Detmmber, 1944
INDUSTRIAL AND ENGINEERING CHEMISTRY
tion, in order t o lessen the superheating caused by a layer of insoluble material that formed on the evaporator. For further tests on this method of solubilisation, samples of bark from salt-water-floated logs, fresh-water-floated logs, and bark peeled in the woods were obtained. Sulfited extracts were prepared from each sample. From the fresh-water-floated and woods-peeled bark, untreated. extracts were prepared for comparison. Liquors obtained by leaching were divided; half was sulfited and the other half reduced to extract without treatment. The analyses of these materials are given in Table 111. All of the spent barks contained approximately the same amount of tannin in these experiments. The insolubles in an aqueous mixture containing 15% of extract from bark peeled in the woods amounted to 8%. Sulfiting reduced this to less than 1%. A similar mixture prepared from fresh-water-floated bark contained 13% insolubles, which was reduced to less than I % by the sulfite treatment. The sulfite treatment was equally effective in solubilizing tannin extract from salt-water-floated bark, samples of which contained 20 to 40y0insolubles without treatment (Table I). The purity of the sulfited extracts was comparable to that of
1149
woods-peeled-bark extracts. The addition of lactic or acetic acid to eolutions of sulfited extracts did not cause precipitation. ACKNOWLEDGMENT
The authors are indebted to Ray Hatch, Weyerhaeuser Timber Company, for providing the pictures and the bark; to t h e Worthihgton Pump and Machinery Corporation for the use of Stacom horn-angle hydraulic press; to M. J. Stacom, Worthington Pump and Machinery Corporation, for operating the press, and Charles H. Binkley of this Laboratory, for processing the bark; to Stephen N. Wyckoff, U. S. Forest Service, for permission to use the map showing the movement and concentration of hemlock logs in pulp manufacture. LITERATURE CITED
(1) Aasoc. of 05cial Agr. Chem., 05cial and Tentative Method8 of Analysis, 5th ed., 1940. (2) Smoot, C. C., and Frey, R. W., U. S. Dept. Agr., Tech. BUZZ. 566 (1937).
(3) Wilson, J. A,, “Modern Practice in Leather Manufacture”. New York, Reinhold Pub. Corp.. 1941.
Gel-Forming Derivative of WHEAT GLUTEN W
HEN wheat gluten is A product obtained from wheat gluten by t h e action of the individual factors inreacted with & l o r e chlorosulfonic acid and pyridine or cold concentrated volved were varied singly. sulfuric acid possesses, after neutralization, t h e property sulfonic acid in pyridine or CHLOROSU LFONIC ACIDPYRIDINE PROCESS with cold concentrated SUIof absorbing rapidly one hundred t o three hundred times i t s weight of cold water t o form a firm, odorless, tasteless, The sulfating agent used acid, a product can be recovered which, a f t e r and nontoxic gel. The Use Of gluten sulfate as a substitute in one procedure ( 6 ) was pre&utralization, possessee an for scarce natural gums In therapeutio jellies, ointments, pared by the dropwise add* and other pharmaceutical Preparations, and as a thickention of 666 ml. (10 moles) unusual and useful property. Upon addition of cold water, ing agent O r emulsifying agent in ice Cream O r other of technical chlorosulfonic foods, I S suggested. The material has already been Used acid t o 3800 ml. (47moles) of this material absorbs almost instantly from one hundred in surgery for t h e absorption Of Postoperative drainage. cold anhydrous technical pyto three hundred times its ridine. The temperature was kept below 10’ C. by cooling weight of water and forms a firm gel. Methods of preparation and some properties of the and effective mechanical stirring. Ventilation was provided to remove the hydrogen chloride evolved. To the reaction mixture sulfated product are described here. were added 454 grams of vacuum-dried wheat gluten. To obtain The only previously reported introduction of sulfate groups into proteins was carried out by Hatano (S),who treated chloroa uniform suspension, it was convenient t o add portions of t h e gluten to 200-500 ml. aliquot8 of the reaction mixture in a Waring form-pyridine suspensions of gelatin and casein with chloroBlendor, the speed of which was reduced by variable resistance. sulfonic acid dissolved in chloroform. The sulfur content of the The homogeneous suspensions were combined in a covered Pyrex (gelatin was incrcased from 0.35 to s.7795,that of the casein from 2-gallon jar, heated with stirring at 75” C. for 2 hours, and poured 0.62 t o 1.37%. I n the present investigation the sulfur content of into 16 liters of a mixture of equal volumes of 1 Nsodium hydroxwheat gluten W&B increased from 0.84 t o as high as 10%. Sulfuric ide and methanol. This mixture wa8 allowed t o stand until groups werc introduced into gluten by two methods. I n the first, the active reagent was the reaction product of chlorosulfonic settling had taken place. The supernatant liquor was siphoned off and the residue washed by decantation, first with B mixture acid and pyridine (6); in the second, cold concent,rated sulfuric consisting of 4 liters of 0.25 N sodium hydroxide and 12 liters of acid was used. The procedures that gave optimal yields and gelforming properties were developed after numerous trials in which methanol, and then with It3 liters of 75% methanol. The residue
H E N R Y C. REITZ, R O E E R T E. F E R R E L , A N D H A R O L D S. OLCOTT W E S T E R N R E Q I O N A L R E B E A R C H L A B O R A T O R Y . U. S. D E P A R T M E N T OF A O R I C U L T U R E . A L B A N Y . C A L I F .
