Amperometric Titration of Sulfhydryl Groups Microgram A nalys is SHELDON ROSENBERG, J. C. PEHRONE, AND PAUL L. KIRK University of California Medical School, Berkeley, Calif, A modification of the amperometric titration method for sulfhydryl groups has been developed for. use with microgram quantities. A vibrating platinum electrode is used as a combination electrode-stirrer. With this technique it i8 possible to determine with reasonable accuracy amounts of sulfh) dry1 as small as about.1 microgram. The method was tested with denatured protein solution and yielded results which were in agreement with previous studies of this type.
K
OLTHOFF and Harris (6) described an argentometric titration for compounds containing the sulfhydryl group, using an amperometric end point and a rotating platinum cathode. I n an effort to adapt the method to determination of mimogram quantities, Perrone and Kirk in 1946 were able to show that a vibrating platinum electrode was equally effective. Using the standard vibrating stirrer ( 6 ) commonly employed in microgram analysis, but substituting a platinum wire for the glass thread and attaching the wire to the electrical system, the end points obtained with the Kolthoff and Harris arrangement were as good as those obtained u ith the rotating electrode, inasmuch as the rapidity of movement of the platinum wire in the solution could be made as great. Since that time, the vibrating electrode has been used for polarography but not amperometry with larger volumes of solution by Harris and Lindsey ( 4 ) . Benesch and Benesch ( 1 ) have recently demonstrated the applicability of the rotating electrode to determination of the sulfhydryl group in rehttively large amounts of biological materials. I n this article is described the modification of standard mic-rogram analysis to the similar determination of microgram quantities of this group in biological and other materials, using the vibrating platinum stirrer-eler trode.
about 45 cm. (I5 inches) long, ti:tving ohms per inch, was satisfactory.
H
resistance of atiout 25
REAGENTS
Silver nitrate solution, about 0.001 M , was made by dilution of a stronger stock solution which was usually 0.1 M . The stock and diluted solutions wcre carefully standardized against potassium chloride on a larger scale, making use of dichlorofluorescein indicator. Supporting electrolytc solution was made to contain 0.25 M ammonium hydroside and 0.1 .I/ :tmmonium nitrate. This solution prevented precipitation of chloride and functioned in the electron transport system. PROCEDURE
A convenient aliquot of the solution being analyzed was measux-ed with a micropipet into the porcelain dish, using two or three rinsings. The half-cell and vibrating platinum electrode were immersed in the solution and the current was allowed to equilibrate while the buret was being filled with silver nitrate solution.
APPARATUS
The vibrating electrode consisted of a standard stirrer, shown in, Figure 1, in which the glass thread was replaced by a platinum wire. Because the vibrator is constructed of steel, electrical connection was made to the screw holding it a t the attached end. The electrode also served aB stirrer, agitation being produced in the small volume of solution by the 60-cycle vibration. Titrations were performed in porcelain titration dishes, carried on the standard titration table ( 5 ) . Titrating liquid was added either from the capillary buret described by Sisco, Cunningham, and Kirk (8) or from a modified horizontal buret ( 5 ) whose total capacity was only about 20 microliters. This buret of very small capacity made possible greater precision in adding small increments when the amounts titrated were in the lower range of the method. As reference cell, a vessel similar to that previously described (8) was filled. I n the bottom of the cell way placed about 0.6 cm. of mercury, on top of which was added an electrolyte solution made by dissolving 4.2 grams of potassium iodide and 1.3 grams of mercuric iodide in 100 nil. of saturated potasRium chloride solution. The salt bridge was filled with a gel of 3y0 agar containing 30%.potassium chloride. After a few weeks' use the agar gel tended to crack and required replacement. This could be retarded by keeping the gel immersed in potassium chloride solution when not in use. Precautions against the inclusion of air bubbles in the agar were necessary a t all times; this was readily accomplished by gently sucking the melted agar wlution into the bottom of the salt bridge tube. The current was measured by a galvanometer of the swinging light-beam type having a sensitivity of 0.21 pa per division. .Z resistance wire was provided across the leads in case the deflection of the instrument became too great. This could be a high resistance radio potentiometer allowing the adjustment of the rcsistance as needed. The substitution of a fine Sichrome wire
Figure 1. Vibrating Cathode Assembly A. G.
Magnetic vibrator Leads to galvanometer
P. Vibrating electrode
Table I .
Sulfhydryl Group,
Y
Y
30.6 15.3 6.06 3.03 1.01 1.01
6.23 3.11 1.029 0,616 0 205 0,205
a
b
1186
Cysteine Solutions of Known Content
cysteine HC1 Taken,
AV-
Time of Standing, Min.
No. of Detns.
10-30 2-3 hours 10-30 10-30 10-30 4-5 hours
Mean * mean deviation. Calculated from formula S.E. =
Cysteine HCI Found', Y
3 0 . 4 t 1.03 11.98 t 0 . 6 4.904 * 0 . 2 2 2.765 k O . 0 8 0.943 + 0 . 0 3 0 . 8 3 t0.05
erage Re- Standard covery, Errorh.
7 0 % 99.6 77.7 07.8 91.2 93.0 82.2
1.6 2.0 2.9 1.3 2.4 4.5
V O L U M E 2 2 , NO. 9, S E P T E M B E R 1 9 5 0
1187
Table 11. Glutathione, Solutions of Known Content Glutathione Taken,
Sulfhydryl Group,
Time of Standing, Y Y Min. 50 5.39 10-30 10-30 21.1 2.27 6-7houra 21.1 2.27 0.58 10-45 5.40 0.58 2-3hours 5.40 a Mean imean deviation. b
Average Re- Standcovard ery, Errorb.
