(1) Goates, J. R., Cole, .4.R., Gray, E. L., J . Am. Cheni. Soc. 73, 3596
(1951). Kolthoff, I. M., Lingane, J. J., “Polarography,” 2nd ed., Interscience, Sew York, 1952. Kolthoff, I. lf,,Miller, C. S., J . Bm. Cheni. SOC.6 3 , 1405 (1941). Thid., p. 2818. Kolthoff, I. hl., Verzijl, E. J. A. H., 12ec. trav. chim. 42, 1055 (1923). Iiury, J. \V., Zielen, J. G., Latimer,
If-. M., J . Electrochem. Soc. 100,
168 (1953). Latimer, K , &I., “Oxidation Poten-
tials,” 2nd ed., Prentice-Hall, New York, 1952. i l l ) Maier. V.. Collection Csechoslov. C‘hem. Communs. 7. 146 11935). (12) Ibid., 9, 360 (1937). (13) Nernst, W., llerriam, E. S., Z . phys. Chem. 52, 235 (1905). (14) Pinkhof. J.. dissertation. Amsterdam, ’1919. (15) Przybylowicz, E. P., Rogers, L. B., ASAL. CHEM.28, 799 (1956). (16) Purdy, W. C., Burns, E. A,, Rogers, L. B Ibid., 27, 1988 (1955). (17) Revenda, J., Collection Czechoslov. Chevi. C o ? m i i o ~ s 6, . 453 (1934). .
I
\
,
~
(18) Scott, ]IK. , , ‘.Standard Methods of Chemical Analysis,” 5th ed., Van Xostrand, Sew York, p. 1443. (19) Spaulding, G. H., MciYabb, W.II., J . Franklin Inst. 237,207 (1944). (20) Strange, J. P., .ZX.~L.CHEX 29, 1878
(1957).
RECEIVEDfor review May 22, 1957. ..iccepted February 6, 1958. Abstracted from a thesis submitted by E. P. Przybylowicz t o the llassachusetts Institute of Technology in partial fulfillment of the requirements for the degree of doctor of philosophy in September 1956.
Dete rminuti o n of Cysteine with Ferricyanide by Amperometric Titration with Two Polarized Electrodes H. GEORGE WADDILL and GEORGE GORIN Deportment o f Chemistry, Oklahoma State University, Stillwoter, Okla.
b Semimicro amounts of cysteine can be determined by direct titration with standard potassium ferricyanide (0.1 to 0.001M) in phosphate buffer of p H 7. Two platinum electrodes connected to a 1 00-mv. direct-current source and to a sensitive galvanometer are used to detect the end point, which is indicated by the onset of a current that increases linearly with the volume of excess reagent. Amounts of cysteine mmole between 1 and 1.5 X can b e determined with a precision The procedure is straightwithin 1%. forward and the stoichiometry is not sensitive to external conditions. The apparatus is simpler than that required for other electrometric methods. Precision also compares favorably with other methods. As other common amino acids do not interfere, the method can be applied to amino acid mixtures.
