Colorimetric Assay for Reaction of Sulfhydryl Groups with Organic Mercurials IRWIN FRlDOVlCH and PHILIP HANDLER Department o f Biochemistry, Duke University School of Medicine, Durham,
b The interaction of p-chloromercuribenzoate with protein sulfhydryl groups in the presence of ultraviolet absorbing materials can be followed by the procedures described, which provide a method for the quantitative determination of millimicromolar amounts of sulfhydryl compounds. The special virtue of this method is its translation of the reaction of sulfhydryl groups and p-chloromercuribenzoate into an absorbance change in the visible range, thus avoiding interference by ultraviolet absorbing species.
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thizone a t 6250 A. diminishing in an essentially linear manner with increasing amounts of the mercurial. The absorbance decrease per pmole of mercurial was the same in neutral or acid media. Under the conditions used, 0.03 pmole of mercurial yielded a decrease in absorbance of 0.160 unit. If an excess of p-mercuribenzoate is previously allowed to react with a sulfhydryl compound, only the unreacted mercurial not bound as mercaptide is available to react with dithizone and bleach the latter. This permits a simple determination of sulfhydryl compounds. Under present conditions, 0.03 pmole of cysteine yielded an absorbance change of 0.155. Linearity of response was obtained over the range from 0.01 to 0.05 pmole of cysteine.
for following the reaction of pmercuribenzoate with sulfhydryl groups has been described by Boyer ( I ) . This method, though both rapid and sensitive, suffers from the fact that the reaction must be followed in the ultraviolet region where many compounds of biochemical interest exhibit intense absorption. Thus, in following the reaction of the sulfhydryl groups of xanthine oxidase with p-mercuribenzoate in the presence of purine substrates, the authors were unable to use Boyer’s method because of interference by the intense purine absorption. Therefore, it seemed desirable to develop a method of following mercaptide formation a t longer wave lengths. Dithizone has been used in the colorimetric determination of mercury (3) and of zinc ( 2 ) . It also reacts with pmercuribenzoate, the absorption of diSPECTROPHOTOMETRIC METHOD
EXPERIMENTAL
Reagents. p-Chloromercuribenzoa t e was prepared according t o Whitmore and Woodward (4). Stock solutions were spectrophotometrically standardized according to Boyer ( I ) , and were prepared by dissolving 16.2 mg. of p-chloromercuribenzoate in water plus a few drops of alkali, addition of 10.0 ml. of 1.OM potassium phosphate buffer (pH 7.0), and 5.0 ml. of 0.50M potassium sulfate, followed by dilution to 100.0 ml. Such solutions were kept in the cold; the concentration of the mercurial did not change over a period of several weeks. Dithizone solution was prepared by dissolving 4.0 mg. of diphenylthiocarba-
zone in 100 ml. of carbon tetrachloride. The solution was clarified by filtration and used promptly. The absorbance of the fresh solution should be approximately 0.840 a t 6250 A. Method. DETERMINATION OF p MERCURIBENZOATE WITH DITHIZONE. T o 4.0 ml. of the dithizone reagent is added 1.0 t o 10 ml. of the mercurial in neutral or acidic solution containing from 0.005 t o 0.050 rmole of pmercuribenzoate. After vigorous shaking for 60 seconds, the phases are allowed to separate and the absorbance of the organic phase a t 6250 A. is determined. The Coleman Junior Spectrophotometer was used in these studies. Blanks containing no mercurial and a standard dithizone solution are included with each run. DETERMINATION OF CYSTEINE. T O 0.15 ml. of the p-mercuribenzoate stock solution containing 0.070 Mmole of the mercurial is added 1.0 to 5.0 ml. of unknown containing 0.005 to 0.050 pmole of sulfhydryl in neutral or weakly acidic solutian. The solutions are mixed and 4.0 ml. of the dithizone reagent is added. The mixture is shaken vigorously for 60 seconds and, after settling, the absorbance of the organic phase is read a t 6250 A Appropriate blanks and standard must be run simultaneously.
DETERMINATION OF PROTEIN SumTo 0.70 ml. of stock p-mercuribenzoate solution were added 0.5 ml. of 1.OM potassium phosphate buffer (pH 7.0), 0.5 mnl of 0.50M potassium sulfate, 1.1 ml. of water, and a t zero time 0.70 ml. of protein solution (containing approximately 0.25 pmole of sulfhydr) 1). At intervals thereafter, 0.50-ml. aliquots HYDRYL.
