January, 1933.
I ND 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
(8) Fieldner, -1.C., Davis, J. D., Thiessen, R., Kester, E:B., Selvig, W.A., Reynolds, D. A., Jung, F. W., and Sprunk, (3. C.. Ibid.. Tech. Paper 525 (1932). (9) Foxwell, G. E., Fuel, 3,229 (1924). (10) Hardgrove, R. M., Trans. Am. SOC.Mech. Eng., 54, 37 (1932). (11) Harkins, IT. B., and Ewing, D. T., J. A m . Chem. Soc., 43, 1790 (1921). (12) Howard, H. C., and Hulett, G. A,, J . Phys. Chem., 28, 1082 (1924). (13) Juettner, B., and Howard, H. C., Carnegie Inst. Tech.. Coal Research Lab., Contrib. 8 (1934). (14) Keppler. G., and Borchers, H., Brennsto.f-Chem., 15, 241 (1934). (15) King, J. G., andEdgeeombe, L. T., Fuel, 9, 213 (1930). (16) Lowry, H. H., I S D . ENG.CHEM., 26,320 (1934). (17) Martin, G., Inst. RubberInd. Trans., 2,125 (1926). (18) Mayers, M . A , . Chem. Rev.,14, 31 (1934).
7:
(19) Rau, O., paper presented before Sect. I11 of Intern. Congr. of Mining, Metallurgy, Applied Mechanics, and Practical Geology, Dusseldorf, June, 1910. (20) Shimmura, T . , and Xomura, H.. J . FueZ SOC.Japan, 11, 132 (1932). (21) Sladek, J., Brennstof-Chem., 15, 1 (1934). (22) Stadnikoff, G., “Chemie der Kohlen,” 1931. (23) Walther, R. V., Steinbrecher, H., and Bielenberg, W., Braunkohlenarch, 8,23 (1924) ; 9, 59 (1925). (24) Warren, W. B., IND. Eiw. CHEM.,Anal. Ed., 5 , 285 (1933). (25) Washburn, E. W., and Bunting, E. M., J . Am. Ceramic Soc., 5 , 55 (1922). RECEIVED October 1, 1934. Presented before t h e Division of Gas and Fuel Chemistry a t the 88th Meeting of t h e American Chemical Society, Cleveland, Ohio, September 10 to 14, 1934.
Effect of Certain Salts on Enzyme Activity Effect of Sodium Selenate, Selenite, Selenide, Tellurite, Sulfate, Sulfite, Sulfide, Arsenite, and Vanadate on Rate of Carbon Dioxide Production during Yeast Fermentation
w.FHANKE,South Dakota State College, Brookings, S. Dak.
ALVINL. MOXON. ~ N DKURT
I
S A FORMER publication
T h e foxicity of sodium salts of selenium, tunaEFFECTO F EQUALd h l O U N T S O F ( 7 ) it was shown that proVARIOUSSALTS dium, arsenic, and tellurium towards the proteins from toxic wheat duction of carbon dioxide during yeast fermentaF i g u r e s 1 a n d 2 s h o w the (laboratory So. 582) and toxic tion of glucose is in the order named. Sulfur effect of equivalent moles and corn (laboratory No, 523) gave equal w e i g h t s of s e l e n i u m , shows some acceleration, depending on the f o r m practically no increase to the rate tellurium, sulfur, arsenic, and of carbon d i o x i d e production used, and probably is in direct correlation with vanadium in the form of sodium during y e a s t f e r m e n t a t i o n , the hydrogen sulJide concentration. selenite, tellurite, sulfite, whereas the proteins from the The toxicity of the sodium salts of selenife, arsenite, and vanadate. The control wheat and control corn selenide, and selenate decreases i n the order curves indicate that the toxicity gave considerable acceleration. of the elements decreabed in the named. The collaborative work of the order: selenium, r a n a d i u m , Bureau of Chemistry and Soils An accelertrting effect is shown by sodium sularsenic, tellurium. Sulfur was of the United States Departfide and, to a lesser degree, by sulfite. The sulthe only one that produced acment of Agriculture with this fate shows a slight retarding Pffect. Sodium sulcelerating effect on the rate of laboratory resulted in the isolafide counieracts the toxic effects of selenium concarbon dioxide production. The tion of selenium from a sample variations between the curves for siderably. Elemental sulfur has no accelerating of toxic wheat (laboratory S o . each element are due mainly to 459) by Robinson (19). Later effect and only slightly counteracts the toxic the differences in concentrations Byers ( 2 ) reported the presence effects of selenium even in the ratio of 20 to 1. which are dependent upon of both selenium and vanadium Sodium sulJite, ammonium sulfate, and sodium a t o m i c w e i g h t s of t h e elein a s a m p l e of toxic w h e a t thiosulfate are also unable to counteract the toxic ments. (laboratory S o . 