d) extraction 10, protein concn. 1.15%; of fractional extraction procedure. Bar angle for (a) 60”, (b)-(d) 65”. hr. periods at room temperature. The volume of solvent used in each of l-9 inclusive, was equal to the volume of the original the extractions, extract (1465 ml.). The protein in extracts 5-9 was recovered by diluting the solutions to The precipitated 0.2 ionic strength and centrifuging off the precipitate. proteins were dissolved in standard buffer to give an approximately 1% solution which was somewhat turbid, but centrifugation at 10000 X g gave water-clear solutions. Figures 4u-c show the ultracentrifuge patterns 5, 6, and 7, respectively. The for the proteins recovered from extractions
proteins from extractions 8 and 9 were identical with Fig. 4~. It appears that the first four extractions ab pH 4.8, 0.2 ionic strength, removed almost all of the 2X fraction and a major part of the 7X fraction since the protein recovered from extract 5 (Fig. 4~) consisted mainly of 11X protein plus some 7X protein and only a trace of 2s fraction (apparent only when a reference base line was fitted to the enlarged pattern).4 In subsequent extractions the relative amount of 7s fraction was reduced to a small but fairly constant value as observed for extractions 7, 8, 9 (Fig.
4c), and 10 (Fig. 4d). Extractions 7-9 yielded the purest preparations of 11s protein yet attained, having the following composition based on area distribution of the ultracentrifuge pattern (Fig. 4~): 2s (3 %), 7S (4%), 11X (87 %), and 15s (6 %). The yield of 11s protein from extracts 7-9 was 3.1 g. A large amount of tan-colored precipitate remained after the 9t,h extraction, and only 52 % of the total nitrogen in the original precipitate has been 4 It is possible that extract 5 contained more 2s and 7s proteins than is indicated in Fig. 4a since they may not have been precipitated completely when the extract was diluted to 0.2 ionic strength. Figures 3b, 3d, and 3e show that these protein fractions are partially soluble at pH 4.8, 0.2 ionic strength.
194
WOLF
AND
BRIGGS
accounted for. An attempt was therefore made to dissolve the insoluble portion in standard buffer so that it could be identified by ultracentrifugal analysis. While a large part of the protein dissolved in the three successive extractions with buffer (extractions 10-12, Table II), 20 % of the total acidprecipitated protein remained insoluble in the buffer. Figure 4d is the pattern for the proteins recovered from extract 10. It reveals that a large amount of 11X protein was still present in the precipitate after the 9th extraction although the progressive decrease in the amount of nitrogen extracted at 0.35 ionic strength (Table II) suggested that most of the 11s protein might have been already extracted. Previous experiments (4) have shown that the 11X protein undergoes an irreversible dissociation at pH 3.0, 0.1 ionic strength, with an accompanying loss of solubility in standard buffer. In the present experiment a part of the 11s protein may have formed an intermediate acid-induced modification at pH 4.8 which was difficultly soluble at 0.35 ionic strength but which was partially reversed at pH 7.6, 0.5 ionic, i.e., under the conditions of extraction 10. The fraction remaining insoluble after the 12th extraction was suspended in 0.5 M sodium chloride, and 1 N sodium hydroxide was added. No appreciable dispersion was noted until a pH of 11 was reached, and the suspension was still extremely turbid at pH 11.5. It would appear that other components, as well as the 11s protein, may have been partially irreversibly modified by the prolonged extraction process at pH 4.8. Estimation of Some of the Physical Characteristics of the 11s Protein The sample of 11s component obtained by fractionation procedure D (pattern shown in Fig. 4c), while only of the order of 90 % pure in this component, was nevertheless utilized to yield the following approximate values for the physical characteristics of this protein. An electrodialyzed sample of this protein had a nitrogen content of 17.12 % (dry basis). Partial Specific Volume. The partial specific volume, V, of the purified 11s protein was determined from the relationship (6),
where p is the density of the solution for which the solute weight fraction is w, and m is the mass of a given volume of the solution. A plot of m against w indicated that no change in dm/dw occurs with solutions of the 11s protein in the standard buffer over the concentration range 1.8-3.6%. The value of V for three concentrations of 11X protein in this range, at 2O”C., was found to be 0.719 f 0.001, which is slightly higher than the 0.710 value reported by Naismith (2) for the unfractionated mixture of soybean proteins.
1lS
COMPONENT
OF
SOYBEAN
PROTEINS
195
13,
IO 0
1.00
0.50 Concentration
1%)
FIG. 5. Plot of SW,,,.versus c.
Sedimentation. The sedimentation constant of the purified 11s protein was measured at room temperature in the model E Spinco ultracentrifuge at 59780 r.p.m. with standard buffer as the solvent. In measuring the values of 2, the distance of the peak from the center of rotation, an allowance of 0.025 cm. was made for stretching of the rotor at 60,000 r.p.m. (7). The sedimentation constant was corrected to the standard state of water at 20°C. (6). Allowance for the cooling resulting from adiabatic stretching of the rotor during acceleration (8) was made by subtracting 1°C. from the average rotor temperature. Calculated s20,Wvalues were plotted against protein concentration as shown in Fig. 5. The equation for the regression line is ~20,~ = 12.20 - 0.62~ where ~20,~ is given in Svedberg units and c is the protein concentration in g./lOO ml. The concentration values have not been corrected for the impurities (about 13 %) estimated to be present from area measurements on the ultracentrifuge patterns, since applying this correction does not change the value of sio,%., altering only the slope of the extrapolation line. Di$usion. Diffusion measurements were made in standard buffer with a Neurath-type diffusion cell (9) at 3.8”C. The concentration gradient of the diffusing protein was photographed periodically with the schlieren scanning optical system of a Tiselius-Klett electrophoresis apparatus. The diffusion constant, D, was calculated from the equation (10)
where K = A/2& A = the average area under the diffusion curves (sq. cm.). H = the maximum ordinate (cm.) of the dn/dx versus x curve at time t.
