116
INDUSTRIAL AND ENGINEERING CHEMISTRY
Figure 11 (left) correlates the power factor for the vacuumdried capacitor after impregnation with the oil refined at 20” C. with sulfuric acid varying in concentration from 96 to 70 per cent. .I n each instance 4.4 pounds of the acid were used per gallon of oil treated. The dielectric instability of the oil-impregnated cellulose after the use of the weaker acid a t 20” C. is not eliminated when the acid-refining treatment is carried out a t a higher temperature. The data in Figure 11 (right) show the behavior of the capacitors impregnated with oil treated with sulfuric acid of varying concentration a t 50” C., using 4.4 pounds of acid per gallon of oil. Fuming sulfuric acid (20 per cent SO3) has not been found advantageous for the treatment of insulating oil. Despite its more efficient action in removing the dielectrically unstable olefin hydrocarbons, its drastic action on aromatic hydrocarbons is difficult to control. The reduction in the aromatic content of the oil has invariably been too severe to obtain the greatest dielectric stability. Typical results are illustrated in Figure 12 which compare the life test behavior of capacitors treated with the distillate oil after refining treatment with 96 per cent concentrated acid and with fuming sulfuric acid. As usual in dielectric tests of this type, the greatest instability of the dielectric appears in the tests a t the higher power factor testing temperature.
Use of Adsorbents Adsorbents are extensively used in the petroleum industry. The use of these materials for the reclamation of contami-
Vol. 34, No. 1
nated insulating oils is well known. One of the common adsorbents in commercial use is fuller’s earth. Under its influence, olefinic compounds can be removed by adsorption without drastic effect on the aromatic hydrocarbon content. Figure 13 compares the dielectric stability of capacitors impregnated with an oil prepared with 4.4 pounds of 96 per cent sulfuric acid per gallon of oil a t 20” C., followed by the “standard” fuller’s earth treatment already described, and the stability of capacitors prepared with the same oil after a similar fuller’s earth treatment applied five consecutive times. The additional earth treatment reduced the olefinic concentration from 3.5 to 2.5 per cent without significant change in aromatic unsaturation. The result is increased dielectric stability for the treated insulation. However, since exhaustive treatment of the oil with an adsorbent such as fuller’s earth has been found to remove, or a t least to reduce, the aromatic hydrocarbon content, excessive and uncontrolled treatment of the oil with the adsorbent must be avoided if the maximum dielectric stability is to be obtained.
Literature Cited (1) Berberioh, L. J., IND.EN@. CHEIII., 30, 280-6 (1938). (2) Clark, F. M., Ibid., 31, 327-33 (1939). (3) Clark, F. M., Proc. Am. Sac. Testing Materials, 40, 1213-34
(1940). (4) Kattwinkel, R., Brenmtof-Chem., 8, 353-8 (1927). (5) Typke, K., Petroleum Z.,22, 751-6, 774-8 (1926). PREISENTED as part of the Symposium on Electrical Insulation Materials before the Division of Industrial and Engineering Chemistry at the 102nd Meeting of the AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J.
Amino Acid Composition of
Cottonseed Globulin Preparations T. D. FONTAINEI, H. S. OLCOTT2, AND ALEXANDER LOWY hlellon Institute and University of Pittsburgh, Pittsburgh, Penna. A CONTINUATION of the studies on the preparation and properties of the cottonseed proteins ( l a , IS,I J ) , a partial analysis of the amino acid composition of the globulin fraction has now been completed. In the light of recent developments in new methods of isolation and new uses for the vegetable proteins, the composition of the protein potentially available in the largest quantities assumes increasing importance. Calculated for the yields here recorded, the 4,000,000 tons of cottonseeds processed in 1940 contained 400,000 tons of globulin. Furthermore, the recent innovations in amino acid procedures have made possible the determination of some components not previously described. #Finally,detailed information on the amino acid make-up of a protein moiety is instructive in the determination of protein structure and nutritive properties. Previous studies on the amino composition of the cottonseed globulins were reported by Abderhalden ( I ) , Friedmann (6),Jones and Csonka (7), and others, and will be referred to in the experimental part. After unsuccessful attempts to 3
Present address, Southern Regional Research Laboratory, New Orleans,
La. Present address, Western Regional Research Laboratory, Albany, Calif.
prepare a crystalline globulin, it was decided that an analysis of the whole globulin would be as useful as a series on preparations obtained by salt fractionation methods. Unpublished observations indicate that the proteins analyzed contained a number of separable globulin fractions. Jones and Csonka (7)described the fractionation of a cottonseed globulin preparation.