582), and seleeffect of selenium. nium, vanadium, arsenic, and S o l l m a n (21) s t a t e s t h a t chromium in the soil on which it “The actions of selenium are mas grown. Since the naturally occurring grains contained identical with those of tellurium and arsenic, but it is much belenium and vanadium, and since the protein of these in- more toxic than tellurium.” He refers to the work of Czapek fluenced the rate of carbon dioxide production during yeast and Weil in which they found that sodium selenite was fermentation, it n-as deemed advisable to determine the effect practically nontoxic for protozoa and ciliated cells, but that of chemically pure salts of these and related elements on it checked alcoholic and bacterial fermentation, being reyeast fermentation. duced t o the red insoluble metallic form. This work was The materials used were small cakes of Fleischmann’s later confirmed by Woodruff and Gies. yeast, hlerck’s c. P. anhydrous glucose, and c. P. reagent The severe inhibiting action of selenium as sodium selenite chemicals. (KaaSOs) is shown in curves B (Figures 1 and 2). Harden The apparatus employed has been described in detail in and Korris (8) state that sodium selenite a t a concentration a former publication (6). The fermentation was carried out of 0.5 per cent almost totally inhibits fermentation of glua t 28” C. for reasons discussed elsen.here ( 7 ) . cose by dried yeast (10 grams of yeast in 100 cc. of 10 per cent The concentrations of yeast and glucose were the same in glucose solution). They also found that the presence of a all experiments reported here. Each fermenting mixture fermentable sugar favors the reduction of sodium selenite by consisted of 0.5 gram of yeast and 1 gram of glucose made up living yeast but ha. little influence on the reducing power of to 20 cc. volume with distilled water. The salts, as men- zymin a t low concentrations. tioned in each experiment, were added to the basic mixture. Levine ( I S ) studied the influence of various selenium com-
I N D U S T R I A L A N D E N G I N E E K I N G C H E MI S T R Y
78
)......,
-----
"
FIGURE1. EFFECT OF EQUIVALENT MOLESOF VARIOUS SALTS RATEOF CARBONDIOXIDE PRODUCTION DURING FERMENTATION OF BASICMIXTURE Basic mixture consisted of 0,.5 gram of yeast and 1 gram of glucose made up to a volume of 20 c r . with distilled water. A . No salt B. 1.26 X 10-5 mole Se (NaiSeOr5HaO) C. I 26 X 10-5 mole S (Na&O?) D. 1126 X Id-5 mole Te (NazTeOa) E. 1.26 X 10-5 mole As (NanH.lsOs) F. 1.26 X IO-. mole V (NaVO )
A . Nosalt B. 1.0 mg. S e (NazSeOa) C.
1 . 0 mg. Se (NazSe)
D. 1.0 m g . S e (NazSeOs)
A. B. C.
D. E. F.
A. B.
0.00 mg. 0.025 mg. 0.05 mg. D. 0.1 mg. C.
E.
0.25 mg.
G.
1.0 mg. 2 . 0 mg.
F. 0.5 mg. H.
pounds upon growth and germination of plants and found that in concentrations of 0.01 per cent or over they were inimical to both growth and germination. Stange (22) found that a t the beginning of fermentation, reducible substances in moderate concentrations may have a stimulating effect (methylene blue) or an inhibiting effect (selenious acid).