196
WOLF
Al’iD
BRIGGS
7
6
I/J+ x 10” (t in seconds) FIG. 6. Plot of H, versus l/d/t. Open circles: protein concn. = 1.06% 2.01 sq. cm.). Closed circles: protein concn. = 0.55% (area = 1.05 sq. cm.).
(area
=
The slope of the H, versus l/& relationship (Fig. 6) was used to evaluate H,d. These data yield values for D 3.8of 1.70 f 0.06 X lo-’ sq. cm./sec. The value of 03.8 was corrected to 20°C. and water as solvent (6) giving a value for D20,W of 2.91 =t 0.10 X lo-’ sq. cm./sec. From this value, the corresponding sedimentation constant, and the partial specific volume, the molecular weight of the 11s protein was calculated to be 363,000 f 13000. Light Scattering. Turbidity measurements of the purified 11s protein dissolved in standard buffer were made as described in a previous publication (11). Turbidity measurements made at angles of 45” and 135” to the incident beam indicated that no dissymmetry corrections were necessary. The specific refractive-index increment of the protein was determined with a Phoenix differential refractometer. Protein concentrations were determined by a semimicro-Kjeldahl procedure. Figure 7 is a plot of HC/T versus c at the two wavelengths of incident
1h-Z COMPOKENT
OF
SOYBEAN
197
PROTEINS
0
3.0A= 436 mp ;
I
2.5,
I
!
I
7
8
9
x
;---Tr--‘; 0
2
3
4
Concentration
5
6
(QmJml. x IO’)
FIG. 7. Plot of HC/T versus c.
light employed. The curves, drawn by the method of least squares, when extrapolated to zero concentration give the reciprocal of the molecular weight, according to the Debye equation (14). The observed intercept values of 3.02 X low6 and 2.83 X 10m6for 546 and 436 rnp, respectively, yield molecular weights of 331,000 and 354,000. Light-scattering measurements with another preparation of 11s protein yielded molecular weights of 335,000 and 359,000 at 546 and 436 rnp, respectively. The average molecular weights by this method are, thus, 333,000 (546 rnp) and 356,500 (436 mb). Apparent depolarization factors of 0.0166 (546 rnp) and 0.0472 (436 rnp) yielded calculated values of the Cabannes factor (15) of 0.965 and 0.902, respectively. However, the recent work of Geiduschek (16) suggests that the real depolarization of proteins is negligible; hence no correction has been made for the apparent depolarization measured. The specific refractive-index increment was found t)o be 0.00185 at 546 rnp and 0.00188 at 436 w. Ultraviolet Absorption. An ultraviolet absorption curve measured on the 11s protein in standard buffer solution through the range of 266-300 rnp was that of a typical protein with a maximum at 279 rnp having an E:?m, value of 9.2 f 0.3. Frictional Ratio. The frictional ratio for the 11X protein was calculated using the constants obtained in the above determinations. These ratios were calculated by equations given by Svedberg and Pedersen (6) which involve : (a) sZO,~,. and M, (t’he molecular weight determined by other than sedimentation velocity, here taken to be 345,000, the average of the light-scattering molecular weight), (b), Dzo,,+.and M, , and (c) ~20,~.and D20,w . The values obtained were 1.51, 1.58, and 1.56 for methods (a), (b), and (c), respectively.
198
WOLF AND BRIGGS DISCUSSION
Figure 1 illustrates that a relatively pure preparation of the 11X protein can be obtained in good yield from the mixture of crude soy proteins by the simple procedureof cooling an aqueous extract of soybean meal. Comparison of Fig. 2b with lb shows that the 11X protein can also be selectively separated from the mixture by salt extraction, ammonium sulfate precipitation, and dialysis. However, the degree of contamination by other components is dependent on the extent of dialysis (Figs. 2b and 2d), and, since this is difficult to determine and control, cold precipitation appears to be preferable to salt precipitation and dialysis for the purification of the 11X protein. Fractionation of the soybean proteins by acid precipitation from an aqueous extract gives a very heterogeneous preparation of the 118 protein (Figs. 3uc), but some purification can be effected by reprecipitation in the presence of 0.2 M sodium chloride, and a possible method for the preparation of the 7X component is indicated (Figs. 3e-g). Fractional extraction of the acidprecipitated protein with sodium chloride solutions of graded ionic strengths yields the 11X protein in the highest purity, free of much of the color and turbidity characteristic of solutions of the unfractionated soybean protein mixture. However, the purified 11X protein is obtained in low yield by this procedure, and the large amount of protein which becomes insoluble on fractional extraction of the acid precipitate suggests that a part of the 11S protein, and/or other proteins, is irreversibly modified in this process. The degree of purification of the 11s protein achieved in the present studies is of the same order as that obtained for other seed globulins in the 11-138 size range where the removal of small amounts of remaining impurities has proven very difficult (18). The physical properties of the purified 118 protein indicate that it is a typical seed globulin. The molecular weight of approximately 350,000 is of the same order as that reported for other seed globulins (18-21). The s&,, value of 12.2X for this component of the soybean proteins is about 7 % lower than the value of 13.1~s’reported by Danielsson (19) using an oil turbine ultracentrifuge. It is not clear from Danielsson’s paper whether this value was obtained by extrapolation to zero concentration. Naismith (2) likewise has presented evidence that Danielsson’s value may be too high but ascribed the discrepancy to differences in ionic conditions employed. A number of workers (22-25) have found that the sedimentation constants of several well-defined proteins, as determined with the Spinco ultracentrifuge, are S-10 % lower than those determined with the oil-turbine ultracentrifuge. SUMMARY