Preparation of Protein Fractions Three globulin preparations were compared. Two were obtained by acid precipitation of an alkaline extract of cottonseed meal in a modification of the method described by Nickerson (12);the second differed from the first only in that further steps of re-solution and re-precipitation were involved. The third preparation was obtained by dilution of a salt extract of cottonseed meal. Dehulled cottonseed meats from the 1939 crop were obtained through the courtesy of W. H. Jasspon of the Perkins Oil Company, Memphis. They were ground in a burr mill to pass a 20-mesh screen and then extracted with ethyl ether in a large-scale Soxhlet-type extractor (18 pounds capacity) for 3 to 4 days. The oil- and gossypol-free meats were dried
January, 1942
INDUSTRIAL AND ENGINEERING CHEMISTRY
in air and reground to pass a 60-mesh sieve. The meals had the following average composition: moisture, 10.4 per cent; residual lipides (by hot chloroform extraction), 2.2 per cent; ash, 9.3 per cent; nitrogen, 9.1 per cent. (The last three analyses were calculated on a moisture-free basis.) GLOBULINPREPARATION I. Fifteen pounds of the above meal were stirred for 45 minutes with 150 pounds of tap water in a 30-gallon stainless steel container. The slurry was separated by centrifugation in a solid basket-type centrifuge, and the centrifugate was discarded. Water-soluble proteins, proteoses, carbohydrates, inorganic salts, etc., are removed by thi$ procedure. The residual meal was transferred to the large container; 200 pounds of warm tap water (38' C.) plus sufficient sodium hydroxide (approximately 1700 ml. of 5 per cent solution) were added to bring the pH to 10.5 (glass electrode). Stirring was continued for 90 minutes, after which the mixture was centrifuged (anti-foam reagent necessary) and the meal discarded. The centrifugate was passed through a bowl centrifuge (6) to remove fines, reheated to 38" C. by passing in steam through a coil with vigorous stirring, and precipitated by the cautious addition of sufficient sulfuric acid (approximately 1400 ml. of 4.4 per cent solution) to bring the pH to 6.0. The precipitate was allowed to settle for 1 hour, and the clear supernatant liquor was removed by siphoning. The precipitate was collected by centrifugation (basket type) and dried in air (fan) by spreading the cake in a thin layer on large pieces of plate glass. The 3.25-pound yield accounted for 21.6 per cent of the meal and 35 per cent of the total nitrogen.
115
Approximately 80 per cent of the amino acids in cottonseed globulin preparations have been qyantitatively estimated. The globulins were prepared by alkaline extraction followed by acid precipitation, and by salt extraction followed by dilution. They contained approximately 18 per cent combined dicarboxylic acids (by amide nitrogen determination), 12 per cent arginine, 9 per cent leucine, 8 per cent phenylalanine, 6 per cent valine, 5 per cent lysine, 3 per cent each of histidine, methionine, tyrosine, serine, and threonine, 2 per cent isoleucine, and 1 per cent each of tryptophane and cystine.
