56
M. L. U R O X , M. E. A D A M S , A N D 4.M. ALTCHUL
ELECTROPHORETIC ANALYSIS O F PEANUT AND COTTONSEED MEALS AKD PROTEIKS1 M. L. KARO?;, MABELLE E. ADAMS, A K D A. M. ALTSCHUL
Southern Regional Research Laboratory,z New Orleans 19, Louisiana Received August 28, 1949 Ih-TRODUCTIOS
The investigations of Johns and Jones (7) have indicated that the total protein of the peanut consists of globulins and a very small amount of heat,-coagulable albumin. They found that it mas possible, by means of ammonium sulfate fractionation of a sodium chloride extract of peanut meal, to separate the globulins into two fractions, arachin and conarachin, which differed in optical rotation and in content of sulfur, basic nitrogen, lysine, methionine, cystine, threonine, tryptophan, and tyrosine (2, 7 , 10). Later, Jones and Horn (11) stated that arachin could be precipitated from the 10 per cent sodium chloride estract by dilution until the extract became cloudy followed by saturation with carbon dioxide, or by the addition of 1 volume of saturated ammonium sulfate to 1.5 volumes of the extract. Conarachin, which was the more soluble fraction, could not be isolated by dilution, but could be precipitated from the filtrates after precipitation of the arachin fraction by dialysis or complete saturation with ammonium sulfate. The fractional precipitation of protein mixtures had been shown to be a n arbitrary procedure (4) and it was not possible to decide whether the arachin and conarachin were single proteins or mixtures in more or less constant proportions. I n order to determine whether these protein fractions were homogeneous, Irving, Fontaine, and Warner (6) conducted electrophoretic analyses on peanut meal, arachin, and conarachin. Their results indicated that peanut meal contained a t least three and probably four components and that the arachin and conarachin fractions each consisted of mixtures of at least two components. Johnson (8) investigated the peanut protein fractions by use of the ultracentrifuge. I t appeared that the arachin fraction obtained by dilution and addition of carbon dioxide consisted of at least two differently sedimenting species of protein molecules, while the fraction obtained by ammonium sulfate fractionation had only one major constituent. The conarachin fractions consisted of two differently sedimenting species. Arachin, prepared by dilution and acidification and analyzed electrophoretically in phosphate buffer, appeared to consist of two components. Later, it was possible to obtain by salt fractionation a peanut protein fraction which appeared to be homogeneous with respect t o molecular size as determined by the ultracentrifuge method, but was not homogeneous when analyzed electrophoretically. 1 Presented a t the Twenty-third Sational Colloid Symposium, which was held under t h e auspices of the Division of Colloid Chemistry of the American Chemical Society at Minneapolis, Minnesota, June 6-8, 1949. * One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U . S. Department of Agriculture.
ELECTROPHORETIC Aii.ILYSIS O F PROTEIM
57
Jones and Csonka (9) made the first extensive investigations of the proteins of cottonseed, in which they determined the solubility of the nitrogenous constituents of solvent-extracted cottonseed using successive exhaustive extract,ions, first with 10 per cent sodium chloride solut'ion, then with 70 per cent ethanol, and finally with 0.5 per cent sodium hydroxide solution. Because 70 per cent ethanol did not extract any nitrogen, they concluded that a prolamine type of protein was absent. ;iceording to the fractionation scheme of these authors, the protein from cottonseed meal could be divided into the following fractions: High-ash-yielding fractions I and 11, a pentose protein, a glutelin, and an a- and P-globulin. They believed the high-ash fractions to be phosphoproteins, RIore recent work by Fontaine, Pons, and Irving ( 5 ) , however, suggested that these fractions were probably impure phytin rather than phosphoproteins. Subsequent to the work of Jones and Csonka, there has been no concerted effort t o differentiate the nitrogenous constituents of cottonseed by use of the new tools of protein chemistry. The present report is concerned with an investigation of peanut and cottonseed meals and derived crude proteins by means of the moving boundary electrophoresis method, with particular reference to the effect of the buffers used a s protein solvents and the p H of the buffer on the electrophoretic patterns. Fractionation by means of saline solutions was successful in effecting separation of the two major components of cottonseed protein extracts, whereas similar procedures did not result in separation of peanut protein extracts. EXPERIMENTAL