Soybean proteins were fractionated by: (a) cooling an aqueous extract; (b) salt extraction, ammonium sulfate precipitation, and dialysis; (c) iso-
IIS
COMPONENT
OF
SOYBEAK
PROTEIM
199
electric precipitation; and (d) acid-precipitation and fractional extraction with sodium chloride solution of graded ionic strength. The purest preparation of the component having the approximate SZO,,,,value of 1lS was obtained by the last-named method. Sedimentation, diffusion, and lightscattering measurements indicate that the 11s protein has a molecular weight of about 350,000. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BRIGGS, D. R., AND MANN, R. L., Cereal Chem. 27, 243 (1950). NAISMITH, W. E. F., Biochim. et Biophys. Acta 16, 203 (1955). BRIGGS, D. R., AND WOLF, W. J., ilrch. Biochem. Biophys. 72, 127 (1957). WOLF, W. J., AND BRIGGS, D. R., Arch. Biochem. Biophys. 76, 377 (1958). WOLF, W. J., AND BRIGGS, D. R., Arch. Biochem. Biophys. 63, 40 (1956). SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge.” Oxford Univ. Press, New York, 1940. TAYLOR, J. F., Arch. Biochem. Biophys. 36, 357 (1952). WAUGH, 1). F., AND YPHANTIS, D. A., Rev. Sci. Instr. 23, 609 (1952). NEURATH, H., Science 93, 431 (1941). NEURATH, H., Chem. Revs. 30, 357 (1942). FULLER, R. A., AND BRIGGS, D. R., J. Am. Chem. Sot. 78, 5253 (1956). OSBORNE, T. B., AND CAMPBELL, G. F., J. Am. Chem. Sot. 20, 419 (1898). SMITH, A. K., SCHUBERT, E. X., AND BELTER, P. A., J. Am. Oil Chemists’ Sot. 32, 274 (1955). DEBYE, P., J. Phys. & Colloid Chem. 61, 18 (1947). CABANNES, J., “La Diffusion Moleculaire de la Lumiere.” Presses Universitaires de France, Paris, 1929. GEIDUSCHEK, E. P., J. Polymer Sci. 13, 408 (1954). SMILEY, W. G., AND SMITH, A. K., Cereal Chem. 23,288 (1946). BRAND, B. P., GORING, D. A. I., AND JOHNSON, P., Trans. Faraday Sot. 61, 872 (1955). DANIELSSON, C. E., Biochem. J. 44, 387 (1949). JOHNSON, P., Trans. Faraday Sot. 42, 28 (1946). SVEDBERG, T., Nature 139, 1051 (1937). TAYLOR, J. F., Arch. Biochem. Biophys. 36, 357 (1952). MILLER, G. L., AND GOLDER, R. H., Arch. Biochem. Biophys. 36, 249 (1952). CECIL, R., AND OGSTON, A. G., Biochem. J. 44, 33 (1949). SHULMAN, S., Arch. Biochem. Biophys. 44, 230 (1953).
OF
BIOCHEMISTRY
AND
86, 1861%
BIOPHYSICS
(1959)
Purification and Characterization of the Soybean Proteins’*’
11s
Component of
W. J. Wolf3 and D. R. Briggs From the Department of Agricultural Biochemistry, University of Minnesota, St. Paul, Minnesota Received
April
27, 1959
INTRODUCTION
Studies on the cold-insoluble fraction of soybean proteins (1) indicate that it consists principally of a component having a sedimentation constant of about 118 (2, 3) which is capable of forming disulfide polymers (3) and of dissociating into subunits (4). The interesting properties exhibited by the 118 component prompted studies directed toward the further purification and characterization of this protein. Several fractionation procedures were investigated, and while they were not uniformly successful in purifying the 11S protein, they are reported in some detail since they may be useful for future work in the purification of this and some of the other soybean protein components. EXPERIMENTAL
AND RESULTS
Materials Soybean meal was prepared as described earlier (5). Salts used were of analytical grade. Mercaptoethanol was an Eastman product. A potassium phosphate-sodium chloride buffer (0.0325 M KzHOPd ,0.0026 M KHtPO( ,0.40 M NaCl) pH 7.6,0.5 ionic strength which was made 0.01 M with respect to mercaptoethanol, was used extensively in these studies as solvent and is referred to as standard buffer. The mercaptoethanol was used to eliminate disulfide polymerization of the 75 and 11s proteins (3).
Ultracentrifugal While fractionations of the soy proteins described, it should be emphasized that
Analyses
were carried all reported
out under the conditions ultracentrifuge patterns
to be were
1 Paper No. 3961 Scientific Journal Series, Minnesota Agricultural Experiment Station. * The contents of this paper constitute a part of a thesis submitted by W. J. Wolf to the Graduate Faculty of the University of Minnesota in partial fulfillment of the requirements for the Doctor of Philosophy Degree, August 1956. 8 Procter and Gamble Predoetoral Fellow 1955-56; present address: Northern Utilization Research and Development Division, U. S. Department of Agriculture, Peoria, Ill. 186
11s
COMPONENT
OF
SOYBEAN
187
PROTEINS
obtained on these fractions while in solution in the standard buffer. Transfers from the solvent conditions used in fractionation procedures to those of the standard buffer were achieved through dialysis, at constant volumes, against large volumes of the standard buffer. By thus obtaining the patterns under uniform conditions which eliminate those aggregation, polymerization, and dissociation changes that some of these components are capable of undergoing (3, 4), a better basis for comparison is made possible than would otherwise prevail. Ultracentrifuge analyses were made at room temperature with a model E Spinco ultracentrifuge, and all photographs of the schlieren patterns were taken after the equivalent of 44 min. centrifugation at 56,100 r.p.m.