,
mu
IUI
min
No salt 1 mg. Se (NazSeOa.5HzO) 1 mg. S (NazSOd 1 mg. T e (NazTeOs) 1 mg. As (NarH-4808) 1 mg. V (NaVOa)
The inhibiting action of vanadium as sodium metavanadate (SaV03) is shown by curves F and indicates that in this form it is more toxic than arsenic as disodium orthoarsenite (Na2HilsO3). In this work, in the comparison of the effect of sodium metavanadate and orthovanadate, it was found that the rate of carbon dioxide production in the presence of either of the two salts varied only by about 2 per cent, which could easily be due to slight experimental differences. Proescher and Sei1 ( 1 7 ) have carried out a series of studies on the toxicities of various vanadium salts. They found that vanadium in concentrations up to 5 per cent vanadium pentoxide (V206) did not interfere with yeast fermentation, but that concentrations above 5 per cent caused a decrease in the fermentation rate. They stated that the metavanadates and orthovanadates are among the most easily reducible as well a3 the most toxic of the vanadium compounds. They also found that 0.2 per cent of vanadium trioxide (V203)in the form of sodium tetravanadite completely inhibited the proteolytic activity of pepsin as well as the amylolytic activity of pancreatin. They concluded that, since vanadium does not precipitate albumin in neutral or slightly alkaline solutions, the enzymes must be directly affected. Ramirez (18) studied the effect of vanadium on plant growth, and his conclusionswere that this element may be absorbed and stored by plants and that they may show anomalies of growth as a result. Arena (1) found that vanadium salts are poisonous to plants, preventing the production of roots on cut stems of Alternanthera spathulata. The inhibiting action of arsenic (sodium orthoarsenite) as shown by curves E is in agreement with the work of Dresel (4). He states that yeast fermentation is inhibited by arsenous oxide (AsnO3). Smorodintzev and Riaboushinskii (20) found that 0.025 N solutions of disodium orthoarsenite (Sa:HAsOs), dipotassium orthoarsenite (K2HAs03), and disodium orthoarsenate (WasHAs04)retard peptic digestion of casein. Harden and Young (9) found that the presence of disodium orthoarsenate in a mixture of glucose, mannose, or fructose and yeast juice causes an immediate increase in the rate of fermentation. This rate increases for a short time, attains a maximum, and then decreases. They also reported that beyond the optimum concentration of arsenate (which varies with each sample of yeast juice) there is an inhibition of fermentation, and, in high enough concentrations of arsenate, fermentation may be completely inhibited. Tellurium in the form of sodium tellurite (NasTeOJ shows (curves D)very little inhibiting effect. Sulfur (0.4047 mg.) in the form of sodium sulfite (NatSOd
-
FIGURE 4. EFFECTOF VARIOUSAMOUNTSOF SELENIUM (AS NazSeOr5H20) ON RATEOF CARBON DIOXIDE PRODUCTION DUAIR'G FERXENTATION OF BASIC MIXTURE
I
so.1
FIGURE 2. EFFECT OF EQUAL WEIGHTS OF S.4LTS ON RATE OF CARBON DIOXIDE PRODUCTION DURING FERMEXTATIOS OF BASIC MIXTURE
ON
FIGURE 3. EFFECTOF EQUAL AMOUNTS OF VARIOUS SELENIUX SALTSON RATEOF CARBONDIOXIDE PRODUCTIOX DURING FERMENTATION OF BASIC MIXTURE
1nl
,
Vol. 2 7 ,
IN DUSTRIAL AND ENGINEER I
January, 1935
ZYO
yno
120
mtn.
FIGURE6. EFFECTOF ELEMENTAL SULFUR PLUSSELENIUM ON RATEOF CARBON DIOXIDE PRODUCTION DURING FERMENTATION OF BASIC MIXTURE
FIGURE5 . EFFECTOF
EQUIVALENT MOLESOF \-ARIOUS SULFUR SALTS ON RATEOF CARBON DIOXIDE PRODUCTION DURING FERMENTATION OF BASIC
MIXTURE
A.
A . X o salt B. 1.26 X 10-5 mole S (Ka:S) C. 1.26 X 10-6 mole S (NazSOs) D. 1.26 X 10-5 mole S (NazSOd
had practically no effect (curve C, Figure I), but rtccelerated the carbon dioxide production when the amoun't of sulfur was 1.0 mg. as shown in curve C, Figure 2 .
600
0
min.
