E A R L F . P O T T E R AND C H A S E B R E E S E JO N E S
W estern R e g io n a l R esearch L ab orato ry , B u re au o f A g r ic u ltu r a l C h e m istry a n d E n g in e e rin g , A lb a n y , C a lif.
D
URING a study by the junior author of the dispersion of certain keratins in solutions of sodium sulfide, it became desirable to determine separately the protein sulfur, sulfide sulfur, and, as a third fraction, other sulfur compounds. A review of the literature failed to provide a method suitable for such a separation. Theis and Ricker (9) studied the inorganic sulfur compounds produced during prolonged action of alkaline sulfide solutions on cowhide, but they reported nowork on the protein sulfur.
Several methods for obtaining the desired separation were tried, including treatment of the dispersion with mineral acids, with lead acetate, and with trichloroacetic acid.
Treatment of the dispersion with mineral acids would re
lease hydrogen sulfide from sulfides present, but this treat
ment would also release sulfur dioxide from sulfites and bisul
fites and, if the acid were sufficiently concentrated, sulfur from thiosulfates. The acid concentration required to pre
cipitate the protein was sufficiently high to release sulfur dioxide from sulfites and precipitate sulfur from thiosulfates.
Precipitation of the dispersion with normal lead acetate, filtering, and washing with hot ammonium acetate and then with hot water separated the other sulfur compounds from the protein and lead sulfide formed but provided no means
■of separating the two latter groups.
• The protein dispersion is colloidal, and mineral salts are effective in flocculating the disperse phase. As aluminum salts are very effective in flocculation (6) a water suspension of basic aluminum acetate was found to precipitate the pro
tein present and also to liberate hydrogen sulfide from the sodium sulfide present. The use of basic aluminum acetate was also found to be applicable to the separation of sulfides from sulfites, thiosulfates, and sulfates.
Three dispersions were analyzed by the method discussed in this paper.
The protein materials used were chicken feathers, hog hair, and wool. The sulfur contents of these materials, based upon their air-dry weight, were: chicken feathers, 2.3 per cent; hog hair, 5.6 per cent; and wool, 3.6 per cent. The keratin dispersions were prepared by a modification of the method described by Goddard and Michaelis (4). These dispersions contained about 7 per cent of the protein material in 0.1 M sodium sulfide. Total sulfur was determined in the protein materials by an alkaline permanganate fusion (8). The official magnesium nitrate igni
tion {2) and the alkaline permanganate fusion (8) were used on the precipitated proteins, and the two methods gave the same results. Magnesium nitrate did not wet the chicken feathers, and so was not used for the determination of total sulfur in this material.
The basic aluminum acetate method was also tried on solutions of sodium sulfide, both by itself and mixed with some other sulfur salts, to determine whether it would be successful in separating sulfide sulfur from other sulfur compounds. Also, sodium sulfide and other sulfur salts were added to the feather dispersion and the sulfide sulfur was separated by the aluminum acetate.
Reagents
Basic aluminum acetate, suspension of 5 grams in 100 ml. of water.
Lead acetate, basic, dry powder, a 1 per cent solution, as for sugar analysis by the Horne method.
Bromine-hydrobromic acid, equal volumes of 48 per cent hydrobromic acid and bromine.
Magnesium nitrate, dissolve 150 grams of magnesium oxide in nitric acid (1 + .1), avoiding an excess of acid. Add a little magnesium oxide in excess, filter from excess of magnesium oxide, iron oxide, etc., and dilute to 1 liter (i).
Potassium permanganate, a saturated solution.
Sodium hydroxide, a 15 per cent solution; also solid.
Nitrogen, compressed.
Hydrochloric acid, concentrated and diluted 1 to 1 with water.
Sodium carbonate, solid, anhydrous.
Methyl orange, water solution, 0.5 gram per liter.
Barium chloride, a 10 per cent solution.
16 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol. 15, No. 1 Carbon disulfide, analytical reagent.
iV-Octadecyl alcohol, a saturated solution in 95 per cent ethyl alcohol.
Ammonium hydroxide, concentrated.
Procedure
A 2-ml. sample (weighed) of the keratin dispersion was washed into a flat-bottomed vial (D. Figure 1) with the minimum amount of water necessary and 5 ml. o f n-octadecyl alcohol reagent were added to minimize foaming. Basic aluminum acetate suspension was added, the mixture was heated, and the hydrogen sulfide evolved was swept with nitrogen into a basic lead acetate solu
tion. This reaction was carried out in the apparatus shown in Figure 1.