Fractionation
Studies
A. Fractionation by Cooling and, Addition of Calcium Chloride. Twenty grams of soybean meal was extracted at room temperature for 30 min. by stirring with 100 ml. water. After clarification by centrifugation (at 10000 X g) and transfer of an aliquot to standard buffer, the pattern shown in Fig. la, containing four peaks having approximate SZO,~values of 2S, 7S, llS, and 15S, was obtained. The remainder of the aqueous extract (pH 6.6) was made 0.01 M to mercaptoethanol, cooled in an ice bath for 3 hr., and centrifuged in the cold to remove the cold-insoluble fraction (1). Figures lb and lc show that cooling of the aqueous extract results in a clear-cut fractionation of the 11s component. The cold-insoluble fraction (Fig. lb) represented 26% of the total protein nitrogen and contained 63 % of the total 11s protein present in the water extract. Area measurements (corrected for radial dilution) indicated that the cold-insoluble fraction had the following composition: 2S protein, 6%; 7S protein, 7 %; 11X protein, 82%; and 15s protein, 5%. Addition of small increments of calcium ion to the aqueous extract (pH
(a) FIG. 1. Ultracentrifuge
(b)
(4
Cd)
6-4
patterns for: (a) The proteins extracted from 20 g. of soybean meal by 100 ml. water, protein concn. 2.92%; (b) the cold-insoluble fraction obtained from (a), protein concn. 0.83%; (c) solution (a) after removal of the coldinsoluble fraction, protein concn. 2.16%; (d) the protein precipitated from (c) by cooling in the presence of 0.025 N calcium chloride, protein concn. 0.36%; (e) solution (c) after removal of fraction (d), protein concn. 1.86%. Patterns were photographed at a bar angle of 65” and are directly comparable. Direction of sedimentation in Fig. 1 and subsequent figures is from left to right.
188
WOLF
AND
BRIGGS
6.6), while causing no precipitation of the proteins at room temperature, increases the amount of precipitate formed at cold temperatures. The nature of this additional protein precipitated in the cold in the presence of added calcium ion was investigated. Calcium chloride was added to the supernatant from the cold-insoluble fraction at room temperature to make it 0.025 N with respect to added calcium ion, and the pH was adjusted to 6.6 with N sodium hydroxide. After standing at 4°C. overnight, the resulting precipitate was removed by centrifugation in the cold. Figures lc-e show that calcium chloride, at 4”C., precipitates an additional amount of the 118 protein but of lower purity, since the 11s peak in Fig. Id accounts for only 65% of the total area of this pattern. The protein precipitated by calcium chloride (Fig. Id) represented 10% of the protein nitrogen and contained 23% of the 11s protein originally present in the water extract. In addition to being a more heterogeneous preparation, the protein precipitated by cooling after calcium chloride addition contained 1.40 % phosphorus compared to a 0.17 % phosphorus content of the initial cold-insoluble fraction. R. Fractionation of a Salt Extract by Ammonium Sulfate Precipitation and Dialysis. Forty grams of soybean meal was stirred overnight at 4°C. with 400 ml. of 1 M sodium chloride buffered at pH 7.0 (0.02 M NaH2P04 , 0.03 M NazHP04 plus sodium hydroxide to give a pH of 7.0). The meal residue was removed by centrifugation at room temperature, and the turbid supernatant was recentrifuged at 10000 X g until clear. The extract was brought to approximately 70% saturation with ammonium sulfate (532 g./l. extract), The precipitated proteins were separated by centrifugation and dissolved in standard buffer. Figure 2a is the pattern for the total proteins precipitated from the extract at 70 % saturation with ammonium
(a)
6)
Cc)
Cd)
(4
patterns for: (a) soybean proteins precipitated with 70% saturated ammonium sulfate from a salt extract, protein concn. 2.82yc; (b) proteins precipitated on dialyzing solution of (a) against water for 40 hr., protein concn. 1.06%; (c) supernatant after removal of (b), protein concn. 1.56%; (d) proteins precipitated on dialyzing solution of (a) against water for 112 hr., protein concn. 2.34yc; (e) supernatant after removal of (d), protein concn. 0.30%. All patterns were photographed at a bar angle of 65” and are directly comparable.
FIG. 2. Ultracentrifuge
11s
COMPONENT
OF
SOYBEAN
TABLE
I
189
PROTEINS
Effect of Time and pH on the Amount of Protein Precipitated by Dialysis against Distilled Water of a Solution (in Standard Buffer) of the Soy Proteins Previously Precipitated by 70% Saturation with Ammonium Sulfate Sample No.
Conditions of dialysis
Dialysis for 40 hr. to pH 6.25 Dialysis for 112 hr. to pH 5.60 Dialysis for 112 hr. and adjusted to pH 4.50 Dialysis for 112 hr. and electrodialysis for 4 hr. to pH 5.12
Per cent of protein N pptd.‘=
Per cent of protein N insolublea,b
% 43 87 86
% 3.5 4.3 4.3
90
4.7
a Based on total protein nitrogen in original salt extract. The nonprotein content of the meal was determined by the loss of nitrogen from the salt extract on dialysis and was found to be 5.2% of the total nitrogen of the meal. b Represents protein nitrogen precipitated on dialysis but insoluble under the standard conditions used for ultracentrifugal analysis.