C
is of great importance in biochemistry, and many methods have been developed for its deterniination (2, 3). In the past the most popular method involved titration with iodine. Ho.c\ever, this gives a somewhat variable stoichiometry unless the conditions are carefully controlled (3, 12). Recently, methods have been proposed which make use of certain metal ions, and in which the end point is determined amperometrically with a rotating platinum electrode and nonpolarizable halfcell (Q-11). The method described here utilizes potassium ferricyanide as reagent and two polarized platinum electrodes for detecting the end point. Kendall and Holst (’7) and Mason ( I S ) have used ferricyanide for the titration of cysteine. I n the latter case the end point was indicated by the appearance of ferricyanide TSTEIXE
color, but in the former the method of end point detection was not specified. Because the color of ferricyanide is rather pale, the end point cannot easily be discerned in dilute solutions, and large blanks may be needed. Mirsky (15) used an excess of ferricyanide and determined the amount reduced to ferrocyanide colorimetrically by converting the latter to Prussian blue; the precision n a s within 5%. dnson ( 1 ) used the same method in several investigations; he determined, more accurately than had been done theretofore, that the stoichiometry a t pH 6.8 \\as 1 to 1; that, at that p H value, other oyidizable amino acids interfered little or none; and that the reaction n.as subject t o catalysis by certain metal ions, among which copper(I1) was quite effective. Use of tmo polarized electrodes for detecting the end point of titrations was initiated by Foulk and Ban-den (6) in connection with the reaction of iodine with thiosulfate. They named the end point “dead stop” because, when thiosulfate is used as titrant, the galvanometer readings decrease rapidly and come to a stop m-hen the end point is reached. Hon-ever, the name is inappropriate when an electroactive species is used as the titrating reagent because the end point is then indicated by the onset of current; this is the case in the method under discussion. The current flows n hen both ferrocyanide and ferricyanide ions are present, and the latter can accumulate only after the end point. The magnitude of the current depends principally on the concentration of these ions, the potential applied, and the size of the elwtrodes. The response of the galvanometer a t the end point, therefore, varies. Indeed, it is an advantage of the method that its sensitivity can be adjusted by
.Q I E
Figure C. E. G.
1.
Circuit
1.5-volt dry cell Electrodes Galvanometer
R1. R2.
S.
diagram 100,000 ohms Decade box Switch
altering these factors. For a more complete discussion of the principles underlying the method, reference may be made to discussions by Delahay ( 4 ) and Kolthoff (8). APPARATUS
The electrical part of the apparatus consisted of a 1 5-volt dry cell-e.g., Eveready, S o . 6-a 100,000-ohm resistor, a 10,000-ohm decade resistance box, and a galvanometer with sensitivity of 0 10 pa. per division (G-11 Laboratories, Inc., Catalog KO. 570-211), connected as shown in Figure 1. Kot shown is the damping resistance of the ga!vanometer, which was connected across its terminals. Because the cell discharges slowly through the high resistance of the circuit, this can he left connected a t all times, except perhaps when a long period of disuse is contemplated. The electrodes were pieces of 22-gag~ platinum wire about 0 5 em. long, fuscd in the end of soft glass tubes, inside which connection was made in t h t uaud way with the aid of mercury. The electrodes were mounted in a S o . 00 twoVOL. 30, NO. 6 , JUNE 1958
*
1069
hole rubber stopper, so that there was a distance of about 5 mm. between the wires. KO special care was required in the preparation and maintenance of these electrodes; this is in contrast to the demanding care sometimes required in other amperometric methods. Titrations were effected in a lOO-ml., tall-form beaker fitted with a rubber stopper, through which were introduced the electrodes, a gas bubbling tube, the buret tip, and a glass stirrer. The stirrer was driven by an ordinary stirring motor (Wilkens-Anderson Co., Catalog No. T10235). The reagent was dispensed from a 10-ml. buret. The desired potential was impressed upon the electrodes by varying the resistance of the decade box. For all the determinations described here, a potential of 100 mv. was used, and this value should be generally applicable. Other investigators may find it convenient to use galvanometers and electrodes of different characteristics, and should determine the optimum potential to be used with a given apparatus and concentration of titrating reagent by increasing the potential gradually until the line of current us. volume of reagent is steep enough to permit accurate extrapolation. REAGENTS