Although the proteins of cottonseed and peanut consist primarily of globulins, their solubility in 0.1 molar salt solutions at 0°C. is too small for electrophoretic analysis in neutral buffers; consequently alkaline buffer solut'ions were used as solvents in these investigations. LongsTvorth (12) had shown that the best separation of the components of blood serum could be effected if 0.10 ionic strength veronal (diethylbarbituric acid) buffer at pH 8.6 was used as the solvent. The slow-moving veronal anion aids in the separation of the components of blood serum during the electrophoresis. This buffer system appears to be inapplicable to the cottonseed and peanut proteins, because slight precipitation of the protein occurs in veronal solution during the electrophoresis and it was very difficult to photograph the boundaries a t the conclusion of an experiment; therefore other buffers were investigated t o determine their effectiveness for use in the analysis of these proteins. Borate and glycine buffers were prepared according to Clarke (3), veronal buffer was prepared according to Longsrvorth (12), and an ammonia buffer was prepared by addding 0.2 molar ammonia to 0.1 molar hydrochloric acid solution. While these buffers were satisfactory solvents for peanut meal and protein, only the glycine buffer v a s satisfactory as a solvent for cottonseed meal. The proteins in cottonseed meal are less soluble than those of the peanut
58
M. L . K A R O S , M . E . ADAMS, AiYD A . M . ALTSCHUL
under the same extraction conditions and more alkaline soluticns are required to solubilize the former. Even in glycine buffer below pH 10, the solubility of cottonseed protein was not sufficient to enable accurate determinations of either mobility or percentage composition. .4buffer composed of 0.2 mole of ethylamine and 0.1 mole of veronal in a liter of solution having a pH of 10.7 had the most desirable characteristics. The protein solutions were prepared by mixing 5 g. of the defatted meal with 100 ml. of the different buffers a t 0°C. and allowing them to equilibrate for 3 hr. The residual meal was separated from the solution by centrifugation and the protein solution dialyzed against 2 liters of the same buffer for 20 hr. on a rockingtype dialyzer. At the conclusion of the dialysis the solution was clarified by centrifugation and placed in the electrophoresis cell. At no time was the temperature allowed to rise above 5°C. The apparatus described by Longsworth (12) for analyzing proteins by the Tiselius moving boundary method was used in the investigation. The duration of an experiment was usually 180 min. a t field strengths from 4.6 to 7.3 volts/cm. The photographic records of refractive index gradients in the boundaries were made by the schlieren scanning method and at a temperature of 0°C. The apparent concentration of the components of either cottonseed or peanut was determined by finding the ratio in each case of the component area to the total area exclusive of the &boundary. The areas were measured on projected tracings of the electrophoretic pattern of the descending boundary with a planimeter, division into components being carried out by the method of Pedersen (13). Mobilities were determined from the center of the &boundary. RESCLTS
Peanut meal and prolein Eflecl o f b u f l e r and p H : The effect of the use of different buffers as the protein solvent on the electrophoretic pattern of deskinned, hesane-extracted Spanish peanuts is shown in table 1. The data in table 1 indicate that the same electrophoretic pattern is obtained over a pH range from 8.3 to 10.2 and with a variety of buffers. Glycine buffer solutions, exhibiting sharper boundaries and allowing greater protein solubility than the other buffer solutions tested, w r e used in the routine electrophoretic esamination of peanut protein solutions. The influence of the pH of a glycine buffer extract of peanut meal on the electrophoretic analysis of the solution was investigated over a range of pH from 8.7 to 11.4 and the results which were obtained are presented in table 2. The data in table 2 indicate that there is no significant difference in the relative percentage of each component in the region of pH 8.i-10.2. I n more alkaline pH solutions there is a significant increase in the amount of components B and C at the expense of component A. As the alkalinity of the buffer is increased above pH 1 1 , an increasing amount of the protein components appears t o become transformed into a single diffuse component which has a mobility
59
E L E C T R O P H O R E T I C ASALTSIS OF P R O T E I N S
between those of components B and C. The exact nature of the effect of alkali is not known, but the process appears to be reversible in solutions less alkaline TABLE 1 Electrophoretac analysis o j different buffer extracts of peanut meal PROTEIN COKPONENT
l P ~ t '
BUFFER'
A
B
1
Per cent!