sulfate. Except for a somewhat lower proportion of the 28 fraction, this pattern is identical with the pattern for the proteins present in the salt extract prior to precipitation with ammonium sulfate and is very similar to Fig. la, the pattern obtained for the proteins present in a water extract of soybean meal. Apparently complete precipitation of the globulin proteins occurs at 70% saturation with ammonium sulfate, while a small fraction of t,he albumins, which appear in the 28 (and 78) peaks (5), remains soluble under this condition. The solution of the 70 % ammonium sulfate-precipitated proteins in standard buffer was divided into four aliquots and dialyzed against cold (4”C.), running dist,illed water for 24 hr. Dialysis was then continued in the cold against large volumes of distilled water saturated with toluene. For these four samples, Table I shows the t’imes and conditions of dialysis, the final pH of the dialyzed sample, the percentage of the total nitrogen present in the original sodium chloride extract which was precipitated during the dialysis, and the percentage which precipitated but remained insoluble in the st,andard buffer. Since each was virtually salt-free after only 24 hr. dialysis, it’ is apparent that t,he pH attained by the protein solution on dialysis is the important factor in determining the amount of protein that is precipitated. Figures 20 and 2c show the patterns for the precipitated and supernatant fractions, respectively, of sample 1. The precipitate consisted mainly of 11’8 protein (70 %), but a considerable part of this component remained in the supernatant under these conditions. Figure 2d is the pattern for the precipitated fraction of sample 2, while
190
WOLF
AND
BRIGGS
Fig. 2e is that for its supernatant. A comparison of Figs. 2d and 2e with Fig. 2a shows that, under these conditions all of the 11X and 15s proteins were precipitated, together with most of the 7S fraction and part of the 2S fraction. Table I shows that after prolonged dialysis (112 hr.) there was no appreciable change in the total amount of protein precipitated when the system was acidified to pH 4.50 or when it was electrodialyzed. Ultracentrifuge patterns for the proteins precipitated at pH’s 4.50 and 5.12 were indistinguishable from Fig. 2d. Osborne and Campbell (12) originally proposed that the fraction which precipitates on dialysis of the proteins which are precipitated by ammonium sulfate from a sodium chloride extract be called “glycinin.” Both of the protein fractions represented by Figs. 2b and 2d could logically be called “glycinin.” It is apparent that the composition of glycinin prepared by dialysis of a buffered solution is dependent on the time allowed for dialysis and on the resultant pH attained by the dialyzing procedure. In addition to the ultracentrifugal differences noted in Figs. 2b and 2d, these fractions also differed in nitrogen content. The proteins precipitated at pH 6.25 (Fig. 2b) contained 16.80% nitrogen (dry basis), while the proteins precipitated at pH 5.60 contained 16.48 % nitrogen (dry basis). This is in confirmation of, and supplies a basis of explanation for earlier reports of variation in the nitrogen content of “glycinin” (17). C. Fractionation by Isoelectric Precipitation. A water extract was prepared by adjusting a suspension of 12.5 g. meal in 100 ml. water to a pH of 7.2, stirring for an hour, and removing the meal residue by centrifugation. The separation of the soybean globulins by adjusting the extract to pH 4.8 is illustrated in Figs. 3a-c. All of the 11s and 15s components are precipitated together with most of the 7X protein and an appreciable amount of the 2X protein. The latter two fractions appear in the supernatant (Fig. 3c) in larger amounts than was noted when the globulins (obtained as the 70% ammonium sulfate-precipitated fraction) were precipitated by dialyzing to a pH of 5.60 (compare Fig. 3c with 2e). Redissolving the acid-precipitated fraction in water by adjusting to pH 7.6 with NaOH and reprecipitating at pH 4.8 with HCl had no effect on the ultracentrifuge pattern (Fig. 36), indicating that the amount of occluded proteins present in this fraction was negligible. [Smith et al. (13) have found that washing or reprecipitation had no effect on the electrophoretic pattern of the protein fraction precipitated at pH 4.5 from an aqueous extract of soybean meal.] If the acid-precipitated protein is dissolved at pH 7.6 and reprecipitated in the presence of 0.2 M sodium chloride at pH 4.8, the 2S and 7s fractions do not precipitate completely, as shown in Figs. 3d and 3e. If the solution containing the 2S and 75 fractions at pH 4.8 in 0.2 M sodium chloride (Fig. 3e) is cooled to O”C., the 7S fraction precipitates preferentially as shown in Figs. 3f and 39.
1153 COMPONENT
64
OF
SOYBEAN
191
PROTEINS
6)
(4
(.f)
67)
6-l)
04
FIG. 3. Ultracentrifuge
patterns for: (a) soybean proteins extracted from 12.5 g. meal by 100 ml. water adjusted to pH 7.2, protein concn. 1.94%; (b) proteins precipitated from (a) at pH 4.8, protein concn. 1.70%; (c) supernatant after removal of (b), protein concn. 0.26%; (d) precipitate obtained at pH 4.8, 0.2 ionic strength after dissolving (b) in water at pH 7.6, protein concn. 1.41%; (e) supernatant after removal of (d), protein concn. 0.28%; (f) proteins in (e) soluble in the cold, protein concn. 0.15%; (g) protein precipitating from fraction (e) on cooling at pH 4.8, 0.2 ionic strength, protein concn. 0.24%; (h) 11s protein isolated from fraction (d) by precipitation at pH 4.8 between 0.4 and 0.2 ionic strength, protein concn. 1.11%. All patterns photographed at a bar angle of 55” except (g) which was at 40”. Patterns (a)-(j) are directly comparable.
