Labile Sulfur The Use of Thallous Nitrate HUGO ZAHND, ROSLYN ALFIN, AND MILTON SCHNEIDER Brooklyn College, Brooklyn, N. Y.
THE
similarity in chemical behavior of lead and thallous salts suggested t h e possible use of thallous nitrate for the qualitative and quantitative determination of labile sulfur in proteins.
TABLE I. LABILESULFUR IN BIOLOGICAL SUBSTANCES Weight of Substance
Cysteine.HC1
32.9 24.5 25.0
Weight of Bas04 Mg. 46.3 34.1 35.1
cystines
20.6 20.6
30.9 30.9
Substance
Me.
Early investi ators [Fleitmann (7), Kruger (8), Osborne (19), and Morner ( 1 4 ] found that all the sulfur of the proteins could not be liberated as sulfide sulfur on boiling the samples with strong alkali and that the values obtained varied within wide limits, depending upon experimental conditions and the protein under investigation. Morner (12),describing a method for the determination of bleischwarzender Schwefel (lead-blackening sulfur), compared the values obtained by the methods of Fleitmann (?'), Malerba (9), Middledorf ( I f ) , Schulz ( 1 4 , and Osborne (IS). Maxwell, Bischoff, and Blatherwick (10)reported a micromethod for the determination of labile and total sulfur in proteins. Sheppard and Hudson (15) developed a colorimetric method for the determination of labile sulfur in gelatin and proteins depending on the blue color produced with p-aminodimethylaniline in the presence of ferric chloride. Zahnd and Clarke (16) devised a method for the quantitative determination of labile sulfur under conditions suitable for the estimation of small amounts of cystine in cystine solutions and protein hydrolyzates. I n a recent paper, Blumenthal and Clarke ( 3 ) described a modification of the alkaline plumbite method.
casein ( pure)
~& ~ i
~~
, 34.1 31.8 33.0
1000 1000
44.4 44.2
Fibrin (Eimer & Amend, pure)
1000 1100 1330
34.3 38.2 47.5
Pepsin (Eimer
1000 1000 1000
18.8 18.8 19.0
1000 1000 1000
28.8 30.8 29.0
''
Amend,
'')
Trypsin (Eimer & Amend, pure)
a
sulfur-containing proteins gave a precipitate of thallous suifide. This qualitative test proved t o be a t least as sensitive if not more so t h a n the conventional plumbite method. It was next decided t o study t h e use of thallous nitrate i n an alkaline medium for the quantitative determination of labile sulfur and to compare this action with t h a t of the plumbite procedure. ~ ~ ~ ~an ~ d i ~ was~ undertaken l ~ , investigation
d
Egg albumin (Merck impalpable powder)
'
I n preliminary qualitative tests i n which t h e alkaline plumbite method was replaced b y an alkaline solution of thallous nitrate, i t was found t h a t solutions of cysteine, cystine, and
~ ~5000~~ 5000 5000
Weight of cystine in each case is 20.6 mg. in 5 00. of
Labile S
%
95.09 93.98 94.69 AV. 94.59 77.80 77.80 .4v. 77.80 0.094 0.087 0.091 Av. 0.091 0.610 0.607 Av. 0.609 0.471 0.476 0.490 Av. 0.479 0.258 0.258 0.261 Av. 0.259 0.395 0.423 0.398 Av. 0.405 stock solution.