INDUSTRIAL AND ENGINEERING CHEMISTRY
1150
was then suspended in 6 liters of 50% methanol, and 1 N sodium hydroxide was added slowly to pH 7.5. About 340 ml. were required. The product was purified by repeated suspension in 50% methanol and removal of the excess solrent in a solid-howl Fletcher centrifuge. Warhing WM oontinued until the filtered wash solution gave only a Blight turbidity with barium chloride. Whenever, during the washing procedure, the gel failed to sepsrate easily, it was washed once with 80% methsnol and then with 50% methanol. The product was dehydrated with acetone, filtered with suction, and dried at room temperature in vacuum. The 227 %ramsrceavered reoresented a yield of 50% based OD the weight of gluten used. ~
for large-scale
prepamtione a commercial vmuumdried wheat gluten was used ("gum" gluten from Kcever Starch Company). Other commercial undenatured glutens were equally satisfactory, as were vacuum-dried gluten snmples prepsred in t h e laboratory. Denatured glutens. especially those which had been subjected to e n c c s s i v e heat during drying, gava inferior products. Figura 1. Both Beaker and Vial With one commerContain 4Cram~ofGlutsnSulfate; cial "devitsliied" Gel It Firm Enough to Hold the gluten, .P eatisfaoClass Rod in an Oblique Position tory product was obtained only after the material had been ground to pass a 200;mesh screen.
Products with gelling properties oould be obtsined by tlie u8e of higher ratios of ehlarosulfonic acid to pyridine, hut they were more difficult to handle. For example, B product with gelling properties was obtained by the use of a 1:2 molar ratio of acid and pyridine, but the reaction product was 80 tough and rubbery that stirring was impossible and the yields were low because of the difficulty in purifying the pmduct. Technical pyridine a p peared to scme equally as well as the reagent grade, hut unless the pyridine was anhydrous, the yield. were markedly reduced. Triethylamine, tri-n-hutylamine, triumylamine. dimethylsniline, ures, formamide, acetamide, and mixtures of the latter three were tried as paasihle substitutes for pyridine. Only with triethylamine was B product with gelling properties obtsined, and this was inferior to those prepared with pyridine. When the reaction mixture was heated for shorter periods than 2 hours, the gel properties were not optimal; with longer periods of heating, the yields tended to be low. SULFURIC ACID PROCESS
T w o liters of reagentgrade concentrated sulfuric acid were placed in B 2-gBUan Pyrex jar and coaled to -2" C. in BD i o 4 t bath; 100 -8 of "gum" gluten wepe then added with thorough etirring. To prevent lumping, the gluten was dusted into the acid through a 10-mesh metal screen. After the mixture had been stirred for an hour, the owlmg bath was removed and
Vol. 36. No. 12
stirring continued until the mixture reached m m temperature (23' CJ, usually ahout 3 hours. The reaction product was then poured slowly into a jar containing about 25 pounds of cracked ice. The mixture was consinuously stirred to prevent local heating. Water was added to make a volume of about 5 gallons. The insoluble msterial was separated by filtering through acidresistant (glass or Vinyon) cloth, washed once by suspension in shout 3 liters of water, and filtered again. It was found that the filtering and washing should he osrried through as rapidly ea pwible; when the acid product was sllowed to stand for 12 hours in aqueous suspension, it was not possible to separate the 6nnl neutralized product by centrifugstion. To secure B grnn&r product that oould be suspended in water without lumping, the moist solid was treated in smell portions with 3 to 4 litera of acetone in a Waring Blendor NU at low speed. The combined acetone suspensions were allowed to stand overnight, and the supernatant acetone was siphoned off. Five gallons of water wcre then slowly added, with stirring, to the residue. The sodium snit was prepared hy bringing the suspension to pH 7.3-7.5 with 0.5 N sodium hydroxide. It was necessary to add the alkdi slowly with efficient stirring in order to avoid the destructive effect of high alkdi concentration. Any s m d l u m p pmcnt were removed hy pouring the suspension through B funnel-shaped wire screen and grinding the lumps in B mortar. The thick aqueous suspension of the sodium gluten sulfate was washed with distilled water in a solid-howl centrifuge until free of inorgsnio sulfates. Washing with aqueous organic solvents instead of water wea somewhat leas effective hut reduced the amount of ncetone required for dehydrsting the final product. T h e wsshed gel was dehydrated with acetone, filtered. snd dried in vacuum at either room temperature or 60' C. The yield of dry salt was ahout 50% based on the weight of gluten uaed. One preparation made with technical grade sulfuric ncid wag found, hy spectroscopic analysis. to contain 1.3% of lead. DETERMINATION OF GEL VOLUME