Repeated precipitation and washing of the acid-precipitated protein fraction (Fig. 36) at pH 4.8 in the presence of 0.2 M sodium chloride and 0.01 M mercaptoethanol yielded an 11s preparation quite similar to the cold-insoluble fraction (Fig. lb); it still contained appreciable amounts of the 2S, 78, and 15s fractions. Further purification of the 11s component was effected by variation of the ionic strength at pH 4.8. For example, a 1% solution of partially purified 11s fraction (obtained by reprecipitation and washing the acid-precipitated protein fraction at pH 4.8 and 0.2 ionic strength) was dissolved in the standard buffer, then adjusted to pH 4.8 with hydrochloric acid and diluted to 0.4 ionic strength. A precipitate formed which consisted primarily of 11s protein plus a high proportion of 15s protein and a small amount of a faster sedimenting material. The protein remaining soluble at 0.4 ionic
192
WOLF
AND
BRIGGS
strength was then recovered as a precipitate by diluting the solution to 0.2 ionic strength. This precipitate was dissolved at pH 7.6, 0.5 ionic strength in a volume of buffer slightly smaller than the original solution, and the entire process was repeated twice. The protein obtained finally, as a precipitate at 0.2 ionic strength, consisted of 11X component contaminated with what appeared to be only traces of 7X and 15X fractions (Fig. 3h). However, when a reference base line was fitted to the enlarged pattern, the following area distribution (corrected for radial dilution) was obtained: 2s (5 %), 7X (5 %), 11s (82 %), and 15X (8 %). While this area distribution is not appreciably different from that for the cold-insoluble fraction described above, the protein obtained by this procedure formed a clear solution in standard buffer as contrasted with the somewhat turbid, yellow solution obtained with the cold-insoluble fraction. D. Fractional Extraction of Acid-Precipitated Protein. Three hundred grams of meal was suspended in 2400 ml. water, and the pH was adjusted to 7.2 with 1 N sodium hydroxide. After stirring for 30 min., the meal residue was removed by centrifugation. Volume of supernatant was 1465 ml. The pH was adjusted to 4.8, which caused 80 % of the total nitrogen in the extract to precipitate. The precipitate (pattern same as Fig. 3b) was then extracted, as indicated in Table II, by stirring with sodium chloride solutions of pH 4.8, made 0.01 M with respect to mercaptoethanol, for lTABLE Practional Extraction 1 2 3 4 5 6 7 8 9 lob lib 126
Extraction
II
of Acid-Precipitated
Soybean Protein
NaCl concn.
N/ml. in extract
Per cent of N extracted’”
moles/l. 0.20 0.20 0.20 0.20 0.35 0.35 0.35 0.35 0.35 -
w. 0.70 0.22 0.11 0.07 0.63 0.39 0.29 0.21 0.16 3.93 0.48 0.14
% 13.2 4.1 2.0 1.3 11.9 7.3 5.4 3.9 3.0 24.2 3.1 0.8
Total a Based on total N in the protein precipitate. b Extraction made with 500 ml. of phosphate-N&l strength. For extraction number 10 the protein-buffer pH 7.6 prior to extraction.
80.2
buffer, pH 7.6, 0.5 ionic suspension was adjusted to
11s
(a)
COMPONENT
(b)
OF
SOYBEAN
PROTEIKS
(4
193
Cd)
FIG. 4. Ultracentrifuge
patterns for 11s protein preparations obtained in (a) extraction5, protein concn. 1.4270; (b) extraction 6, protein concn. 1.15%; (c) extraction 7, protein concn. 1.16%; (d) extraction 10, protein concn. 1.15%; of fractional extraction procedure. Bar angle for (a) 60”, (b)-(d) 65”. hr. periods at room temperature. The volume of solvent used in each of l-9 inclusive, was equal to the volume of the original the extractions, extract (1465 ml.). The protein in extracts 5-9 was recovered by diluting the solutions to The precipitated 0.2 ionic strength and centrifuging off the precipitate. proteins were dissolved in standard buffer to give an approximately 1% solution which was somewhat turbid, but centrifugation at 10000 X g gave water-clear solutions. Figures 4u-c show the ultracentrifuge patterns 5, 6, and 7, respectively. The for the proteins recovered from extractions
proteins from extractions 8 and 9 were identical with Fig. 4~. It appears that the first four extractions ab pH 4.8, 0.2 ionic strength, removed almost all of the 2X fraction and a major part of the 7X fraction since the protein recovered from extract 5 (Fig. 4~) consisted mainly of 11X protein plus some 7X protein and only a trace of 2s fraction (apparent only when a reference base line was fitted to the enlarged pattern).4 In subsequent extractions the relative amount of 7s fraction was reduced to a small but fairly constant value as observed for extractions 7, 8, 9 (Fig.
4c), and 10 (Fig. 4d). Extractions 7-9 yielded the purest preparations of 11s protein yet attained, having the following composition based on area distribution of the ultracentrifuge pattern (Fig. 4~): 2s (3 %), 7S (4%), 11X (87 %), and 15s (6 %). The yield of 11s protein from extracts 7-9 was 3.1 g. A large amount of tan-colored precipitate remained after the 9t,h extraction, and only 52 % of the total nitrogen in the original precipitate has been 4 It is possible that extract 5 contained more 2s and 7s proteins than is indicated in Fig. 4a since they may not have been precipitated completely when the extract was diluted to 0.2 ionic strength. Figures 3b, 3d, and 3e show that these protein fractions are partially soluble at pH 4.8, 0.2 ionic strength.