__ Borate Verona1 Ammonia Ammonia' Glycine Glycine
1
-1
8.36 1849 i 870 926 920 1 10 21
i
-
C
IMobil1 ityt
-1-
-6.3 1 76 -5.1 -6.9 84 -5.7 -7.1 81 , -5.4
Per :entg
-2.5 -3.6 -3.5 -3.7 5 -2.9 6 , -3.3 4
11 -4'7 7 , -4.0 9 I -4.5 ~
1
,
1 __D __ Per Mobilitytt _ I_ _ cent5 -.
I
9 9 8 13 4 4 __
* All buffers were of 0.10 ionic strength. t p H determined a t 25OC.,using a glass electrode. t Mobility in cm?/volt/sec. X 106. $ Percentage of the total area exclusive of the &boundary occupied by the area of component. D a t a of Irving, Fontaine, and Warner (6). TABLE 2 Effect of p H of glycine buffer eztracts of peanut meal on the electrophoretic analysis'
I PHt
I ~
8.73 9.20 9.51 9.66 10.21 10.77 10.95 11.15 11.40
'I
PROIEIN COYPONENIS
A
-7.2 I -7.4 -7.1 I -8.0 -7.7 -8.3
_
_
Per cents 1 Mobility$
Mobility:
~
84 83
81 65 63 33 12
B
-5.3 6 -5.7 7 -5.6 7 -6.4 6 9 1 -5.4 -6.4 13 -6.4 17 -6.2 40 -6.8 46 __ ___ ~
D
C
~ Per cent!
Mobility:
Per centl
-3.4 -4.0 -4.8 -4.9 -4.5 -5.4 -5.5 -5.2 -5.2
6 5 6 7 6 16 17 10 29
1
i i ,
1 11
Mobility:
-2.4 -2.9 -3.9 -3.8 3.3 14.0 -4.1 -4.1 -3.8
Per cent$
4 4 3 4 4 6 6 11 7
* Ionic strength of buffer maintained a t 0.1; field strength approximately 5.0 volts/cm.; protein concentration approximately 1 .O per cent. t p H determined a t 25OC. with a glass electrode. t Mobility expressed as cm.*/volt/sec. X 106. 8 Percentage of the total area exclusive of the &boundary occupied by the area of component. than pH 11. As shown in table 3, a protein solution prepared in a buffer at pH 11 and reequilibrated in buffer of pH 9 had an electrophoretic pattern characteristic of a protein solution originally prepared a t pH 9.
60
M. L. KARON, M. E. ADAMS, .4ND A . M. ALTSCHUL
Eject of method of extraction and precipitation of protein: Samples of peanut meal were extracted as previously described, using 0.1 N solutions of sodium chloride, sodium sulfite, sodium trichloroacetate, and sodium hydroxide adjusted to p R 8.0. After equilibration for 3 hr., the residual meals were separated by centrifugation and the protein liquor was dialyzed against the glycine buffer of pH 10.5 until equilibrium was obtained. The electrophoretic analysis indicated that the extracts had identical composition irrespective of the solvent used for extracting the protein. If, however, one part of peanut meal was washed with 10 volumes of water adjusted to pH 5.0 prior t o the extraction of the protein, appreciable soluble sugars Eind phytin were removed and the protein isolated from the washed meal had an electrophoretic pattern which was different from that of the usual salt- or alkali-extracted protein. TABLE 3 Reversible V H effect of glycine buffer extracts of peanut meal on the electophoretic analysis PPOTEIX COYPONENT
C _____
PH
hours
9.20 9.17 I O . 95 10.95 * n n? n
*-
1U.YO-Y.Ii
ne+
YO+
/
D
~
- 1 . 0
,
00
-U.L
I
-0.a
I
-*'I
1
=
* Mobility expressed
as cm?/volt/sec. X lo5. t Percentage of the total area exclusive of the 8-boundary occupied by the area of component. $ Equilibrated for 24 hr. at p H 10.95 and then reequilibrated for 72 hr. a t pH 9.17. t o be present to the extent of 83 per cent of the entire protein concentration. I n the protein isolated from the washed meal, however, component A was no longer present, but appeared to be replaced by two components, A, and A,, Tyhich were present to the extent of 45 and 37 per cent, respectively, This effect of washing the meal is clearly shown in figure 1. Protein concentrates were prepared from fat-free peanut meal by first extracting the meal with either salt solution or mild alkali solution adjusted t o pH 7.5, then clarifying the extract by centrifugation and precipitating the protein by acidifying the clarified extract to pH 4.5 and drying the wet curd. Protein prepared in this manner had a distribution of components as determined in the electrophoresis apparatus similar to that of extracts of unwashed peanut meal. If, however, the protein concentrate was prepared from pre-washed meal or if the crude protein curd mas washed with water prior to the other prepurification operations, t n o components, A, and A?. were obtained just as in the case of protein extracts of pre-washed meal.