All chemicals were of ACS reagent grade unless otherwise specified. Buffer solution was prepared by titrating phosphoric acid, somewhat more concentrated than 0.6iCf, with concentrated sodium hydroxide to pH 7.0, then adjusting the final volume to give the molarity indicated. L-Cysteine was obtained from the California Foundation for Biochemical Research, Los Angeles (33, Calif. (CFP grade, sample I), and from Schwarz Laboratories, -Mount Vernon, N. 1'. (sample 11). The purity and handling of these samples are discussed below. L-Cystine, CFP; [a]& (c = 1 in 1.11 hydrochloric acid) -208'. Nitrogen was of commercial, waterpumped grade, and was passed through a solution of vanadous ion (14) to remove oxygen. Sodium amalgam was prepared by weighing freshly cut sodium pieces under xylene, heating on a hot plate until the sodium melted, and adding 100 parts by weight of mercury (Care!). After the vigorous reaction which ensued had subsided, the amalgam was rinsed with iso-octane and passed through a pinhole made in a sheet of filter paper. The amalgam could be made more conveniently by grinding commercially available 5% amalgam with 4 parts of mercury in a mortar, but not all such samples gave a satisfactorily clean product. Potassium ferricyanide was prepared volumetrically by dissolving accurately weighed samples of solid reagent. The solutions were freshly prepared each day because they deteriorate appreciably in 24 hours; exposure to direct sunlight, even for a few minutes, must be avoided. Solutions of 0.1 to 0.001M were used with satisfactory results. 1070
ANALYTICAL CHEMISTRY
Water which was used in preparing all solutions was distilled, deionized by passage through a mixed-bed ion exchange resin (Amberlite MB-1), and deaerated by boiling for a few minutes and then passing a brisk stream of nitrogen through it while cooling. The water was stored in and dispensed from a siphon flash, into which air was admitted only after passage through a solution of alkaline pyrogallol. CYSTEINE SAMPLES
The samples of cysteine hydrochloride monohydrate available from the sources indicated were of good quality. They were both crystalline, free flowing material, which was largely immune to the difficulties caused by the hygroscopic properties of anhydrous samples. However, the samples could not be considered analytically pure. It is unreasonable to expect that they might be, owing to the chemical properties of cysteine, especially its susceptibility to oxidation. Attempts to purify the cysteine samples further were not successful; therefore, they were used without further purification. Standard solutions of cysteine were prepared volumetrically by dissolving weighed samples in water or in 1M hydrochloric acid, which had been freshly purged with nitrogen, followed by dilution. The presence of hydrochloric acid retards alteration of the cysteine titer, and its use was found to be essential in preparing 0.001M solutions which would be stable for a few hours. More concentrated solutions were less demanding in care, and satisfactorily stable solutions of 0.1M concentration could be prepared without added hydrochloric acid. The standard cysteine solutions were kept in stoppered containers under nitrogen, and a stream of this gas was passed over the surface of the solutions while samples were withdrawn. Solutions were freshly prepared each day. REDUCED CYSTINE SAMPLES
An accurately weighed amount of cystine to give approximately a 0.05M solution was dissolved volumetrically in 1M hydrochloric acid. The solution was placed in a dry centrifuge bottle of 250-ml. capacity. This was closed with a two-hole rubber stopper, into which were fitted a capillary vent and a 1-mm. capillary tube, the tip of which dipped into the solution. The other end of the capillary tube was connected to a separatory funnel with a shortened stem. Sodium amalgam, under a layer of iso-octane, was placed in the funnel and the stopcock was opened to allow dropwise flow of the amalgam into the solution. This was continued until samples withdran-n a t intervals had attained a constant titer; about 100 ml. of amalgam and 2 . 5 hours were required. PROCEDURE