194
WOLF
AND
BRIGGS
accounted for. An attempt was therefore made to dissolve the insoluble portion in standard buffer so that it could be identified by ultracentrifugal analysis. While a large part of the protein dissolved in the three successive extractions with buffer (extractions 10-12, Table II), 20 % of the total acidprecipitated protein remained insoluble in the buffer. Figure 4d is the pattern for the proteins recovered from extract 10. It reveals that a large amount of 11X protein was still present in the precipitate after the 9th extraction although the progressive decrease in the amount of nitrogen extracted at 0.35 ionic strength (Table II) suggested that most of the 11s protein might have been already extracted. Previous experiments (4) have shown that the 11X protein undergoes an irreversible dissociation at pH 3.0, 0.1 ionic strength, with an accompanying loss of solubility in standard buffer. In the present experiment a part of the 11s protein may have formed an intermediate acid-induced modification at pH 4.8 which was difficultly soluble at 0.35 ionic strength but which was partially reversed at pH 7.6, 0.5 ionic, i.e., under the conditions of extraction 10. The fraction remaining insoluble after the 12th extraction was suspended in 0.5 M sodium chloride, and 1 N sodium hydroxide was added. No appreciable dispersion was noted until a pH of 11 was reached, and the suspension was still extremely turbid at pH 11.5. It would appear that other components, as well as the 11s protein, may have been partially irreversibly modified by the prolonged extraction process at pH 4.8. Estimation of Some of the Physical Characteristics of the 11s Protein The sample of 11s component obtained by fractionation procedure D (pattern shown in Fig. 4c), while only of the order of 90 % pure in this component, was nevertheless utilized to yield the following approximate values for the physical characteristics of this protein. An electrodialyzed sample of this protein had a nitrogen content of 17.12 % (dry basis). Partial Specific Volume. The partial specific volume, V, of the purified 11s protein was determined from the relationship (6),
where p is the density of the solution for which the solute weight fraction is w, and m is the mass of a given volume of the solution. A plot of m against w indicated that no change in dm/dw occurs with solutions of the 11s protein in the standard buffer over the concentration range 1.8-3.6%. The value of V for three concentrations of 11X protein in this range, at 2O”C., was found to be 0.719 f 0.001, which is slightly higher than the 0.710 value reported by Naismith (2) for the unfractionated mixture of soybean proteins.
1lS
COMPONENT
OF
SOYBEAN
PROTEINS
195
13,
IO 0
1.00
0.50 Concentration
1%)
FIG. 5. Plot of SW,,,.versus c.
Sedimentation. The sedimentation constant of the purified 11s protein was measured at room temperature in the model E Spinco ultracentrifuge at 59780 r.p.m. with standard buffer as the solvent. In measuring the values of 2, the distance of the peak from the center of rotation, an allowance of 0.025 cm. was made for stretching of the rotor at 60,000 r.p.m. (7). The sedimentation constant was corrected to the standard state of water at 20°C. (6). Allowance for the cooling resulting from adiabatic stretching of the rotor during acceleration (8) was made by subtracting 1°C. from the average rotor temperature. Calculated s20,Wvalues were plotted against protein concentration as shown in Fig. 5. The equation for the regression line is ~20,~ = 12.20 - 0.62~ where ~20,~ is given in Svedberg units and c is the protein concentration in g./lOO ml. The concentration values have not been corrected for the impurities (about 13 %) estimated to be present from area measurements on the ultracentrifuge patterns, since applying this correction does not change the value of sio,%., altering only the slope of the extrapolation line. Di$usion. Diffusion measurements were made in standard buffer with a Neurath-type diffusion cell (9) at 3.8”C. The concentration gradient of the diffusing protein was photographed periodically with the schlieren scanning optical system of a Tiselius-Klett electrophoresis apparatus. The diffusion constant, D, was calculated from the equation (10)
where K = A/2& A = the average area under the diffusion curves (sq. cm.). H = the maximum ordinate (cm.) of the dn/dx versus x curve at time t.
196
WOLF
Al’iD
BRIGGS
7
6
I/J+ x 10” (t in seconds) FIG. 6. Plot of H, versus l/d/t. Open circles: protein concn. = 1.06% 2.01 sq. cm.). Closed circles: protein concn. = 0.55% (area = 1.05 sq. cm.).
(area
=
The slope of the H, versus l/& relationship (Fig. 6) was used to evaluate H,d. These data yield values for D 3.8of 1.70 f 0.06 X lo-’ sq. cm./sec. The value of 03.8 was corrected to 20°C. and water as solvent (6) giving a value for D20,W of 2.91 =t 0.10 X lo-’ sq. cm./sec. From this value, the corresponding sedimentation constant, and the partial specific volume, the molecular weight of the 11s protein was calculated to be 363,000 f 13000. Light Scattering. Turbidity measurements of the purified 11s protein dissolved in standard buffer were made as described in a previous publication (11). Turbidity measurements made at angles of 45” and 135” to the incident beam indicated that no dissymmetry corrections were necessary. The specific refractive-index increment of the protein was determined with a Phoenix differential refractometer. Protein concentrations were determined by a semimicro-Kjeldahl procedure. Figure 7 is a plot of HC/T versus c at the two wavelengths of incident
1h-Z COMPOKENT
OF
SOYBEAN
197
PROTEINS
0
3.0A= 436 mp ;
I
2.5,
I
!
I
7
8
9
x
;---Tr--‘; 0
2
3
4
Concentration
5
6
(QmJml. x IO’)
FIG. 7. Plot of HC/T versus c.
light employed. The curves, drawn by the method of least squares, when extrapolated to zero concentration give the reciprocal of the molecular weight, according to the Debye equation (14). The observed intercept values of 3.02 X low6 and 2.83 X 10m6for 546 and 436 rnp, respectively, yield molecular weights of 331,000 and 354,000. Light-scattering measurements with another preparation of 11s protein yielded molecular weights of 335,000 and 359,000 at 546 and 436 rnp, respectively. The average molecular weights by this method are, thus, 333,000 (546 rnp) and 356,500 (436 mb). Apparent depolarization factors of 0.0166 (546 rnp) and 0.0472 (436 rnp) yielded calculated values of the Cabannes factor (15) of 0.965 and 0.902, respectively. However, the recent work of Geiduschek (16) suggests that the real depolarization of proteins is negligible; hence no correction has been made for the apparent depolarization measured. The specific refractive-index increment was found t)o be 0.00185 at 546 rnp and 0.00188 at 436 w. Ultraviolet Absorption. An ultraviolet absorption curve measured on the 11s protein in standard buffer solution through the range of 266-300 rnp was that of a typical protein with a maximum at 279 rnp having an E:?m, value of 9.2 f 0.3. Frictional Ratio. The frictional ratio for the 11X protein was calculated using the constants obtained in the above determinations. These ratios were calculated by equations given by Svedberg and Pedersen (6) which involve : (a) sZO,~,. and M, (t’he molecular weight determined by other than sedimentation velocity, here taken to be 345,000, the average of the light-scattering molecular weight), (b), Dzo,,+.and M, , and (c) ~20,~.and D20,w . The values obtained were 1.51, 1.58, and 1.56 for methods (a), (b), and (c), respectively.