7Y
CHEMISTRY
;I' G
N o salt
B. 1 mg. Se (NazSeOa) C
10 mg. S (elemental) 0'.1 mg. Se (NnzSeOa) E. 1 m g . Se (NazSeOs)
I
!
++ 20 10 m g . S (elemental) mg. S (elemental)
!
'
r
EFFECT OF VARIOUS SELENIUM SALTS Figure 3 4iows the effect of equal amounts of selenium as sodium Gelenate (Sa2SeOa), sodium selenite (NanSeOa), and sodium selenide iSa2Se). The selenate wa5 not reduced to metallic selenium and is not nearly as toxic as the selenite; both of theqe results are in agreement with the findings of other workers. The selenide is a n unstable compound when added to water. Some of the selenium is precipitated in the metallic form Therefore the concentration of soluble selenium is loner than in tlie other two solutions. Joachimoglu (11) found that the isolated frog heart had the power of reducing the selenium in sodium selenite but not in sodium selenate. Levine (14) states that sodium selenate is not reduced t o irietallic selenium by bacteria, and that sodium selenite retards growth, while sodium selenate has but a slight retarding effect. In his studies of the effect of selenium compounds upon growth and germination of plants, Levine ( I S ) arranged the compounds in the order of diminishing toxicity as follows : selenious acid, selenic acid, sodium selenite, sodium selenate, and potassium selenocyanide.
OF SODTGM SULFIDE PLUS SELENIUM FIGURE 7. EFFECT ON RATE OF CARBON DIOXIDE PRODUCTION DURING FERMENTATIOS OF B a s ~ cMIXTURE
A. N o salt B. 1 mg. S e (NazSeOa) C. 1 mg. S (Na2S)
D.
10 mg.
S (NazS)
E. 1 mg. Se (NazSeOa) F. 1 mg. Se (NazSeOa) G. 1 mg. Se (NazSeOs) H . 1 mg. Se (NazSeOa)
+ 51 mg. mg. S (NazS) S (NazS) +++ 10 mu.S (NazS) 15 mg. S (NazS)
EFFECT OF VARIOVS CONCENTRATIONS OF SODIUM SELENITE Figure 4 definitely shows the inhibiting action of selenium as sodium selenite in various concentrations. In all of the experiments where sodium selenite or tellurite was used, it was found that the yeast reduced the selenium or tellurium to the insoluble metallic form. The change in color of the fermenting mixture, owing to precipitation of red metallic selenium, was very noticeable after about 30 minutes in the case of the higher concentrations of selenium. The reduction of sodium selenite by living yeast cells appears to be an intracellular process, since it is impossible t o separate the reduced selenium from the yeast cells by repeated washing and centrifugalizing. dfter fermenting for 13 hours, the fermenting mixtures in Figure 4 appeared a. follows: P.p .
P. p.
m.
1.25 2.50 5.00 11,50
N o color N o color Slight tinge of pink DeEnite pink
rn.
25.00 50.00 100.00
Definite pinkish red Definjte red Definite red
OF AMMONIUM SULFATE PLUS SELEFIGURE 8. EFFECT NIUM ON RATEOF PRODUCTION OF C.4RBON DIOXIDE DURING FERMENTATION OF BASIC MIXTURE
A.
B.
$.
N o salt 1 mg. S [(NHa)zSOa) 1 m g . Se (NaxSeOa) 1 mg. S [(NHdzSO4 1 mg. Se (NazSeOa) 10 mg. S [(NH4)2SCh]
++
Living cells in general appear to possess the power of reducing selenium and tellurium of the -ite salts to the insoluble metallic form. Luchetti (16) reported that bacterial cells always reduce the -ite salts of selenium and tellurium. Levine (14) reported that, where there is no bacterial growth,
ISDUSTR I AL AN D EN G IN EER IN G C HE MI STR Y
80
Vol. 27, No. 1
TABLEI. SALTCONCENTRATIONS OF VARIOUSEXPERIMENTS FIQCRE
ELECURVE MENT .. A 0 B
C D
E F A B
C D
..
Se Se Se Se Se Se
Se
..
S E S
C D 1 B
..
Se
I
G
H A B C
..
Ee 8
[
S
11: ..
NazSOa
...
NanHrisOa
NaVOa
...
SanSeOa KanSe SazSeOa
...