experiments would have been sufficient in amount for the amino acid determinations, it appeared of greater interest to determine the composition of materials obtained in pilot-plant scale operations since they would be directly comparable to industrial protein preparations.* The final products contained approximately 10 per cent COMPOSITION OF COTTONSIRID GLOBULIN moisture and had the composition shown in Table I (calTABLE I. ELEMENTARY PREPARATIONS culated on a moisture-free basis). ---Preparation-Jones and Csonks (7)I n preliminary runs it was determined that maximal hyConstituent I I1 I11 a-Globulin &Globulin drolysis was accomplished by boiling with 8 N sulfuric acid for Carbon ... 49.3 49.6 62.7 60.3 approximately 24 hours. For those amino acids determinable 8.0 8.0 7.6 6.6 Hydrogen Nitrogen li:i 16.9 17.9 18.2 17.8 in such a hydrolyzate, 50 grams of protein were refluxed with Sulfur 0.79 0.76 0.69 0.93 0.78 Phosphorus 0.45 0.38 0.23 ... 200 cc. of the 8 N acid. Aeh 1.28 0.95 0.75 0:37 0.25
Determination of Amino Acids GLOBULIN PREPARATION 11. The procedure used was the same as that already described, up to the separation of the precipitated globulin. I n this preparation the wet protein was redispersed in 100 pounds of water and dissolved by adding 5 per cent sodium hydroxide solution (approximately 800 ml.) t o pH 10.5. The solution was clarified by centrifugation (bowl type), the insoluble fraction was discarded, and the protein was reprecipitated with sulfuric acid (1140 ml. of 4.4 per cent solution) to pH 6.0 and recovered as previously described. The yield was 3.18 pounds. GLOBULINPREPARATION 111. Extraction was effected with salt instead of with alkali. The water-washed meal was dispersed in 200 pounds of water containing sufficient salt t o bring the concentration to 3 per cent and stirred for 3 hours. The clarified centrifugate was then precipitated by diluting with 4 volumes of water, and allowed to settle for 2 hours before siphoning off most of the supernatant liquor. The residue was stirred with an additional 50 gallons of water to wash out residual salt, again allowed to settle, siphoned, and centrifuged. The yield was 1.88 pounds or 12.5 per cent of the meal. Each procedure up to and including the step of spreading the wet protein on the glass plate was carried out in one 16hour working day, in order to minimize denaturation and bacterial fermentation. The conditions used in the preparations were those found to be most suitable in numerous previous laboratory-scale extractions. Whereas protein samples prepared during such
HISTIDINE, ARGININE,AND LYSINE. The methods used were those recommended by Block and Bolling (2). Histidine and arginine were quantitatively precipitated as the silver salts, and then converted to the nitranilate and monoflavianate, respectively. The filtrate from which the arginine and histidine had been removed was treated with phosphotungstic acid t o precipitate the lysine. The lysine phosphotungstate was decomposed by an amyl alcohol-ether mixture and the lysine remaining in the aqueous phase was estimated by amino and total nitrogen determinations (ratio, 0.98). The values recorded probably represent the maximum limit, inasmuch as traces of other .amino acids were possibly present. M~THIONINE, CYSTINE,AND SULFATESULFUR. The reagents and:methods were those employed by Kassel and Brand (9). Standardization with pure methionine gave 92 to 93 per cent recovery. The protein preparations were extracted with petroleum ether (boiling at 28-30' C.) in a Soxhlet extractor for 24 hours and desiccated over phosphorus pentoxide in a high vacuum for one week. Samples of 500 to 600 mg. were used in each determination, and the methyl iodide, cystine, and sulfate sulfur were determined as directed. Sulfate sulfur was present to the extent of 0.072,0.086, and 0.022 per cent in the three preparations. PHENYLALANINE. Phenylalanine was determined by the method described by Jervis et al. (6). A Coleman 10s spectrophotometer was used, and comparisons were made against a calibration curve obtained with pure phenylalanine a t 560 mp. I n coniirmation of the observation of Block
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TABLE 11.
Vol. 34, No. 1
AMIDEKITROGES. Amide nitrogen was determined by ACIDCOMPOSITION O F COTTONSEED GLOBULIN adding solid magnesium oxide to an aliquot of an acid hyPREPARATIONS (IN PER CENT) drolyzate and distilling the released ammonia. The constant -Preparation-. amount determinable during the early stages of the hydrolyPrevious Analysis I I1 I11 sis was used as an estimate of the total dicarboxylic acids 11.5 11.65 13.6 13.5 (IS),14.9 (19) 2.9 3.2 (6)",3.8 (7)'' 2 . 8 3.2 present (17).
AhfINO
Amino Acid Arginine Histidine Lysine Cystine Methionine. Phenylalanine Tyrosine Tryptophan Serine Threonine Leucine Isoleucine Valine Dicarboxylic acids NHt-N) otr;;sm
6.4
1.1
2.5 8.1 3.6 1.3 2.9 2.9 8.5 2.2 6.1
6.4 1.1 3.2 7.9 3.5
1.35
2.9 2.9 9.0
2.4 6.1
4.9 1.2 1.7 9.1
4 2 6)" 4 0
1: 1 [8),'1.f ...
(7)O
(6)a, 0.8 ( 7 ) O
3.2 1.5 2.9 2.9 8.4 2.5 7.5
1 (l)b 187 ( b ) = 21.8 (7)5 Glycine 1:2, alanini4.5, ... 19.7 ... 20 proline 2.3 (f) - - -
17.5 16.8 I . .