Two milliliters of the basic aluminum acetate were drawn into the pointed tube, C, a clamp at A was tightened and another at B loosened, the T-tube was connected, and C was lowered into the solution in vial D. Nitrogen was then turned on so that 3 or 4 bubbles a second passed through the water in bottle II. Con
nections were completed with test tubes F and G; vial D was lowered into boiling water in the 400-ml. Griffin beaker, E; the clamp at A was loosened and that at B tightened, whereupon the stream of nitrogen drove the basic aluminum acetate into the dispersion, precipitating the protein and evolving hydrogen sulfide from sulfides present. The hydrogen sulfide was carried into the basic lead acetate solution in F, precipitating lead sul
fide. Tube G also contained basic lead acetate and was used only as a precaution. All the hydrogen sulfide was driven off in 5 to 10 minutes and further heating was avoided, since it caused the protein to stick to vial D. The stopper containing tube C was removed from D while the nitrogen was still bubbling through it to prevent protein material from collecting inside the tube.
The protein was broken up with a rubber-tipped stirring rod, transferred to filter paper in a Biichner funnel, and washed thor
oughly with hot water. The final volume of the filtrate was usually about 250 to 300 ml. The protein was then ignited with about 15 ml. of magnesium nitrate solution or with 10 ml. of permanganate solution plus 1 ml. of 15 per cent sodium hydroxide.
The alkaline permanganate fusion was made at 600 ° C. instead of at 500° C. as in the original method (S), but after fusion the mixture was treated as directed by Pollock and Partansky (8).
The magnesium nitrate ignition mixture was analyzed by the A. O. A. C. method (2). After the ignition mixture was dissolved, the solution was made just acid to methyl orange by the proper use of ammonium hydroxide and hydrochloric acid. About 0.5 ml. of hydrochloric acid per 100 ml. of solution was then
added and the solution was heated to boiling. Two milliliters of the barium chloride solution were then slowly added from a pipet, with constant stirring of the solution. The solutions were kept at about 40° C. overnight, filtered through weighed Gooch crucibles, and the precipitated barium sulfate was washed with
hot water. The barium sulfate was then ignited at a cherry red heat in a muffle furnace (about 650° C.) to constant weight, cooled for an hour in a desiccator, and weighed.
The filtrate from the precipitated protein contained the other sulfur compounds. To it were added about 5 grams of sodium hydroxide and 5 ml. of bromine water. The solution was kept alkaline (3). It was heated on a steam bath for an hour, acidified with hydrochloric acid (1 + 1), and boiled to expel bromine, and the sulfur was determined as before.
The sulfide sulfur was determined by dissolving the lead sulfide in tube F (Figure 1) in 3 ml. of bromine-h^drobromic acid mix
ture, heating gently until all bromine was driven off, then cooling and neutralizing with sodium carbonate. Tall-form beakers were used to avoid loss by effervescence. An excess of about 1 gram of sodium carbonate was added and the solution was boiled for about 15 minutes. Lead carbonate w'as precipitated and sodium sulfate remained in solution. The lead carbonate was filtered off and freed from sodium sulfate by washing. The filtrate was then made slightly acid with hydrochloric acid (1 + 1) and the sulfur was determined as before.
The amount of basic aluminum acetate necessary to precipitate the protein and drive off the hydrogen sulfide was determined by treating the dispersion with measured quantities of basic alu
minum acetate, boiling until the hydrogen sulfide was all evolved, and testing the solution with a drop of 10 per cent normal ace
tate. The time necessary to remove the hydrogen sulfide was also determined by testing at intervals with lead acetate paper.
Care was taken to have all rubber and glass connections per
fectly dry before the operation was started, so that no hydrogen sulfide would be dissolved in the water present.
Preliminary determinations of the sulfur content of methionine and cystine were made by the official magnesium nitrate method and the alkaline permanganate fusion method.
The results, given in Table I, show that a fusion temperature of 600° C. must be used with the alkaline permanganate fusion method to obtain the true amount of sulfur in methio
nine. The results obtained by this method compared favor
ably with those obtained by the magnesium nitrate ignition.
R esults a n d Discussion
Three sodium sulfide dispersions of keratins were analyzed for total sulfur by fusion and the three fractions of sulfur compounds separated from each dispersion by the use of basic aluminum acetate were also analyzed for sulfur. The analyses were made in duplicate or triplicate on freshly pre
pared dispersions. Analyses were also made of dispersions
(Comparison
Ta b l e I.
of
De t e r m i n a t i o n o f Su l f u r
magnesium nitrate method and alkaline permanganate method)
method 500° C. 600° C. theoretical
% % % %
Feather dispersion, 2.3 2.1 1.1 5 .5 5.3
freshly prepared 2.4 1.9 1.1 5.4 5.5
Feather dispersion,
stood 1 m onth 1.8 None • 3 .6 5 .4
Hog hair dispersion, 4.1 1.5 0.7 6.3 6.1
freshly prepared 4.4 1.3 0.7 6.4 6.1
4.1 1.4 0.6 6.1
H og hair dispersion, 4 .2 None 2 .2 6.4
stood 15 days 4.1 None 2.2 6.3
W ool dispersion, freshly 3.2 1.5 0 .6 5 .3 5.6
prepared 3.4 1.7 0 .5 5 .6 5.5
that had stood in the laboratory for some time, of these analyses are given in Table II.