NatSeOs NazSeOs NazSeOa NazSeOa NanSeOa NanSeOa NanSeOa
...
TOTAL MOLES OF
ELEMEXT
..........
1.262 1.262 1.262 1.262 1.262
X 10-5 X 10-6 X 10-5 X 10-6 X 10-6
..........
1 . 2 6 2 X 10-5 3.119 x 10-6 7 . 8 4 3 X 10-6 1.334 X 10-5 1.9607 X 10-6
10-6 10-6 10-6 10-5 10-5
..........
1.262 X 10-5
...
..........
.......... 1.262 x 10-5 .......... 1 . 2 6 2 X 10-5 .......... ..........
1.262 3.119 3.119 1.262 3.119 1.262 1.559 1.262 3.119 1.262 4.678
X 10-3
X 10-5 X 10-4 X 10-5 X 14-6 X 10-6 x 10-4 x 10-5 X 10-4 X 10-5 X 10-4
..........
....
1.0 1.0 10.0 1.0 1.0 1.0 5.0 1.0 10.0 1.0 15.0
....
1.0 1.0 1.0 1.0 10.0
there is no reduction, but, when the concentration of selenium is small, there may be growth without visible reduction. The lack of visible reduced selenium may be due to its removal by volatilization. With higher concentrations of selenium compounds the activity of reduction outbalances that of alkylation. Korsakov (12) found that fermentation of glucose solutions by living yeast was accelerated by sodium selenite in concentrations up to 0.5 per cent, which is contrary to the results reported in Figures 1 and 2. It was found (Figure 4, curve B ) that concentrations as low as 0.000125 per cent selenium (equivalent to a concentration of 0.000274 per cent sodium selenite) show distinct inhibition. The other curves of Figure 4 show that increased concentrations of selenium cause an increase of inhibitory effect. However, after a concentration of about 0.0025 to 0.005 per cent of selenium (sodium selenite) is reached, there appears to be no further reduction of the rate of fermentation with increased concentration of selenium, as it was found that 0.5 per cent of selenium gave practically the same rate of fermentation as 0.005 per cent. Preliminary studies indicate that a concentration of 0.005 per cent selenium (sodium selenite) completely destroys the fermenting power of cell-free yeast juice. The yeast juice lacks the ability to reduce the selenium t o the elemental form. Therefore, the power of the living cell to reduce the selenium probably protects the enzyme from complete damage in the case of yeast fermentation.
0.005 0.00202 0.00805 0.004733 0.0032
0,005 0.005
......
0.005 0,005 0.005
......
0,000125 0.00025 0.0005 0,00125 0.0025 0.005 0.01
0.4047 0.4047 0.4047
....
......
0.005
....
1.0 10 1.0 10 1.0 20.0
I N SOLN.
0.005 0.005
. . .
....
PERCENTOF ELEMENT
.....
,..
1.0 1.0 1.0 1.0 1.0
0.025 0.05 0.1 0.25 0.50 1.0 2 0
10-1 10-7
SanSeOa Elemental NanSeOa Elemental ?*ia?SeOa Elemental NazSeOa Na?S NanS NazSeOa NazS Nan Se 0s Na2E NarSeOa NazS Na zSe 0a NanS
1.0 0.4047 1.610 0.9465 0.6439
3:iii'x 6.310 X 1.262 X 3.155 X 6.310 X 1.262 X 2.524 x
1.262 X 10-6 1 . 2 6 2 X 10-5 1.262 x 10-5
...
....
1.0 1.0 1.0
NazS NazS03
Na?SOa . .
110. OF
ELEMENT
i:isz x '10-5 1.262 X 10-5 1.262 X 10-6
3.119 x 10-6 ("4)?SO4 NanSeOs 1.262 X 10-6 ("4)2S04 3.119 X 10-5 NanSeOs 1 . 2 6 2 X 10-6 D :e (NH4)ZSOI 3.119 x 10-4 Controls contain no salts. b Control = 100 per cent activity. S
{? {
a
S
{E
2C F
NazSeOa NanSOs Na2TeOa
Se
B
D E
Se S
Se
H A
E
NarTeOa Na2HAsOa NaVOs
Se
E F G
D
$@
..