centrifu ing and it according to the method of Zahnd and Clarke $16). After complete oxidation, when all the sulfur is dissolved, gradually warm the mixture on a water bath until there is no further evolution of chlorine. Then cool the solution and add a 20 per cent solution of sodium hydroxide in slight excess of the amount necessary to precipitate the reddish-brown thallic oxide. Separate the thallic oxide by centrifuging and transfer the supernatant liquid t o a small beaker. I n this process, most of the excess thallic ion is removed in order to prevent its interfering seriously with the subsequent precipitation of the sulfate as barium sulfate. Adjust the pH of the solution to the optimum for barium sulfate recipitation, the final volume of the solution being about 100 CC. Frecipitate the sulfate quantitatively with barium chloride, a t e r it off, wash, and ignite. The results are very inconsistent and much above the expected recovery. Treat the ash with 4 to 5 drops of concentrated sulfuric acid, heat the whole very carefully on an air bath, and subsequently heat to constant weight. A distinct increase in weight is noted. Wash the ash with small quantities of hot water, using a porcelain spatula to break up the small particles of barium sulfate, filter, and ignite. A loss in weight is noted. After repeating the sulfuric acid treatment, no further gain or loss in weight is observed. Follow the same procedure, substituting solutions of cystine for the cysteine hydrochloride, 5 cc. of the cystine solution being equivalent to 20 mg. of cystine. The amount of protein used in the determinations is dependent upon the per cent of labile sulfur expected (Table I). To the dried sample add 50 cc. of a 20 per cent solution of sodium hydroxide and heat until solution of the protein takes place. Add 10 cc. of a 0.4 N solution of thallous nitrate, and reflux the sample for known intervals of tirne in order to determine the rate at which the thallous sulfide is formed and to ensure maximum recovery. The rest of the procedure is identical with that followed for cysteine and cystine.
t o determine whether the thallous ion would quantitatively remove the labile sulfur from cystine and cysteine, and to compare the rate of removal of the sulfur from these two amino acids using both the thallous and plumbite procedures. Furthermore, several proteins were investigated with respect to the rate of formation of thallous sulfide b y the action of thallous nitrate in a strongly alkaline medium. In the of the work, a technique was developed for a practical working method when using thallous nitrate.
Experimental To 2 cc. of a 20 per cent soluQUALITATIVE DETERMINATION. tion of sodium hydroxide, add a small amount of the substance to be examined. Heat to boiling, then add 3 drops of a saturated solution of thallous nitrate. Labile sulfur is indicated by the darkening of the solution or the appearance of a black color on the surface of the suspended material. If the substance upon examination is suspected to be low in labile sulfur, permit the solution or suspension to stand for several minutes, as the appearance of the black color may develop slowly.
In addition to the proteins investigated quantitatively, blood albumin, edestin, globulin, and zein gave positive results when subjected t o this qualitative examination. QUANTITATIVE DETERMINATIOX. Dissolve small quantities of cysteine hydrochloride, ranging from 23.1 to 32.9 mg., in 5 CC. of a 1N hydrochloric acid solution, add 50 cc. of a 20 per cent solution of sodium hydroxide and 10 cc. of a 0.4 N solution of thallous nitrate, and reflux the solution for known intervals of time. At the end of these periods separate the thallous sulfide formed by
I n Table I are indicated the maximum labile sulfur values obtained for cysteine, cystine, and commercial grades Of casein, egg albumin, fibrin, Pepsin, and trypsin. 44
ANALYTICAL EDITION
January 15, 1941
I n Table I1 are reported the rates of formation of the labile sulfur. Each value represents the average of three individual determinations. In Figure 1 the action of thallous nitrate on cysteine hydrochloride and on cystine is compared with that of lead nitrate. The concentrations of the lead nitrate and alkali are identical with that used in the case of the thallous nitrate procedure. Each point plotted represents the average value of three determinations.
4s
TABLE11. ACTIONOF ALKALINESOLUTION OF THALLOUS NITRATE ON BIOLOGICAL SUBSTANCES Substance
Refluxing Houras 2 4
Cysteine. HC1
8 12 24 1 2 3 5 8 12
Cystine
Discussion The qualitative procedure described for labile sulfur proved to be a t least as sensitive as the conventional lead method. Furthermore, it is felt that sufficient quantitative results of good reproducibility have been obtained to permit the use of the new quantitative technique reported. An examination of Figure 1 indicates that under the same experimental conditions the action of thallous and lead ions on cystine and cysteine is essentially the same. Cystine yields but three fourths of its sulfur as labile sulfur, whereas practically all of the sulfur of cysteine can be recovered in this form. These observations are in agreement with the results of Clarke and Inouye (5). Bergmann and Stather ( 2 ) as well as Brand and Sandburg (4) found that the lability of the sulfur atom towards alkali differs enormously in the different compounds of cystine. Furthermore, Abel and Geiling (1) and du Vigneaud (6) showed that when cystine is a constitutent of the insulin molecule, the ease of lability is enhanced considerably as compared with cystine itself. An examination of the results reported in Table I1 indicates this increased sulfur lability of the cystine and cysteine sulfur when these are structural units of the protein molecule.
r r 00-
0
TI-CYSTEINE-HCI
0
Pb CYSTElNE * HCI
0
Pb-CYSTINE TI-CYSTINE
Casein (Eimer & Amend, pure)
1
2 3 6 E g g albumin (Merck, impalpable 0.5 powder) 1 4 6.5 Fibrin (Eimer & Amend, pure) 1 3 6 Pepsin (Eimer & Amend, U. S. P.) 2 4 6 Trypsin (Eimer & Amend, pure) 2 4 6 a Does not include time necessary to dissolve samples.