Glutathione No. of Founda, Detns. Y % 5 50.46 t 0 . 2 4 100.1 4 21.55 1 0 . 5 5 102.0 5 ‘19.3 * 0 . 4 91.5 5 4.84 1 0 . 2 9 88.50 6 4.64 AO.49 86.0
%
0.27 1.7 1.5 3.4 4.5
Calculated from formula S.E. =
Titration was not started until the current remained steady,. At times this required as long as 10 minutes when a new reference cell was first employed. The vibrating electrode required a shorter time for equilibration than the rotating electrode, usually operating a t the very beginning of its movement. Titration was carried out by adding increments of silver nitrate of convenient size and noting the galvanometer deflections after each addition. When the first large deflection was observed, the current was recorded for a t least three more additions of reagent. Blank determinations were made under each set of experimental conditions. Blanks were uniformly low, corresponding to 0.2 to 0.8 microliter of titrating solution.
A graph of the results showing galvanometer deflections as ordinate versus increment as abscissa was constructed. The shape of the curves was the same as those of Kolthoff and Harris and others. RESULTS
Using the technique described, analyses were made of cysteine hydrochloride, glutathione, and eventually protein solutions, some of which had been denatured. The range of the method was tested by analysis of cysteine solutions of known content (Table I). The recoveries were satisfactory when the experimental conditions were controlled with reasonable accuracy. When long times of standing were employed, the recovery fell uniformly, as shown by the small standard error combined with considerable lack of recovery. The smallest amounts of cysteine when run rapidly still yielded a reasonably high recovery, though not uniformly theoretical. As the amounts became larger the theoretical recovery was approached more closely. Similar results were obtained with known quantities of glutathione (Table-11). Here, also, the time of standing was significant, any time over a half hour causing low recovery. The smaller quantities here also were not completely recovered. Considering that the amount of sulfhydryl group present in glutathione
is only 10.77%, the lower limjt of accurate analysis is a t least as low as 1 to 2 micrograms of sulfhydryl group. After the procedure had been tested with known concentrations of simple compounds, it was thought desirable to test its utility with denatured proteins. For this purpose, bovine serum albumin was titrated to determine the amount of sulfhydryl normally present. It was also denatured by various denaturing agents, as shown in Table 111. The amounts of sulfhydryl obtained with denatured proteins by this procedure were found ta be reasonably constant. The values were in good agreement with the value of 0.34% fouqd by Greenstein (3). There is no method by which the exact amount of sulfhydryl which should be present may be demonstrated. The agreement between denaturation with various concentrations of alcohol and that with guanidine hydrochloride was striking, whereas the value with urea wassomewhat lower.
Table 111. Sulfhydryl Group in Bovine Serum Albumin Denaturing Agent No. of Sulfhydryl as a n d Solvent Detns. Cysteine y/100 y Protein Alcohola, % 0.32 t 0 . 0 2 31.6 4 0.29 = t O . O l 42.5 3 4 0.30 * 0.02 63.3 4 No titration None Guanidine hydrochloride b Distilled water 6 0.33 0.03 6 3 . 3 % alcohol 4 0.31 * O . O Z Urea 5 N o titration Distilled water 5 0.21 r O . O 1 4 3 . 5 % alcohol a Protein placed in alcohol solution a n d allowed to stand a t room temperature 0.5 hour before titration. b Protein solution added to guanidine hydrochloride according t o method of Greenstein (21. C Denatured according t o method of Anson (7).
LITERATURE CITED
(1) Benesch, R., and Benesch. R. E.. Arch. Biochem., 19,35 (1948). (2) Greenstein, J. C., J . Bid. Chem., 128,501 (1938). (3) Ibid., 136,798 (1940).
(4) Harris, E. D., and Lindsey, A. J., .Vatwe, 162,413 (1948). (5) Kirk, P.L., “Quantitative Ultramicroanalysls,” New York, John Wiley & Sons, 1950. (6) Kolthoff, I. M., and Harris, W. E., IND. ENG.CHEM.,ANAL. ED., 18, 161 (1946):ANAL.CHEM..21,963 (1949). (7) Northrup, J. H., Kunitz, M., and Herriot, R. M., “Crystalline Enzymes,” New York, Columbia University Press, 1948. (8) Sisco, R. C., Cunningham, B., and Kirk, P. L., J . B i d . Chem., 139,1 (1941).
RECEIVED M a y 1, 1950. Aided by granta from t h e American Cancer Society recommended b y the Committee o n Growth, a n d t h e Research Board of t h e University of California.
Microbiological Determination of Sulfur in Yeast DORIS K. MCMANUS, ALFRED S. SCHULTZ AND WAYNE E. MAYNARD The Fleischmann Laboratories, Standard Brands Znc., New York, N . Y .
T
HE recent investigation of the ability of various yeasts to use the sulfur of amino acids and other sulfur compounds (I I ) suggested the possible development of a microbiological assay method for sulfur. A search of the literature revealed that most of the values given for the sulfur content of yeast were obtained by direct ashing of \he stmples. Fink and Just (9) used the Eschka mixture as a fixative during the ashing of yeast samples and obtained much higher values than ‘those obtainable by direct ashing. Highest total sulfur values found were obtained by Block and Bolling ( 4 ) using the Pregl method. These authors reported that sulfur analyses on dried yeasts are difficult to carry out and may a t times be low. In view of the widely divergent values f x sul-
fur in yeast reported by different investigators, an attempt has been made to clarify this problem by the development of a microbiological method. The success of such a microbiological assay method is partially dependent upon the utilization pf a test organism capable of responding equally well to all forms of available sulfur in the sample. I n previous work ( I f ) Torula utilis gave indications of responding to both organic and inorganic sulfur sources in this manner. Further investigation has revealed that although its growth responses t o small increments of various sulfur sources are not ide,iticnl, they are more alike than those of any other or-
