ISDC;STRIAL A S D ESGISEERING CHE-VISTRY
January 15, 1930
i3
Determination of Labile Sulfur in Gelatin and Proteins’ S. E. Sheppard a n d J. H. Hudson EASTXAX KODAKRESEARCHLABORATORIES, ROCHESTER,N. Y .
T
HE nature of the combination of sulfur in proteins, and
These particulars are given to indicate that two arbitrary indeed in living tissues in general, has become of increas- definitions of labile sulfur are made-namely, (1:1sulfur sepaing importance since the discoveries of glutathione and rated as hydrogen sulfide by heating for 30 minutes at 98” C. insulin ( I , 8). The position prior to these discoveries has in 0.1 9sodium carbonate and ( 2 ) sulfur separated as hydrogen sulfide by heating for b e e n f u l l y discussed by Johnson ( I O ) who points out 30 minutes at 98” C. in 10 in summation that “it apper cent potassium hydrosThe definition a n d estimation of labile sulfur i n pears very probable * * * ide. The lahile sulfur values proteins are discussed. A method for determining that there are other sulfur are espressed as percentages very small quantities of labile sulfur, defined by combiof the total sulfur. On heatcombinations in proteins benation with silver i n the presence of ammonia, is deside the cystine group which ing proteins with alkaline scribed. Applications of this method i n t h e analysis can break down in hydrolylead acetate, more sulfur i q of protein sulfur are considered. ,is with formation of hydroseparated as lead sulfide Sen sulfide.” As an example than would be separated, 8John.on sugeeded the DOSin the same time, as hydrosible existence of tliiopolypeptides, containing a gen sulfide by the second of theqe procedures. On the other hand, the amounts of sulfur fixed as lead sulfide from -SH,CH2.Ccystine itself, as described in the literature, vary from about 50 to 83 per cent of the total sulfur present in cystine (3). S Determinations of sulfur separated as lead sulfide from a progroupiiig \ , l l ) . W t h the discoveries of glutathione and in- tein or similar material, therefore, can give no reliable value sulin, the question of the lability of sulfur in proteins became for cystine sulfur therein, even supposing no other sources of acute (1). Before this, Brand and Sandberg (.5) drew atten- labile sulfur were present, because the lability of the cystine tion to the fact that, although the sulfur in cystine itself is sulfur varies with the mode of its binding in peptide form and fairly stable (to alkalies), the linkage of cystine with other because the determination of labile sulfur in cystine itself is amino acids in peptides makes the sulfur much more labile. subject to unknown sources of variation. Thus, under the same conditions of boiling with 0.1 11’ sodium * A given sample of protein material, more specifically of carbonate for 45 minutes, cystine gave off only 2.8 per cent of gelatin, may contain sulfur in a t least, the following forms: its total sulfur, whereas dialanylcystine gave 18.6 per cent -S-(0) (as in sulfates); -SH (as in mercaptans) ; -S-Sand dialanylcystine anhydride 91.8 per cent. (as in disulfides); =S (as in thioamides or isothiocyanates); A lability of sulfur much greater than that of cystine is and =S (as in sulfides). It is evident that a single detershown by many proteins, and is no evidence that the sulfur mination of labile sulfur under one set of conditions, together is not present in cystine combination (.5). with a determination of total sulfur, cannot give definite The term “labile sulfur” is arbitrary. A differentiation evidence as to the amount of cystine or other source of labile was made early in protein chemistry between loosely and sulfur in the material. Separate determinations of the diffirmly bound sulfur (G,l a ) , and again between unoxidized and ferent groups present in the mixture are necessary before oxidized sulfur, although these two differentiations cannot deductions as t o the nature of the components furnishing the be regarded as synonymous. Reaction with alkaline lead sulfur can be accepted. acetate (lead plumbite), giving lead sulfide, was taken as The cystine fraction giving labile sulfur is complicated bedistinguishing labile sulfur, the total sulfur being determined, cause of the mobility of the cystine: cysteine system. for example, by oxidat,ion and estimation as Sod. The recogc y
I
nition of degrees of reactivity of the labile sulfur led to a definition by Maxwell, Bischoff, and Blatherwick (13) of labile sulfur as the amount given off under the following conditions: Principle. The sample in weak alkali is heated in a current of nitrogen for a specified time, The solution is then acidified, and the liberated hydrogen sulfide oxidized, and the sulfur estimated a s barium sulfate with the nephelometer, Procedure. The solution containing from 0.1 t o 0.2 mg. sulfur is placed in a test tube, and t h e air displaced by a slow current of nitrogen. An equal volume of 0.2 N Na2C03, or 20 per cent NaOH, is added through a dropping funnel t o give a concentration of 0.1 iV sodium carbonate, or 10 per cent sodium hydroxide, depending upon the type of labile sulfur t o be determined. A slow current of nitrogen is bubbled through the solution while it is being heated on a boiling water bath * * * for 30 minutes. 1 Received October 18, 1929. Presented before t h e Division of Leather and Gelatin Chemistry a t t h e 78th Meeting of t h e American Chemical Society, Minneapolis, M i n n , September 9 t o 13, 1929.