Products made by both methods swell in water to form pein insoluble in water. The volume of gel in millititem obtainable from 1 gram of product is arbitrarily designated the "hydration capacity". This wea determined in the following manner: 50 mg. of the air-dried preparation were weighed into a 25-d. Erlenmeyer flask, 10 ml. of distilled water were added, and the mixture was allowed to stand for 2 hours. The contents of the flask were then traderred to 8 12-ml. graduated centrifuge tube and centrifuged at 4oM) r.p.m. for 5 minutes in s six-place sngle head ccntdfuge. Most of the samples of sulfated gluten varied in hydration capacity within the range of 100 to 300. USK OF OTHER PROTEINS
When the glutenin component of gluten wasaulfated by either of the pracedwres describal above, the product consistently showed higher hydration capacity than did that obtained from gluten alone. Gliadin, on the other hand. yielded only wster-soluble products. These observatioas suggest that glutenin b the fnction of gluten chiefly responsible for the gelling properties of gluten aulfste. The protein obtained from feathers by auljide dispersion and acid preeipitation (4) was found to yield a gel-forming product. T h e most satisfactory pmedure involved heating B mixture of the protein with the pyridine-chlorosulfonic acid resetion product an a steam bath for 3 hours and pouring the reaction mixture into n-propanol. The product was washed twice more with the same solvent. This material showed no tendency to gel upon neutralization; but when the produet wea suapended in water, brought to pH IO, and hested in a boiling water bath for 5 minutes, it formed a atiff gel, with a simultaneous drop in pH to approximately 7. This material wea further washed and dried
1151
INDUSTRIAL A N D ENGINEERING CHEMISTRY
December, 1944
by the same methods used for gluten. Inferior producta were obtained from this protein by the sulfuric acid proms. Among other proteins subjected t o the sulfation procedures were soybean protein, casein, cottonseed protein, peanut protein, egg white, hoof meal, and zein. The derivatives obtained were wholly or largely soluble in water. Only the product from egg white treated by the sulfuric acid process showed any appreciable tendency toward gel formation. PROPERTIES AND USES
Table I contains comparative analytical data for a gluten derivative prepared with chlorosulfonic acid and pyridine, one prepared with concentrated sulfuric acid, and the original glutan. The total number of acid groups ($3) were proportional t o the amount of sulfur introduced. Sodium gluten sulfate prepared by either of the two methods is a cream colored solid which is not hygroscopic but, when brought in contact with cold water, absorbs immediately one hundred t o three hundred times its weight of water t o form an odorless, tasteless, and almost water-clear gel (Figure 1). The ammonium, potassium, and lithium salts of gluten sulfate are similar t o the sodium salt in properties, but the calcium salt appears t o have a slightly lower hydration capacity. The barium, aluminum, and ferric salts exhibit no tendency t o form gels. It has not been found possible t o dissolve sodium gluten sulfate by any means that permits subsequent recovery of the gel. Within the p H range of 3 t o 10, less than 3% of the nitrogen was soluble in aqueous solution. The gels were liquefied by trypsin, but the results for pepsin and papain were inconclusive aa a result of wide variation in their action on individual preparations. When dry gluten sulfate waa treated in an autoclave (120" C.) for 15 minutes, its gelling property waa reduced. However, heating the gel at 80' for 30 minutes or at 100" for 5 minutes appeared t o have no effect.