Total 76.3 77.4 82.0 5 Calculated from N distribution data. b By isolation methods, e The value for leucine probably represents a combined leucine, Isoleucine, and valine fraotion.
et al. (S),it was found that approximately 10 per cent more phenylalanine was determinable after alkaline as compared with acid hydrolysis. The data in Table I1 were obtained from samples hydrolyzed by refluxing for 16 hours with 5 N sodium hydroxide. TYROSINE AND TRYPTOPHAN. The method used was that described by Lugg (10). Calibration curves for tyrosine and tryptophan a t 450 mp were determined. Maximum intensities developed after 90 seconds in the tyrosine and 20 seconds in the tryptophan determination. The protein preparations (300 mg.) were hydrolyzed with 4 ml. of 5 N sodium hydroxide for 16 hours a t 125" C. (oil bath). Five ml. of 5 N sulfuric acid were then added, and the solution was diluted to 25 ml. Aliquots were treated in the same manner as the control solutions. The transmittances were determined at the time of maximum color intensity, and the amounts of tyrosine and tryptophan were computed from the calibration curves. THREONINE AND SERINE. Hydrochloric acid hydrolysates containing the equivalent of 300 to 450 mg. of protein were used for the determination of threonine and serine by the method described by Shinn and Nicolet (11, 18). Periodic acid was employed to oxidize the two quantitatively to acetaldehyde and formaldehyde, respectively. Acetaldehyde was removed by passing carbon dioxide through the solution buffered a t pH 7.15, and absorbed by bisulfite solution in which it was determined iodometrically. The remaining solution was adjusted to pH 4.6, as suggested by Yoe and Reid (do), and the formaldehyde was precipitated with dimedon (5,5-dimethyl-l,3-~yclohexanedione)and weighed. With the exception of hydroxylysine, other amino acids are reported not to interfere3. LEUCINE,ISOLEUCINE, AKD VALINE. The procedures used were modifications by Block and Bolling of methods described in their book (,%')*. The a-hydroxy acids arising from the deamination of leucine, valine, and isoleucine were oxidized t o acetone (leucine and valine) and ethyl methyl ketone (isoleucine). Differences in the yields of acetone by oxidation with potassium dichromate and by potassium permanganate were used to calculate the relative amounts of leucine and valine. The spectral curves for the acetone and ethyl methyl ketone determinations (with salicylaldehyde) showed that the former had maximum absorption a t 470 mp and the latter a t 500 mp. Subsequent measurements were made a t these wave lengths. 8 Recently, cottonseed globulin was reported t o contain 0.23 per cent hydroxylysine [Van Slyke, D. D., Hiller, A,, and MacFadyen, D. A,, J . Biol. Chem., 141, 681 (1941)). 4 Detaile reoeived a8 a personal oommunioation from R. J.,Block.
Discussion The results obtained in the present investigation are compared in Table I1 with those available from previous reports. Methionine, threonine, isoleucine, and valine are recorded for the first time. More striking, perhaps, than any differences is the general conformity of results, except those obtained by the isolation methods used by Abderhalden and Rostoski (1). Comparing preparation I with preparation 11, possibly significant differences were noticeable only with the amide nitrogen, histidine, and methionine values. A slight hydrolysis of preparation I1 induced by the added exposure to alkali might explain the loss in amide nitrogen. The other two determinations were each run in triplicate, and the differences between the two preparations were greater than errors inherent in the method. Preparation 111 apparently differed only slightly in analysis from the other two. Obviously some slight fractionation had taken place. Preparations I and I1 probably contained traces of glutelins not present in preparation 111. All the essential amino acids were present in amounts in excess of 1 per cent. Preliminary growth experiments designed to indicate the nutritive value of these preparations showed that they were complete proteins but that they did not permit as rapid growth as did the unfractionated cottonseed meal when compared a t the same protein level. Osborne and Mendel (16) first demonstrated the high nutritive values of isolated cottonseed globulin.