61
E L E C T R O P H O R E T I C AKALTSIS O F P R O T E I N S
A
b FIG.1. (a) Sormal pattern of a glycine buffer extract of peanut protein after electrophoresis for 180 min. in glycine buffer at pH 9.50. (b) Pattern of a glycine buffer extract of peanut protein isolated from washed peanut meal after electrophoresis for 180 min. a t p H 9 50. TABLE 4 Effect of p H of glycine buffer* o n the electrophoretic pattern of cottonseed meal eztracts P R O I E l l i COMPONENTS
I
PHt
A
I I Mobility$
10.25 10.40 10.65 10 75 10 85 11.03
-6.8 -6.8 1
1 ~
B
,I -~ Per cent! 10 14
D
C
lrlabilityt Per cents
1
hlob~lity: Per cents
~
Per cent!
-2.1 -2.5
3 6
~~
--I
-5.4 -5.5 -5.8 -6.4 -5 7 -5.6
Mobility$
32 35 95 91 95 92
-3.7 -4 2
50 39
~
* Buffer of 0.1 ionic strength; protein concentration, 1.2 per cent.
t pH
determined a t 25°C. with a glass electrode. expressed as cm.*/volt/sec. X lo5. C Percentage of the total area exclusive of the 8-boundary occupied by the area of component.
2 Mobility
Cottonseed meal and protein
Eflect of bufler and p H : The low solubility of cottonseed proteins in buffer solutions less alkaline than p H 9 limited the number of buffers available for use in electrophoretic investigations of these proteins. A comparison of electro-
62
Y. L. KARON, M. E. ADAMS, .4ND
.4. M. ALTSCHUL
phoretic analyses of cottonseed protein solutions in glycine and ethylamineveronal buffers is given in tables 4 and 5 . The data indicate that glycine buffer is suitable for electrophoresis analysis of cottonseed proteins only over a very limited range of pH. Ethylamine-veronal buffer, however, can be used in the electrophoretic analysis of cottonseed proteins over a much wider range of pH. TABLE 5 Effect of pH of ethylamine-veronal* buffer o n the electrophoretic pattern of cottonseed meal extracts I
PROTEIN COYPONEXIS
,
PHt
1
1
9.16 9.71 10.16 10.54 10.62 10.72
I
I
A
I
B
Mobility:
Per cents
Mobility!
-5.6 -5.7 -6.5 -6.7 -6.8 -6.4
25 35 36 49 35 27
-4.1 -3.9
-1-1
Per cents
'
1
54 51
'
42 58
C Mobility$
D
Mobility$
10
-1.8 -2.0
, 1
I Per cent$ 1 - 1 - 1 -
-1.0
Per centt
I
11
-4.0 -3.6
2
~
* Buffer is of 0.1 ionic strength; protein concentration t pH determined at 25°C. with a glass electrode.
is 1.2per cent.
$ Mobility expressed as cm.*/volt/sec. X lo6. 5 Percentage of the total area exclusive of the &boundary occupied by the area of component.
TABLE 6 Effect of protein extraclant on the electrophoretic analysas of cottonseed meal extracts in ethylamine-veronal buffer at pH 10 7 PROTEIN COMPONENTS
A
E X I R A C I I N G SOLUTION
Per centt
Mobil-
ity' __
0.2 N NalSOa.. . . . . . . . . . . . . . -6.2 0.2 N N a C l . . . . . . . . . . . . . . . . . . -6.6 0.2 N NaOOCCCla (trichloro acetate) . . . . . . . . . . . . . . . . . . . NaOH to pH 10.0.. . . . . . . . . .