Twenty milliliters of buffer were placed in the beaker (as much as 5-mL
samples of 1M hydrochloric acid could be added to this without necessitating adjustment of pH) and 2 ml. of 10-4M copper(I1) sulfate were added. Nitrogen gas was bubbled through for 10 minutes with stirring; then, the gas bubbling tube was raised above the surface of the solution and the nitrogen was allowed to stream over the surface during the titration. The cysteine sample was pipetted into the titration beaker through a hole in the stopper. The buret tip was lowered into the liquid through the same hole, and the ferricyanide solution was added slowly. Nearness of the end point was indicated by fluctuations of the galvanometer needle. After there was a permanent deviation from the zero point, four or five small portions of reagents were added, and the galvanometer was read after each addition. DISCUSSION OF RESULTS
The results of a typical titration indicate that a linear increase in current is obtained after the end point. There is little difficulty in locating the point of intersection with the line representing the current before the end point (practically a t zero). Vhen less concentrated titrating solutions were used, the line became less steep and the uncertainty in locating the point of intersection became correspondingly greater; this can be counteracted to some extent by increasing the potential applied to the electrodes. Steady galvanometer readings were obtained quickly in the presence of copper sulfate. In the absence of this catalyst the reaction was slower, especially in the vicinity of the end point, and steady readings were obtained only after 2 or 3 minutes following each addition of ferricyanide. Variation by a factor of 2 in the amount of catalyst added did not affect the amount of ferricyanide consumed. I n accordance with the findings of Anson ( I ) , a stoichiometry of 1 mole of ferricyanide per mole of cysteine was assumed. The reaction may be represented by the equation:
+
2HSCH2CH(NH3+)COO- 2 Fe( C N ) B - ~ + [-SCH&H(KH3+) COO-]* 2 Fe(CN)e-4 2H+
+ +
Because the samples of cysteine available were not analytically pure, the precision of the method must be estimated from the consistency of the values for per cent cysteine found in each sample under different conditions. Many standard solutions were prepared in the general ranges of concentration indicated. and samples of varying sizes were taken from each solution. Table I summarizes the results obtained with samples I and 11; the average deviation of single determinations from the mean indicates the precision that mas attained. Sample I1 was analyzed by the iodimetric method of Lavine ( 1 2 ) ; the
cysteine found was 96.9%. Both Samples I and I1 were analyzed by direct iodimetric titration in a medium of phosphoric acid-sodium dihydrogen phosphate of p H 2; the results were 96.2 and 97.5%, respectively (16, 1 7 ) . While these comparisons are instructive, especially in view of the fact that iodimetric methods have been so extensively used, the results cannot be considered as giving a reliable indication of accuracy. Doubts concerning the stoichiometric exactness of the reaction with iodine have been expressed many times, and the experience of these authors confirms them. As a check on the absolute accuracy of the method could not be obtained in this way, samples of cystine n-ere reduced and analyzed. Cystine is available commercially in nearly pure form, and, in contrast t o cysteine, is stable. The optical rotation of the sample used (-208”) indicates a purity of 97.770 (6). The cysteine solution in 1X hydrochloric acid was reduced n-ith sodium amalgam, a procedure which has been used before (10, 18) for the same purpose. The technique used in this work was somewhat different from that previously described, and may be found advantageous. The results are shown in Table I, sample 111; the -SH titer is in excellent agreement with that indicated by the optical rotation. The work of Anson (1) had indicated that other amino acids would not interfere, but this was checked directly. The -SH titer found in the presence of a mixture of amino acids (aspartic acid, isoleucine, leucine, lysine, methionine phenylalanine, threonine, tryptophan. and valine), each in the same concentration as the cysteine, gave 96.6% cysteine, compared with 97.0% in the absence of added amino acids. Serine and tyrosine were tested individually
Table 1.