198
WOLF AND BRIGGS DISCUSSION
Figure 1 illustrates that a relatively pure preparation of the 11X protein can be obtained in good yield from the mixture of crude soy proteins by the simple procedureof cooling an aqueous extract of soybean meal. Comparison of Fig. 2b with lb shows that the 11X protein can also be selectively separated from the mixture by salt extraction, ammonium sulfate precipitation, and dialysis. However, the degree of contamination by other components is dependent on the extent of dialysis (Figs. 2b and 2d), and, since this is difficult to determine and control, cold precipitation appears to be preferable to salt precipitation and dialysis for the purification of the 11X protein. Fractionation of the soybean proteins by acid precipitation from an aqueous extract gives a very heterogeneous preparation of the 118 protein (Figs. 3uc), but some purification can be effected by reprecipitation in the presence of 0.2 M sodium chloride, and a possible method for the preparation of the 7X component is indicated (Figs. 3e-g). Fractional extraction of the acidprecipitated protein with sodium chloride solutions of graded ionic strengths yields the 11X protein in the highest purity, free of much of the color and turbidity characteristic of solutions of the unfractionated soybean protein mixture. However, the purified 11X protein is obtained in low yield by this procedure, and the large amount of protein which becomes insoluble on fractional extraction of the acid precipitate suggests that a part of the 11S protein, and/or other proteins, is irreversibly modified in this process. The degree of purification of the 11s protein achieved in the present studies is of the same order as that obtained for other seed globulins in the 11-138 size range where the removal of small amounts of remaining impurities has proven very difficult (18). The physical properties of the purified 118 protein indicate that it is a typical seed globulin. The molecular weight of approximately 350,000 is of the same order as that reported for other seed globulins (18-21). The s&,, value of 12.2X for this component of the soybean proteins is about 7 % lower than the value of 13.1~s’reported by Danielsson (19) using an oil turbine ultracentrifuge. It is not clear from Danielsson’s paper whether this value was obtained by extrapolation to zero concentration. Naismith (2) likewise has presented evidence that Danielsson’s value may be too high but ascribed the discrepancy to differences in ionic conditions employed. A number of workers (22-25) have found that the sedimentation constants of several well-defined proteins, as determined with the Spinco ultracentrifuge, are S-10 % lower than those determined with the oil-turbine ultracentrifuge. SUMMARY
Soybean proteins were fractionated by: (a) cooling an aqueous extract; (b) salt extraction, ammonium sulfate precipitation, and dialysis; (c) iso-
IIS
COMPONENT
OF
SOYBEAK
PROTEIM
199
electric precipitation; and (d) acid-precipitation and fractional extraction with sodium chloride solution of graded ionic strength. The purest preparation of the component having the approximate SZO,,,,value of 1lS was obtained by the last-named method. Sedimentation, diffusion, and lightscattering measurements indicate that the 11s protein has a molecular weight of about 350,000. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BRIGGS, D. R., AND MANN, R. L., Cereal Chem. 27, 243 (1950). NAISMITH, W. E. F., Biochim. et Biophys. Acta 16, 203 (1955). BRIGGS, D. R., AND WOLF, W. J., ilrch. Biochem. Biophys. 72, 127 (1957). WOLF, W. J., AND BRIGGS, D. R., Arch. Biochem. Biophys. 76, 377 (1958). WOLF, W. J., AND BRIGGS, D. R., Arch. Biochem. Biophys. 63, 40 (1956). SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge.” Oxford Univ. Press, New York, 1940. TAYLOR, J. F., Arch. Biochem. Biophys. 36, 357 (1952). WAUGH, 1). F., AND YPHANTIS, D. A., Rev. Sci. Instr. 23, 609 (1952). NEURATH, H., Science 93, 431 (1941). NEURATH, H., Chem. Revs. 30, 357 (1942). FULLER, R. A., AND BRIGGS, D. R., J. Am. Chem. Sot. 78, 5253 (1956). OSBORNE, T. B., AND CAMPBELL, G. F., J. Am. Chem. Sot. 20, 419 (1898). SMITH, A. K., SCHUBERT, E. X., AND BELTER, P. A., J. Am. Oil Chemists’ Sot. 32, 274 (1955). DEBYE, P., J. Phys. & Colloid Chem. 61, 18 (1947). CABANNES, J., “La Diffusion Moleculaire de la Lumiere.” Presses Universitaires de France, Paris, 1929. GEIDUSCHEK, E. P., J. Polymer Sci. 13, 408 (1954). SMILEY, W. G., AND SMITH, A. K., Cereal Chem. 23,288 (1946). BRAND, B. P., GORING, D. A. I., AND JOHNSON, P., Trans. Faraday Sot. 61, 872 (1955). DANIELSSON, C. E., Biochem. J. 44, 387 (1949). JOHNSON, P., Trans. Faraday Sot. 42, 28 (1946). SVEDBERG, T., Nature 139, 1051 (1937). TAYLOR, J. F., Arch. Biochem. Biophys. 36, 357 (1952). MILLER, G. L., AND GOLDER, R. H., Arch. Biochem. Biophys. 36, 249 (1952). CECIL, R., AND OGSTON, A. G., Biochem. J. 44, 33 (1949). SHULMAN, S., Arch. Biochem. Biophys. 44, 230 (1953).