R-S R-S
1
2RSH
+ 2H = 2RSH
+0
(RS)z 4-HzO
The reduced cystine, or cysteine, does not react for labile sulfur as does cystine. Hence the labile sulfur determination depends upon the r H (15) as well as upon pH. But this again affects the actual amount of sulfide ions, or hydrogen sulfide, as these readily undergo irreversible oxidations. All operations, therefore, must be carried out anaerobically, and preferably in a current of purified nitrogen. In advance of a more complete investigation of methods for separately determining the labile sulfur component, a method has been developed which lends itself to (1) determination of total sulfur in proteins in small quantities (2) determination of a given labile sulfur figure, namely, the sulfur forming silver sulfide under given fised conditions, and (3) micro-
A NB L Y TICA L ED1 T I 0 S
74
chemical determination of sulfide ion, respectively, of hydrogen sulfide, produced by any previous reactions. The principle of the method consists in decomposing the sulfur-containing body with an ammoniacal solution of a silver salt, usually silver chloride, to form silver sulfide. The silver sulfide can later be quantitatively decomposed with concentrated aqueous hydrochloric acid to form hydrogen sulfide. This hydrogen sulfide is then removed by aeration with nitrogen and estimated as methylene blue according to the method of Mecklenburg and Rosenkrtinzer (Id), as modified by Almy ( 2 ) .
Figure 1-Apparatus
for Determining Sulfur in Gelatins and Proteins
Apparatus
The apparatus (Figure 1) provides for the aeration of several samples of gelatin simultaneously. The aerating gas. which may be either nitrogen or carbon dioxide, is washed by passing it through a 1 per cent solution of silver nitrate in bottle F . This removes traces of hydrogen sulfide which may be present in the gas. If oxygen be present, wash bottles, containing pyro or hydroquinone in strong alkaline solution, are used. I n our experience hydrogen sulfide did not appear t o be present in either of the aerating gases recommended. The first 100-cc. graduate ( E ) contains mercury. This acts as a safety valve and prevents damage to the apparatus, should all the stopcocks be closed. The 100-cc. graduates (D) can be filled with either water or a heavier-than-water solution. They act both as safety valves and rough manometers. The desired flow of gas through any unit of the apparatus can be determined by displacing water in an aspirator bottle connected to the outlet of C, and maintained b y adjusting the height of the solution in the graduate D to counterbalance the desired pressure. The pressure should then be slightly increased so that the excess bubbles out through D. The sample tube (A) with ground connections is blown from Pyrex or hard glass. The tube measures 19.685 cm. by 3.81 cm. inside. The funnel ( B ) is blown to contain 50 cc. The absorbing bottle (C), similar t o F , is known as the Xlligan gas washing bottle. Owing to the spiral, the bubbles make 8 successive revolutions before reaching the upper liquid. The bottle is about 22.86 cm. high with an inside diameter of 5.08 em.
Vol. 2,
KO.