TABLE^ I. COMPARISON OF PROPEIRTIES OF GLUTEN SULFATE
PRJCPARED BY Two PROCESSES
Determination N (dry basis). % 8 (dry basis), % H dration capecity %% groups I (per 104 g. protein, SI
Chlorwulfopic Acid-Pyndme Process 8.6 10.0
130
6.8 89
Sulfuric
Acid
Process 16.0 3.7
172
6.6
14
Originat Gluten 14.8 0.8
... ... 6
Figure 2 shows the influence of various concentrations of sodium chloride on gel volume. Other electrolytes of the same ionic strength have about the same effect as sodium chloride, although multivalent cations tend more than the monovalent cations t o reduce the gel volume. The fact that sodium gluten sulfate possesses a strong negative charge waa readily demonstrable. During electrodialysis, the gel migrated rapidly toward the positive electrode. A suspension of the electrodialyaed product had a pH of 3.5. The marked effect of salts on gel volume made it necessary t o increase the gluten sulfate concentration from 0.5 t o 4,5% t o secure firm gels in physiological saline solution. Nonelectrolytes such as glucose and urea, even in solutions of one molar concentration, had no effect on gel volume. Practical uses for a substance having the unusual properties of gluten sulfate will be obvious to workers in various fields. Tests at this Laboratory on experimental animals have failed to'show toxic effects from oral administration or subcutaneous injection; nor was it possible t o demonstrate antigenic properties. It haa been used aa an absorbing agent for body fluids in cystostomies and other cases involving postoperative drainage. The dressings required changing in these cases only about half as often as when no gluten sulfate was used. When used in surgery it has been
OQmENTRATlDll OP
Figure 2.
N a a , PEROENT
Effeeot of Sodium Chloride on Gel Volume of Gluten Sulfate
combined with sulfa drugs, since no means of sterilization without destruction of gelling properties has yet been found. Gluten sulfate is suggested as a vehicle and suspending agent in pharmaceutical preparations, and aa a base for therapeutic jellies and ointments. It haa been shown t o be superior to tragacanth and karaya gums as an emulsifying agent for mineral oil, olive oil, and cottonseed oil. The method of Merrill (6)waa used in making comparisons of emulsion stability. Gluten sulfate preparations made by the chlorosulfonic acid-pyridine process appeared t o give smoother and more stable emulsions than did those by the sulfuric acid process. Since the gels are not affected by freezing, the use of gluten sulfate aa an emulsifying or thickening agent in ice cream, as well as in other foods, seems possible. CHEMICAL STRUCTURE
Baumgarten, Marggraff, and Dammann ( 1 ) treated amino acids, peptides, and diketopiperazine wlth ethyl chlorosulfonate in dilute alkali and in pyridine. I n the experiments with amino acids and peptides, sulfates and sulfamates were formed with hydroxyl and amino groups, respectively. The disulfamate of diketopiperazine could be synthesized but only in a nonaqueous medium. For the sulfated proteins discussed in this paper, proof of the positions of the added Sulfate or sulfamate groups is being sought. Coverage of the free amino and hydroxyl groups could not account for more than a fraction of the total amount of sulfur introduced. For example, in the derivatives containing 10% of sulfur, one sulfate group would be present for every two or three amino acid residues. It thus appears that reaction with amide or p e p tide nitrogen or both may occur. ACKNOWLEDGMENT
The authors wish t o thank H. Fraenkel-Conrat and M. Cooper for the determination of total acid groups, C. B. Jones and D. K. Mecham for the modified feather protein, L. M. White for analytical data, and H. L. Fevold and R. H. Wilson for antigenicity and toxicity tests. They are indebted to J. W. Brand. M.D. for making available his clinical results. LITERATURE CITED
(I) Baumgarten,MarggrsfI, and Dammann, 2. phgswl. Chem., 209, 146 (1932). (2) Fraenkei-Conrat and Cooper, J . Biol. C h . ,154,239 (1944). (3) Hatano, BCooohem. Z.,145, 182 (1924). (4) Jones and Mecham, Arch. Bwchem.,2,209 (1943). (6) Merrill, IND.ENG.CHEM., ANAL.ED., 15,743 (1943). (6) Reits, U. 8.Patent 2,344,267(1944).