* Mobility
~
~
16 44
-4.8 -5.7 -5.0
__ -~ 57 -3.5 46 -3.5 56
~
-3.6
ILqX~ 11 1
~
5
-1.7
16
-2.0
~
~
5 11
expressed as cm.l/volt/sec. X lo6. of the total area exclusive of the 8-boundary occupied by the area of com-
t Percentage ponent.
Efect of method of ertraction of protein: A series of experiments were conducted in which 0.2 N sodium sulfite, sodium trichloroacetate, sodium chloride, and sodium hydroxide solutions which had been adjusted to pH 10.0 were each used to extract the protein from samples of cottonseed meal. The protein liquors were equilibrated against ethylamine-veronal buffer and analyzed in the electrophoresis apparatus. The results are given in table 6 .
63
ELECTROPHORETIC ANALYSIS O F PHOTEINS
TABLE 7 Electrophoretic analysis of major components of sodium chloride extracts of cottonseed meat i n ethylamine-veronal buffer at p H 10.69
1.0-0.0 1.04.9
17.0
j
-6.0
~
63
-5.1
1
18
-4.2
16
* Mobility expressed as cm.z/volt/sec. X 106. t Percentage of the total area exclusive of the &boundary occupied by the area of component.
Concentration of Sodium Chloride,Moler/Liter FIG.2. Distribution of the major components, A and B , in the precipitates removed after equilibrium was reached at each final concentration of sodium chloride solution used.
Unlike the behavior of peanut proteins, the type of anion used in the estractant had a marked effect upon the distribution of protein components in the cottonseed meal extract. The greatest difference \vas observed in the distribution of
64
M. L. KARON, X I . E. .4D.4MS, APiD A. hI. ALTSCHUL
components A, C, and D. Component B was equally well extracted by all protein extractants. In a previous publication (1) it was shonn that extraction of proteins with sodium sulfite yields a protein product which can more easily be spun into a fiber than one extracted with alkali. I t is entirely possible that the properties of a protein concentrate n-hich determine its usefulness in industrial applications
B
a
B
B
C
FIG.3. Effect of iractional dialysis of a sodium chloride extract of cottonseed meal on the electrophoretic pattern oi purified fractions. (a) Whole cottonseed meal in ethylamineveronal buffer p H 10.5, 120 min. at a field strength of 7.0 volts/cm. (b) Cottonseed protein fraction precipitated from 0.4 t o 0.3 N sodium chloride in ethylamine-veronal buffer p H 10.5,160min. at a field strength of 7.0volts/cm. ( e ) Cottonseed protein fraction precipitated from 0.9 t o 0.8 N sodium chloride in ethylamine-veronal buffer pH 10 5 , 180 min a t a field strength of 7.0 volts/cm.
are dependent upon the nature of the distribution of the various protein components. Fractionation of protein components: The partial fractionation achieved simply by difference in type of extraction anion (table 6) suggested that it might be possible to obtain pure protein components from cottonseed by relatively simple means. Cottonseed meal (50 g.) was reextracted eight times u ith 200-ml. portions
ELECTROPHORETIC ANALYSIS O F PROTEIKS
e5
of normal sodium chloride and the extract solutions combined. The combined solution was analyzed in the electrophoresis apparatus and thenmas dialyzed against an equal volume of 0.8 .V sodium chloride solution. After equilibrium was attained, the resulting cloudy mixture was centrifuged, the precipitate collected, and the sodium chloride solution. This process was supernatant dialyzed against 0.7 repeated using lower and lower normalities of sodium chloride solution as the dialyzing medium. The precipitates obtained after each dialysis operation were analyzed in the electrophoresis apparatus with the results given in table 7 . The distribution of the major components, A and B, in the precipitates removed after equilibrium !vas reached at each final concentration of sodium chloride solution used, is plotted in figure 2. I t can be seen that the precipitate obtained by lowering the concentration of sodium chloride solution from 0.9 to 0.8 W had a high concentration of component A and that the precipitate formed by reducing the concentration from 0.3 to 0.2 S sodium chloride was almost pure component B. Electrophoretic patterns of extract of cottonseed meal and of the purified fractions obtained by atepivise dialysis Tvith sodium chloride solution are shown in figure 3. DISCUSSIOK