Literature Cited (1) Abderhalden, E., and Rostoski, O., 2. physiol. Chem., 44, 265 (1905). (2) Block,'R. J:, and Bolling, D., "Determination of Amino Acids", 2nd ed., Minneapolis, Burgess Publishing Go., 1941. (3) Block, R. J., Jervis, G. A., Bolling, D., and Webb, M., J . B i d . Chem., 134,567 (1940). (4) Folin, O., and Marenzi, A. D., Ibid., 83,89 (1929). (5) Friedmann, W.G.,Ibid., 51, 17 (1922). (6) Jervis, G. A.. Block, R. J., Bolling, D., and Kanze, E.,Zbid.. 134, 105 (1940). (7) Jones, D. B., and Csonka, F. A., Ibid., 64,673 (1925). (8) Jones, D.B., Gersdorff, C. E. F., and Moeller, O., Ibid., 62, 183 (1924). (9) Kassell; H.,and Brand, E.,Ibid., 125, 145 (1938). (10) Lugg, J. W. H., Biochem. J., 31, 1422 (1937);32,775 (1938). (11) Martin, A. J. P., and Synge, R. L. M., Ibid., 35,294 (1941). (12) Nickerson, R.F., U.S. Patent 2,194,835(1940). (13) Olcott, H.S.,Ibid., 2,194,867(1940). (14) Olcott, H. S., and Fontaine, T. D., J . Am. Chem. Soc., 61, 2037 (1939); 62, 1334 (1940). (15) Osborne, T.B., Leavenworth, C. S., and Brantlecht, C . A.,Am. J . Physiol., 23, 180 (1908). (16) Osborne, T. B., and Mendel, L. B., J . Bid. Chem., 29, 289 (1917). (17) Schmidt, C. L. A., "Chemistry of Amino Acids and Proteins", p. 204,Springfield, Ill., Charles C. Thomas, 1940. (18) . . Shinn. L. A.. and Nicolet, B. H.. J . Bid. Chem., 138,91 (1940): 139, 687 (1941). (19) Viokery, H.B., Ibid., 132,325 (1940). (20) Yoe, J. H.,and Reid, L. C., IND.ENG.CHEM.,ANAL.ED., 13, 238 (1941). CONTRIBUTION No. 433 from the Department of Chemistry. Univeraity of Pittsburgh, and No. 25 from t h e Multiple Fellowship of the Cotton Research Foundation, Mellon Institute. The material in this paper is t o be used for a dissertation by T. a.Fontaine in partial fulfillment of the requirementa for the degree of doctor of philosophy in the Graduate School of the University of Pittsburgh.
January, 1942
INDUSTRIAL A N D ENGINEERING CHEMISTRY
119
THE STUDIOUS ALCHEMIST Artist Unknown
No. 133 in the Berolaheimer Series of Alcheg&al and Historical Reproductions comes to us from Mr. Arthur L i e , who presented the original to the undersigned. It has been cleaned, revarnished, and framed, and presented to The Chemists’ Club, New York, and hangs there in Room B. Absolutely nothing is known concerning this painting. It is 1 5 l / ~by 21 inches in size and is on a plywood panel, hence must be modern. It may be that this is a copy from an older original unknown to us. It has the general appearance of German work. Any one having a copy of a picture similar to this will confer a favor by communicating details to the address as below. D. D. BEROLZFIEIMER 50 East 4 l s t Street New York, N. Y.
BEROLZHEIMER ALCHEMICAL AND HISTORICAL REPRODUCTIONS
THESE prints of famous paintings and engravings were started in the August, 1931, issue, and appear monthly thereafter in our volumes for the subsequent years. Orders for photographic prints 8 by 10 inches at $1.50, and 16 by 20 inches at $4, specifying the numbers here indicated and titles as given, should be sent with advance payment to D. D. Berolzheimer, 50 East 41st St., New York, N. Y. These photographs are not carried in stock, but are ordered only on receipt of remittance. Purchase of these photographs does not confer any rights of publication of these reproductions. Special arrangements must be made with Mr. Berolzheimer to obtain such rights. Prints and enlargements can be supplied in black and white only; velvet, matt, or glossy finish. Prints in color cannot be supplied, nor of any size smaller than 8 by 10 inches. An additional reproduction will be published each month. The list of reproductions published during 1941 follows. The figures in parentheses following the name of the artist denote the pages on which the respective reproductions will be found. A list of the first 96 reproductions was published in the January, 1939, issue, page 124, and of Nos. 97 to 120 in the January, 1941, issue, page 114. 1941 121. 122. 123. 124. 125. 126.
Le Chimiste, Mieris (114) Chinese Alchemist, Ts’ao (256) The Alchemist, Webb (374) Chymischer Goldt Narr, Weigel (549) Suitland Alchemist, Artist Unknown (581) Goldmacher Narr, Weigel (786)
127. 128. 129. 130. 131. 132.
De Scheider, Luyken (943) Two Alchemists, Artist Unknown (1048) The Alchemist, After Teniers (1153) Alchymist, Wijck (1330) Distillation, Stradano (1439) The Sorcerer, Forestier (1528)