D A B C D
C
ie Te
v
2C
...
NaiSeOa
Te A8
E F
SALT
......
0.00202 0.00202 0.00202
......
P.P. OF
.....
50.000 20.237 81.5 47.325 32.195
...... 50.0 50.0 50.0
.....
....
50.0 50.0 50.0
.....
1.25 2.50
5,oo 12.5 25.0 50.0 100.0
.....
20.237 20.237 20.237
.....
0:005.'
50.0
0.005
......
50.0
......
.....
0.05 0.005 0.075
......
0,005 0.005 0.005 0.005 0.05
....
2,000 2.000 2,000 2,000 2.000
50.0
0,005 0.005 0.05 0.005 0,005 0.005 0.025 0.005
....
2.000 0,809 3.220 1.893 1.288
50.0 50.0
0,005
......
M.
ELEM~N hfQ. T P E R GRAM SOLN YEAST
AV.
COZ
.....
..... .....
50.0 50.0 500.0 50.0 50.0
50.0 250.0 50.0 500,o 50.0 750.0
.....
50.0 50.0 50.0 50.0 500.0
2,000 2,000 2,000
....
0.050 0.100 0.200 0.500 1,000 2.000 4.000
....
0,809 0.809 0.809
....
2.000 20.000 2.000 20.000
I
4;:00:1
....
1,000 1,000 10.000
:::E)
1.000 5,000)
1;:
% ! I
1
1::;1
....
2,000
I::::;
220:
1
cc.
PER MIN.
IN
0.332 0.069 0.323 0.282 0.207 0.096 0.324 0.075 0.388 0.316 0.249 0.143 0.278 0.262 0.191 0.038 0.279 0.263 0.248 0.244 0.163 0.093 0.083 0.065 0.333 0.399 0.320 0.315 0.301 0.074 0.290 0.091
PER CENT ACTIVITY b 100.00 20.78 97.28 84.9 62.34 28.92
100.00 23.15 119.75 97.53
76,85
44.14 100.00 94.24 68.70 13.67 100.00 94.27 58.89 87.46 58.42 33.33 29.75 23.30 100.00 119.82 96.10 94.59 100.00 24.58 96.35 30.23
0.102
33.89
0.300
O.Oi4 0.362 0.296 0.209
100.00 34.67 120.67 98.67 69.67
0.242
80.67
0.257
84.67
0.240
80.00
0.287 0.072 0.074
100.00 25.09 25.78
0.085
29.62
The relative effects of different amounts of selenium (sodium selenite) are still evident, even when the amount of yeast is two and four times that used in the fermentations as reported in Figure 4.
EFFECT OF SULFUR SALTS Figure 5 shows the effect of three different sulfur compounds in the same concentration as the molar equivalent of selenium used in Figure 1. Sulfur in the form of sodium sulfide has an accelerating effect on the rate of carbon dioxide production as shown by curve B, Figure 5 . Curves C of Figures 2 and 5 show that sulfur in the form of sodium sulfite has a slight accelerating effect. (Curve C of Figure 1 started to go above the control curve but dropped back again.) One milligram of sulfur (sodium sulfite) was used in Figure 2 while only 0.4047 mg. was used in Figures 1 and 5, which accounts for the greater accelerating effect shown in Figure 2. Sulfur added in the form of sodium sulfate showed a slight inhibitory effect in a concentration of 0.4047 mg. of sulfur. Based on the color developed on moistened lead acetate paper inserted into the outlet tubes of the fermentation bottles, the amount of hydrogen sulfide produced was found to be in the following order: sodium sulfide, > sodium sulfite, > sodium sulfate (trace), and a slight trace in the case of the control. Since the rate of hydrogen sulfide production of the compound was in the same order as the accelerating effect, it seems plausible that the hydrogen sulfide x a s responsible for the increased activity.