Sample I I1 I11
Taken, Mmole 0 . 4 to 0.9 0.015 to 0,055 0 . 3 to 0 . 6 0.03 to 0.05 0.0015 to 0.0025 0.0016 to 0 . 6
Summary of Analyses
No. of Determinations
because they are more likely to interfere. Cysteine found, in the presence and absence of added serine, was 97.0 and 96.7%, respectively; in the presence and absence of added tyrosine, 97.7 and 97.9%, respectively. Adequate protection of the samples from oxidation is essential for satisfactory analytical work in this field. I n the authors’ experience, this requires considerable practice. It is xell t o standardize and continually to check the technique employed with cysteine samples as small as the lowest amounts it is desired to determine. ACKNOWLEDGMENT
The authors would like to acknowledge the contribution made to this work by the experiments of R. G. Pilmer on the use of polarized electrodes in the iodimetric titration of cysteine (16). The work was supported by Grant RG4669 of the Division of Research Grants. ;National Institutes of Health, administered b y the Research Foundation of Oklahoma State University. LITERATURE CITED
(1) Anson, M. L., J . Gen. Physiol. 23, 321 (1940): 24, 709 (1941). (2) Block, F. P., Bolling, B., “Amino Acid Composition Gf Proteins and Food8,” pp. 186-223, C. C Thomas, Springfield, Ill., 1951. (3) Chinard, F. P., Hellerman, L., in
Cysteine 96.8 95.8 96.4 96.9 96.9 97.5
8 6
15 6 4
13
Av. Dev., % 0.18 0.52 0.47 0.21 0.15 0.35
‘Wethods of Biochemical Analysis,” Vol. I, pp. 1-26, Interscience, New York, 1954. (4) Delahay, P., “New Instrumental Methods in Electrochemistrv,” pp. 258-64, Interscience, Hew York, 1954. ( 5 ) Dunn, M. S., Rockland, L. B., Advances in Protein Chem. 3, 354 (1947). (6) Foulk, C. W., Bawden, A. T.,J . Am. Chem. SOC.48,2045 (1926). (7) Kendall, E. C., Holst, J. E., J . Biol. Chem. 91, 435 (1931). (8) Kolthoff. I. M..ANAL.CHEST.26, 1685 (1954). ’ Kolthoff, I. M., Stricks, IT., Ibid., 23, 763 (1951). Kolthoff, I. &I.,Stricks, W.,J . Am. Chem. SOC.72, 1952 (1950). Kolthoff, I. hl., Stricks, W.,Morren, L., ANAL.CHEM.26, 366 (1954). Lavine, T. F., J . Biol.Chem. 109,141 (1935). Mason, H. L., Ibid., 86, 623 (1930). “Polarographic TechMeites, niaues. Interscience, New York, 1965. ’ Mirsk; A. E., J . Gen. Physiol. 24, Mirsky, 725 (1941). Pilmer, R. G., M. 8. thesis, University of Oregon, 1955. Pilmer. R. G., Pilmer, G.. Gorin, Gorin. G., G.. “Abstracts,” “Abstracts,” e. ACB, 128th National Meeting, ACS, Minneapolis, &$inn., September 19.5.5. n. 17R. 1955, p. r . 17B. ~. Sullivan, SuiGGan, RI. X., Hess, W. C., Howard, H. IT,, J . Biol. Chem. 145, 621 (1941). RECEIVED for review July 29, 1957. Accepted January 10, 1958. Presented in part, Division of Analytical Chemistry, 129th meeting, ..iCS, Dallas, Tex., April 1956.
k.,
I
Polarographic Study of Cytotoxic Nitrogen Mustards ROGER MANTSAVINOS’ and JOHN E. CHRISTIAN Department of Pharmaceutical Chemistry, Purdue University, West lafayetfe, Ind.
Pharmacologically active ethylenimonium ions formed by the cyclization of P-chloroethyl groups of aliphatic nitrogen mustards are reducible a t the dropping mercury electrode. The diffusion current obtained upon reduction is linearly related to the concentration of the electroactive species over certain ranges of concentration. Polarographic procedures are described for the quantitative estimation of ethylenimonium intermediates, and for determining the rate a t which the
initial cyclization process occurs. The polarographic waves appear to b e irreversible and a two-electron reduction is postulated.
T
HE cytotoxic nitrogen mustards have enjoyed some degree of SUCcess in the chemotherapy of cancer (11). The pharmacologically active intermediate of this class of compounds is believed to be the ethylenimonium (aziridinium) cation, which is capable of alkylating the functional group of many
compounds of biological importance
(1%. Ethylenimonium ions are formed from parent compounds b y the internal cyclization of 0-chloroethyl groups in near neutral aqueous solutions (6). The initial cyclization process undergone by methyl-bis(p-chloroethyl)amine, a typical nitrogen mustard, is illustrated b y the following equation: Present address, Department of Pharmacology, Yale University School of Medicine, New Haven, Conn. VOL. 30, NO. 6, JUNE 1958
0
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