1
Analytical Procedure
Cut 5 grams of gelatin into pieces not over 0.635 cm. long, and place them in tube A . Add 25 cc. of 1 per cent silver chloride solution, prepared by dissolving the silver chloride in strong ammonium hydroxide, nnd let the gelatin swell for 1 hour. Effect solution by heating the tube in a beaker of water at about 50” C., regulating the gas flame under the beaker so that the temperature gradually increases. Continue the heating of the tube for about 2 hours. As too rapid evolution of the ammonia will cause loss of the sample by foaming, it is necessary to start heating a t the temperature stated. Shake the sample tube a t regular intervals to break the skin which forms on top of the solution. A t the end of about 2 hours the sample should be well blackened, owing to the formation of silver sulfide, silver oxide, and metallic silver. Evaporate the volume to 10 cc. or less. Cool the tube and connect it to the aerating train. Grease all ground joints and use rubber bands, 8.89 em. long and 1.27 em. wide, to hold the greased joints together when the apparatus is under pressure. Charge the absorbing bottle (C) with 130 cc, of 1 per cent zinc acetate and 5 cc. of 10 per cent sodium hydroxide. Turn on the aerating gas for about 5 minutes and sweep the apparatus free of air. ?Tow stop the gas temporarily by turning the stopcock nearest to the sample tube, and allow 50 CC. of special hydrochloric acid to run in through the funnel. Stop the flow in time to prevent the last few drops from leaving the bulb. If the acid does not flow fast enough, a gentle suction on the outlet of C will remedy this Allow the aerating gas to pass through the apparatus for about 1 hour. The amount of aerating gas passed does not appear to be the important consideration, but rather the time it takes to decompose all the silver sulfide. This time varies with the amount of silver oxide and silver formed, and this, in turn, varies n-ith the grade of gelatin analyzed. I n 1 hour of aerating time, passing about 3 liters of gas, enough hydrochloric acid gas will be carried over to nearly neutralize the zinc hydroxide which is present in C. Disconnect C from the apparatus and treat its contents with 25 cc. of diamine reagent. Mix this reagent and solution of the remaining zinc hydroxide by a rapid whirling of the bottle, after which add 5 cc. of 0.02 mol ferric chloride and mix. The blue color develops at once and is proportional (after a 2-hour reaction) to the amount of hydrogen sulfide present. Transfer the blue solution to a 250-cc. volumetric flask. When the hydrogen sulfide amounts to 0.0001 gram or more, a blue color, which can be matched very readily with standards after 2 hours, is obtained. Quantities of hydrogen sulfide less than 0.0001 gram have a tendency to allow a greenish tinge to predominate in the solutions after standing overnight. As the standard colors are prepared in advance and kept for several weeks, it appears necessary to allow the colors of the unknown of low hydrogen sulfide content to stand overnight before matching v d h the standards. Preparation of Color Standards
The standards may be prepared by dissolving c. P . sodium sulfide in distilled water. After standing several hours, it is analyzed by iodoinetric methods. When the concentration has thus been found, dilute a portion with boiled and cooled distilled water so that each cubic centimeter contains 0.0001 gram of sulfur. I n preparing the standard colors, place 130 cc. of zinc acetate solution and 5 cc. of 10 per cent sodium hydroxide in a 250-02. volumetric flask. Pipet in the requisite amount of standard sodium sulfide solution, followed immediately by 25 cc. of diamine reagent and 5 cc. of ferric chloride solution. dfter the colors are developed, dilute the
I S D CSTRIAL A N D EXGINEERING CHEMISTRY
January 1.5, 1930
solutions to the mark. The colors may be compared in a colorimeter or in Nessler tubes, but the standard used should not be over 100 per cent stronger than the unknown under analysis. The following solutions and reagents are necessary: (1) 0.02 mol ferric chloride hexahydrate in 4 per cent hydrochloric acid; ( 2 ) diamine reagent, prepared by dissolving 0.1 gram of p-aminodimethylaniline sulfate (Eastman Kodak Company No. 1333) in 100 cc. of hydrochloric acid (1 : 1); (3) 10 per cent sodium hydroxide; (4) special hydrochloric acid, prepared by dissolving 0.5 gram of hydroquinone in 100 cc. of concentrated hydrochloric acid. This hydroquinone is necessary, as the c. P. hydrochloric acid contains hypochlorous acid, or other oxidizer, which oxidizes some of the hydrogen sulfide. I n place of sodium sulfide, which requires troublesome protection against oxidation, a solution of allylthiourea can be employed with advantage. This is checked by gravimetric determination of silver sulfide on treatment with the ammoniacal silver chloride solution. The procedure described applies to a single direct determination of “labile to silver” sulfur in gelatin or a protein. There are indications that this figure is frequently lower than the corresponding figure for “labile to lead” sulfur, which may be largely due to the difference in hydrolytic efficiency of the ammonia and the caustic alkali solutions. I n the case of egg albumin, however, the value 0.49 per cent was obtained, agreeing well with Osborne’s ( 1 7 ) determination. Determination of total sulfur in a protein by the method described depends upon its combination with Muellen’s (16) procedure. This consists in heating the protein in a current of hydrogen and absorbing the hydrogen sulfide. I n the present procedure, the hydrogen sulfide is absorbed by zinc acetate and estimated as already described. Similarly, for the sulfide produced by hydrolysis in the absence of lead or silver, the hydrogen sulfide is discharged by acid and absorbed by zinc acetate. The hydrolysis of cystine, or of cystine containing proteins, to give sulfide ions, in the absence of lead or silver, is complicated by the secondary reaction of the sulfide with cystine itself, whereby cystine is reduced to cysteine, R-S R-S