;ilthough peanut and cottonseed have little in common, it is interesting to compare the properties of their proteins as determined by electrophoretic measurements. I n both cases the protein nitrogen is distributed over two major and several minor components. Peanut proteins resist fractionation and seem to be bound together by complex-forming agents. The fact that component A can be separated into two components by pre-washing the meal or washing the wet protein curd suggests that the binding agents may be carbohydrate and phytin components in the meal. There is considerable asymmetry between the patterns of the ascending and descending boundaries of peanut protein solutions and probably ronsiderahle protein-protein interaction. More evidence for the close relationship between the protein components of the peanut is the reversible interconversion from one component to the other which takes place in alkaline buffer solutions. Cottonseed proteins do not seem to be as closely bound as peanut proteins. There is less asymmetry in the patterns of the two boundaries, no marked effect of alkalinity on the relative abundance of the protein components, and a much greater ease in separation of the individual components by methods of extraction and salt fractionation. SUMM?.RY
Cottonseed and peanut meals have been analyzed electrophoretically. The factors investigated were ( 1 ) the effect of buffer used in the electrophoretic analysis, ( 2 ) the pH of the buffer, and (5)the effect of the solvent used to extract the protein from the meal. Peanut protein has been found t o consist of two major and several minor components. The major components migrate as a single entity under most
GG
SIDNEY SHULMAN AKD JOHN D. FERRY
conditions; however, if the protein was separated from a meal which had been washed with water adjusted t o pH 5 to remove soluble sugars and phytin, the major components separated into two almost equal fractions. Cottonseed meal protein has two major and two minor components. By fractional precipitation involving a change of ionic strength of the sodium chloride solution which was the solvent, purified fractions of the major components could be obtained. REFERENCES ARTHUR, JETTC., J R . , AND KARON,MELVINL.: J. Am. Oil Chemists' SOC.26, 99-102
(1948). BROWN, W. L . : J. Biol. Chem. 142, 299 (1942);164, 57 (1944). CLARK,W. M . : The Determination of Hydrogen Ions, 3rd edition, p. 206. The Williams & Wilkins Company, Baltimore, Maryland (1928). COHN,E . J . , MCMEEKIN, T. L., OXCLEY, J. L., NEWELL,J. M., AND HUGHES,W. L.: J. Am. Chem. SOC.62, 3386 (1940). FONTAINE, T. D., PONS,W. A., JR., AND IRVING,G. W., JR.:J. Biol. Chem. 164. 487507 (1946). IRVING, G.W., JR., FONTAIXE, T. D., A N D WARNER, R . C . : Arch. Biochem. 7, 475-89
(1945). JOHNS,C. O.,AND JONES, D. B.: J . Biol. Chem. 26, 77 (1916);SO, 33 (1917). JOHNSON, P.: Trans. Faraday Soc. 42,28-45 (1946). JONES,D.B., A N D CSONKA, F. A.: J. Biol. Chem. 84, 673-83 (1925). JONES,D.B., GERSDORFF, C. E . F . , . ~ N DMOELLER, 0.:J. Biol. Chem. 62,183 (1924-25). JONES, D.B.,AND HORN,M.J.: J. Agr. Research 40,673 (1930). LONGSWORTH, L . G.:Chem. Revs. SO, 423 (1942);Ind. Eng. Chem., Anal. E d . 18, 219
(1946). SVEDBERG, T., AND PEDERSEN, K. 0.: The UltracenlTifuge, p. 296. Oxford University Press, London (1940).
T H E CONVERSIOS OF FIBRINOGEN TO FIBRIN. I1
INFLUENCE OF PH AND IONICSTREXGTH ON CLOTTING TIMEAND CLOTOPACITY' SIDNEY SHULMAN
AND
JOHN D. F E R R Y
Department of Chemistry, University of Wisconsin, Madison, Wisconsin Received August 88, 194.5
The time required for the clotting of fibrinogen by thrombin is prolonged by a variety of hydroxyl compounds, as described in the first paper of this series (8), by increasing concentration of electrolyte (1, 7 , 9), and by shift of the pH either above or below the neutral zone, as noted by a number of investigators (1, 7 , 16, 23). Under all these circumstances except decreased pH, the prolongation of clotting time is accompanied by a lowering of clot opacity, indi1 Presented a t the Twenty-third Yational Colloid Symposium, which was held under the auspices of the Division of Colloid Chemistry of the American Chemical Society a t Minneapolis, Minnesota, June 6-8, 1949.