Labile S
% 52.75 68.21 84.38 89.10 94.59 40.92 63.91 71.10 73,89 77.55 77.80 0.075 0,083 0.089 0.091 0.483 0.608 0.596 0.609 0.479 0.474 0.467 0.224 0.259 0.255 0.355 0.391 0.405
Conclusions 1. Methods for the qualitative and quantitative determination of labile sulfur in biological substances have been devised. 2. The action of lead and thallous ions in alkaline medium on cystine or cysteine is essentially the same, cystine yielding but three fourths of its sulfur, whereas cysteine yields all of its sulfur in the labile form. 3. The labile sulfur values as determined by the thallous nitrate procedure of casein (Eimer & Amend pure), egg albumin (Merck, impalpable powder), fibrin (Eimer & Amend, pure), pepsin (Eimer &Amend, U. S. P.), and trypsin (Eimer & Amend, pure) are, respectively, 0.091, 0.609, 0.479, 0.259, and 0.405 per cent.
Acknowledgment Some years ago, the possibility of using thallous nitrate for the qualitative and quantitative estimation of labile sulfur was sumested to the senior author bv H. T. Clarke of the Collegecbf Physicians and Surgeons, “Columbia University, New York, N. Y.
Literature Cited 4
12 I6 TIME (HOURS)
8
20
24
FIGURE 1. ACTIONOF THALLOUS AND LEADNITRATE
Finally, the labile sulfur values of the proteins were obtained by the direct action on the commercial grades, as indicated in Tables I and 11, rather than on the crystalline products. As such, the reported labile sulfur values of the proteins are primarily important in that they are illustrative of the reproducibility of the results. Nevertheless the values for casein and fibrin are in close agreement with the values reported by Zahnd and Clarke (16). The value for egg albumin is much higher and must be ascribed to the differences in the commercial product examined (16). The values for pepsin and trypsin could not be found in the literature.
(1) Abel, J. J., and Geiling, E. M. K., J . Phurmcol., 25, 425-48 (1925). (2) Bergmann, M., and Stather, F., 2. physiol. Chem., 152, 189201 (1926). (3) Blumenthal, D., and Clarke, H. T., J . Biol. Chem. 110, 343-9 (1935). (4) Brand, E., and Sandburg, M., Ibid., 70, 381-95 (1926). (5) Clarke, H. T., and Inouye, J. M., Ibid., 89, 399-419 (1930). (6) du Vigneaud, V., Ibid., 75, 393-405 (1927). (7) Fleitmann, T., Ann., 61, 121-6 (1847); 66, 380-1 (1848). (8) Kriiger, A., Pfliioers Archiv. ges. Physiol. 43, 244-64 (1888). (9) Malerba, P., Rend. accad. sn’. Napoli, [ 2 ]8, 59-62 (1894). (10) Maxwell, L. C., Bischoff, F., and Blatherwick, N. R., J. Biol. Chem.. 72. 51-6 (1927). (11) Middledorf,’ E., V e r h a h l . Phys. med. Gesellschaft, Wiirzburg, N. F. 31 Bd., No. 9,393-422 (1897). (12) Morner, K. A. H., 2. physiol. Chem., 34, 207-338 (1901). (13) Osborne, T. B., Ann. Rept. Conn. Agr. Expt. Sta., No. 24,443-71 (1900) * (14) Schuls, F. N., 2. physiol. Chem., 25, 16-35 (1898). (15) Sheppard, 5. E., and Hudson, J. H., IND. ENG.CHEW,Anal. Ed., 2,73-5 (1930). (16) Zahnd, H., and Clarke, H. T., J . Biol.Chem., 102, 171-86 (1933)