The results
To test for the possible formation of elemental sulfur in the protein fraction upon standing, the protein was separated from duplicate samples of a hog hair dispersion that had stood in the laboratory for 15 days. One protein fraction was analyzed as usual; the other was washed twice with boiling carbon disulfide, after which it was dried and analyzed for sulfur. The results were nearly identical, showing that no sulfur had been removed from the second protein fraction; moreover, the residue from the carbon disulfide used in washing contained no sulfur.
The pH of a freshly prepared feather dispersion was 12.0.
One milliliter of this material was diluted with 5 ml. of water (resulting pH 10.6) and boiled with 2 ml. of basic aluminum acetate (pH 4.5) until all the hydrogen sulfide was driven off.
The solution was then cooled and again tested for pH, which was found to be 4.5.
Mixtures of the feather dispersion and various sulfur com
pounds were analyzed by the basic aluminum acetate method as described. Recovery of the added sulfur was complete and the amounts of protein sulfur and sulfide sulfur in the dis
persion were not affected. When sodium sulfide was added to the dispersion, the protein sulfur remained unchanged, the sulfide sulfur increased by the amount present in the sodium sulfide, and the other sulfur fraction also increased by the amount present in the added salt. When solutions of sodium sulfite, sodium thiosulfate, and potassium sulfate were added to the protein dispersion, analyses showed that the protein and sulfide sulfur of the dispersion remained unchanged, the sulfur of the added salt being included in the remaining sulfur of the dispersion. Sodium bisulfite, when added to the dis
persion, did not behave like the other added salts; an increase was observed in both the protein and sulfide sulfur fractions.
Results of the analyses of mixtures of the protein dispersions with added sulfur compounds are given in Table III.
The sulfur distribution of a keratin dispersion in sodium ulfide solution is probably the result of an equilibrium be- een several reactions involving the inorganic sulfide ion
and the disulfide sulfur of the protein. Part of the original sulfide is oxidized to disulfide or to a polysulfide during reduction of the cystine residues of the keratin U). Another reaction that would also result in a decrease of the sulfide-ion concentration is that described by Nicolet and Shinn (7).
In this reaction a part of the original sulfide sulfur is incor
porated into the protein molecule by addition to the double bonds formed by the action of alkali on the hydroxy amino acid residues of the original protein. On the other hand, it is possible that the final sulfide sulfur might include that part of the cystine sulfur which appears as inorganic sulfide during the conversion of cystine to lanthionine—a conversion which, as Horn and Jones (5) have shown, occurs upon treatment of proteins with sodium sulfide.
It is evident from the foregoing discussion that no con
clusion can be drawn from the data in Table I I as to the sources of the sulfur in the three fractions. Indeed, this problem lies outside the scope of the present paper. How
ever, it is hoped that the method of separation and deter
mination of the sulfur fractions described here may be a useful tool (with cystine analyses, for example) in studies of the reactions involved in the dispersion of keratins by sodium sulfide and of the action of alkali on the sulfur of proteins.
T a b l e I I I . B a s i c A l u m i n u m A c e t a t e S e p a r a t i o n o f S u l f u r i n K e r a t i n D i s p e r s i o n s P l u s A d d e d S u l f u r C o m p o u n d s
Protein Sulfide Other T otal
Sulfur Sulfur Sulfur Sum S u lfu r“
M g. M g. Mg. M g. M g.
Protein dispersion (1 gram) 2 .3 2.1 1.1 5.5 5.4
2.4 1.9 1.1 5.4 5 .5
Sodium sulfide (1 ml.) None 2 .2 1.4 3 .6 3 .4
None 2 .3 1.4 3 .7 3 .6
Protein dispersion (1 gram) 2 .3 4 .2 2 .4 8 .9 9 .4 + sodium sulfide (1 ml.) 2.4 4.1 2 .5 9 .0 9 .2
Protein dispersion (1 gram) 2 .5 1.9 7 .7 12.1 11.9
+ sodium thiosulfate (1
° A lkaline perm anganate fusion method.
S u m m a r y o f Results
The sulfur in sodium sulfide dispersions of keratins may be separated into three parts: protein sulfur, sulfide sulfur, and other sulfur compounds by means of basic aluminum acetate.
Sulfides may be separated from sulfites, thiosulfates, and sulfates, but not from bisulfites, by treatment with basic 190-1, New York, John Wiley & Sons, 1927.
(7) Nicolet, B. H., and Shinn, L. A., J . Am. Chem. Soc., 63, 2284