January, 1935
INDUSTRIAL AND ENGINEERING
Chovrenko (3) found that hydrogenization of sulfur is brought about by all yeasts, which could account for the hydrogen sulfide in the case of sulfite and sulfate. Segelein (16),in his work on the effect of hydrogen sulfide upon respiration and fermentation of yeast, found that hydrogen sulfide in a concentration of 10- molar completely stops the respiration of fermenting yeast but has practically no effect upon the fermenting process. Euler and Nilsson ( 5 ) suspended a top yeast in 3 per cent sucrose solution, saturated the solution of hydrogen sulfide, and let it stand for 24 hours a t room temperature. The yeast was then centrifugalized off, and fermentation tests showed it to be about one-fourth as active as untreated yeast, in terms of both carbon dioxide production and oxygen consumption. Waldschmidt-Leitz and others (24) found that hydrogen sulfide accelerated the rate of reaction of papain, arginase, phosphotase, and catalase. EFFECTO F SULFUR AKD SELEKIUM Figure 6 shows the effect of adding elemental sulfur to fermenting mixtures containing selenium. Curves D and E show that elemental sulfur added in the proportion of 10 to 1 and 20 to 1 of selenium has only a slight effect in counteracting the toxic action of selenium. Hurd-Karrer (10) has reported that plants grown in selenium-containing water cultures were uninjured by the selenium if sulfur was added so as to make the proportion of selenium to sulfur 1 to 12 or less. She also stated that elemental sulfur as well as ammonium sulfate completely inhibited visible injury to wheat plants to which sodium selenate had been added.
EFFECT OF SODIUM SULFIDE AND SELENIUM Figure 7 shows that sulfur in the form of sodium sulfide is able to counteract much of the toxicity of selenium. However, sodium sulfide is unstable in water and results in the formation of hydrogen sulfide. Hydrogen sulfide produces a lemon-yellow precipitate of selenium and sulfur when added to solutions of selenious acid or sodium selenite. Therefore, the protective action of sodium sulfide as shown in Figure 7 is probably caused by the precipitation of the selenium in an insoluble form. EFFECTOF A X M O ~ I USULFATE M AS-D SELENIUM The curves in Figure 8 show that ammonium sulfate has little influence in overcoming the toxic action of selenium in the concentration of 10 of sulfur to 1 of selenium. Experiments, in which sulfur in the form of sodium sulfite or sodium thiosulfate were used, gave results practically identical with those shown in Figure 8 because of their failure to counteract the toxic effect of selenium. As mentioned earlier in the paper, observations indicate that all yeasts bring about the hydrogenization of sulfur, for all fermenting mixtures containing sulfur compounds or elemental sulfur give a distinct odor of hydrogen sulfide. Therefore, any sulfur compound probably would show slight protective action against selenium. Preliminary studies on the growth and multiplication of yeast during the fermentation periods, based on the dry
CHEMISTRY
81
weight of the washed yeast, would indicate that gain in weight decreases as the concentration of the selenium increases. It is well known that the hydrogen-ion concentration plays an important part in enzymatic reactions, and studies already made show the need of further studies with buffered solutions. It is of interest to note that the living cell can remove the toxic forms of selenium by reducing them, but that there seems t o be a definite limit. As the work progresses, it is hoped t o determine these limits. Table I gives the concentration of salts used in these experiments in terms of total moles of the element, milligrams of the element, parts per million of the element in the solution, percentage concentration of the element in solution, milligrams of element per gram of yeast, average number of cubic centimeters of carbon dioxide produced per minute, and percentage activity with the control as 100 per cent. The average number of cubic centimeters of carbon dioxide produced was calculated by taking the total cubic centimeters of carbon dioxide produced before the curve breaks, owing to lack of glucose, and dividing by the time in minutes. A fairly close agreement in the average number of cubic centimeters of carbon dioxide per minute is shown by the controls. This is also true when one compares the various activity percentages of similar salt concentrations. BIBLIOQRAPHY (1) (2) (3) (4) (6)
(6)
(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
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RECEIVED August 18, 1934. Published with the permission of the Director of the South Dakota Agricultural Experiment Station as Communication No. 10 from the Department of Experiment Station Chemistry, a n d is p a r t VI11 of “A New Toxicant Occurring Naturally in Certain Samples of Plant Foodstuffs.” These investigations are being carried out under the Purnell F u n d a n d with the cooperation of the Bureau of Chemistry and Soils, Bureau of Plant Industry, Bureau of Animal Industry. a n d Bureau of Home Economics of the United States Department of Agriculture.
The seeds sown by research have proved unusually productive during the past few lean years. S. K. COLBY