I
+ Hk3 = 2R.SH f S
where R stands for HOOC.CH.1\’H2.CH,--. Hence, measures of labile sulfur by alkaline reaction have a n approximate character. I t appears that this secondary reaction may vitiate the conclusions drawn by Treadwell (19) from his study of the alkaline hydrolysis of egg albumin, in which the sulfide ion present a t any stage of hydrolysis was determined electrometrically. The exact reactions involved in the hydrolysis of cystine itself are not known (8). I n the presence of lead, whereby the secondary reduction to cysteine should be prevented, the best results seem to be those of Andrews (S),which closely approach 75 per cent of the total sulfur in cystine. This would be attained if the reactions in hydrolysis were R-S
I
c w.n
= R
Evidence for the hydrolysis of elementary sulfur, particularly as separating in a nascent state, is given by Bassett (4). I n the presence of oxygen the yield would be lower and elementary sulfur separated. Folin and Marenzi’s ( 7 ) recent improvement of their phosphotungstate reagent, which can now be prepared free from
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phenol (tyrosine) reagent,, makes possible better direct determinations of cystine as cysteine, and we are applying this to the analysis of the total sulfur in proteins in conjunction with the procedure described. The reaction between sulfide ions and cystine, producing cysteine, is probably connected with the deliming of hides (18, SO)in lime baths, or in lime baths sharpened with sulfides. We have found, however, that hide which has been completely dehaired continues to give off sulfur, estimated as methylene blue via silver sulfide, to successive fresh lime baths, as illustrated by the results in Table I. T a b l e I-Sulfur
in S a t u r a t e d L i m e S o l u t i o n in W h i c h H i d e H a s Been Soaked
TIMP Hours 24 24 24 24 24 24 24 96
SULFUR IN SOLN. IN HID^ PH
12.1 12.1 12.1 11.8 11.8
11.8
11.8
..
STOCK
Grams per million parts 33.4 26.7 30.0 11.2 10.0 7.4 3.0 Trace
The hide which gave u p this sulfur to the lime treatments, however, gave off no hydrogen sulfide when decomposed with hydrochloric acid in the cold. Either the labile sulfur was not present in a cystine complex or the cystine was not hydrolyzed under the conditions operative. L i t e r a t u r e Cited Abeland Geiling, J , Pharmacol., 26, 423 (1915); Scienctr, 67, 169 (1925). Almy, J. A m . Chem. Sac., 47, 1381 (1925). Andrews, J . Bioi. Chem., 80, 191 (1928). Bassett and Durrant, J. Chem. SOC.,1401 (1927), Brand and Sandberg, J. B i d . Chem., 7 0 , 381 (1926). Fleitmann, Ann., 61, 121 (1847). Folin and Marenzi, J . Bioi. Chem., 83, 89 (1929). Gortner and Hoffman, J. A m . Chem. Sac., 44, 341 (1922). Hopkins, Biochcm. J., 16, 286 (1921). Johnson, J . Bid. Chem., 9, 439 (1911). Johnson, I b i d . , 9, 449 (1911). Kriiger, Pfluger’s Archiv., 45, 243. Maxwell, Bischoff, a n d Blatherwick, J. B i d . Chem., 7 2 , 51 (1927). Mecklenburg and Rosenkranzer, Z . anoyg. Chem., 86, 143 (1914). Michaelis, “Monographien Physiologie der Pflanzen u n d Tiere.” Vol. 17, p. 98, Springer, 1929. Muellen, Rcc. Iraw. chim., 41, 1 (1922). Osborne, J . Am. Chem. SOC.,‘24, 140 (1902). Sheppard, Eastman Rodak Co., Research Laboratory Monograph 3 (19231. Treadwell and Eppenberger, Helo. Chim. Acta, 11, 1035 (1928). Wilson, “Chemistry of Leather Manufacture,” p. 151, Chemical Catalog, 1923.
Wallboard to Soundproof Motion Picture Studios The motion picture industry is directing its attention toward the use of wallboard in an effort to solve the problem of sound insulation in making talking pictures, according to Axel H. Oxholm, director of the National Committee on Wood Utilization. Uses of wallboard have increased greatly in recent years, because of recognition of its effectiveness as an insulator, and many thousand square feet are also used for body parts in automobiles. Exports in 1927 totaled 45,908,731 square feet, valued a t $1,506,512, almost twice as much as the previous year. The largest factors in the increase of exports have been sound and temperature insulation and the ease with which the material is applied to practically any kind of frame wood. I t is estimated that between 75 and 80 per cent of wallboard is being produced by a mechanical process, 15 to 20 per cent by the sulfate or a carbonation process, and about 2 per cent by the sulfite process. A recently developed process makes it possible to use hogged wood waste or wood chips by reducing them to fiber by means of steam pressure. Today approximately 100,000 cords of wood waste are being utilized for pulp and paper products, but millions of cords of by-product wood and other fibers are still available for the manufacture of wallboard and similar products.