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Figure 1 . Rate of reaction of xanthine oxidase with p-mercuribenzoate 0 Dithizone method 0 Boyer method
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Figure 2. Rates of reaction of triosephosphate dehydrogenase and bovine serum albumin with p-mercuribenzoate 0, 0 Boyer method A, A Dithizone method VOL. 29, NO. 8, AUGUST 1957
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of this reaction mixture mere delivered into 4.0 ml. of the dithizone reagent in an ice bath, and the usual procedure was followed. Two blank tubes, one in which the mercurial was replaced by an equal volume of mater and the other in which the protein was replaced by an equal volume of water, were required and the method was calibrated by comparison with a standard cysteine solution. RESULTS
Figure 1 compares the results obtained with this method and the Boyer assay in following the reaction of p mercuribenzoate with partially purified milk xanthine oxidase. The initial rate given by the dithizone method was somewhat more rapid than that given by the Boyer procedure, b u t the end points were in excellent agreement. Because aliquots of the reaction mixture
are shaken with dithizone in carbon tetrachloride, i t is possible that the greater initial rate observed with the dithizone method reflects the “unco~-ering” of protein sulfhydryl groups by the denaturation attendant upon shaking with the organic phase. Figure 2 shows the excellent agreement between the present procedure and that of Boyer ( 1 ) when applied to crystalline bovine serum albumin and to a commercial, trricecrystallized preparation of muscle triosephosphate dehydrogenaee which had been incubated with 0.002M cysteine at p H 7.0 for 5 hours and then dialyzed against 0.01% Versene (pH 7.0) to remove the cysteine. A possible source of variability in the dithizone method is incomplete separation of the organic from the aqueous phase. m i t h care, however, good replication of results mag be obtained. Thus, the data shown in Figure 1 repre-
sent the means of triplicate deterniinations p;hich agreed to within 3%. LITERATURE CITED
(1) Boyer, P. D., J . Am. Chem. SOC.76,
4331 (1954). (2) lIalmstrom, Bo G., “Methods of Biochemical Analysis,” D. Click, ed., Vol. 3, p. 327, Interscience, Sew York, 1956. (3) Sandell, E. B., “Colorimetric Determination of Trace Metals,” p. 321, Interscience, Sew York, 1944. ( 4 ) Whitmore, F. C., Woodward, G. E., “Organic Syntheses,” Coll. Vol. I, p. 159, Wiley, Xem York, 1941.
RECEIVED for review September 4, 1966. Accepted February 2, 1957. rlssisted in part b y Contract AT-(40-1)-289 between Duke University and the U. S. Atomic Energy Commission and Grant RG-91 from the National Institutes of Health. Work done during the tenure of a U. S. Public Health Service Postdoctoral Fellowship of the National Heart Institute to Irlvin Fridovich.
Separation of Rhodium from Platinum, Palladium, and Iridium by Ion Exchange WILLIAM
M. MacNEVlN
and EDWARD S. McKAYI
McPherson Chemical laboratory, The Ohio State University, Columbus, Ohio
b The occurtence of rhodium as a yellow cation form and its conversion to a pink anion form are not well understood and cause erratic results during separations. Study of the preparation of these two forms shows that the cation form can b e prepared exclusively by oxidizing the rhodium to the quadrivalent state, reducing it to trivalent with hydroquinone, precipitating the yellow hydroxide, and dissolving it in hydrochloric acid. Platinum, palladium, and iridium treated in the same way behave as anions, if palladium is allowed to age at the hydroxide stage. This principle of cation-anion differentiation was used in the separation by ion exchange of rhodium from mixtures with platinum, palladium, and iridium. Rhodium chloride of high purity was obtained.
S
of milligram quantities of rhodium(III), platinum(IV), palladium(II), and iridium(1V) by ion exchange was reported in 1953 by MacNevin and Crummett (7), who used the principle of cation-anion differentiaEPARATION
1 Present address, Department of Chemistry, University of Tulsa, Tulsa, Okla.
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ANALYTICAL CHEMISTRY
tion to form the cationic Pd(NH3)ZtT complex and separate palladium(I1) from the stable chloride anions of the other three metals. Separation of rhodium and iridium by elution as anions was only 957, complete and iridium could not be removed from the resin. I n the s u n e year, Stevenson and associates (1 I ) reported successful separation of the four metals by elution of the perchlorates from a n ion exchange resin. Repeated attempts in this laboratory to bring mixtures of milligram amounts of these four metals into solution by fuming with perchloric, and nitric acid preparatory to separation have not been successful. Palladium remains insoluble, apparently as perchlorate. Berg and Senn ( 1 ) also used cationanion differenti‘ition between rhodium and iridium bv converting rhodium to a cation by heating with thiourea. The rhodium is retained by a cation resin and the anionic iridium appears in the eluate. The nature of the thiourea complex was not reported. Large amounts of organic material must be destroyed before the separated metals are used further. Cluett, Berman, and McBryde (S) have reported the separation of rhodium and iridium by elution in chloride solu-
tion. Very large volumes of eluate containing a high salt concentration i r e eluted. Several years’ experience in this laboratory have shown that the behavior of rhodium is uncertain and erratic. This suggested that the reactions of rhodium should be more fully understood, if its behavior was to be controlled. lleyer and Kawczyk (IO) and Tuthill (12) had observed the change in color of freshly prepared rhodium chloride solutions from pink to yellow. Kraus and Umbacli (6) reported a yellow ionic form and pink un-ionized form of rhodium sulfate. When the yellow form of the chloride is treated with silver nitrate, silver chloride precipitates; no precipitate is obtained with the pink form. The change from pink to yellow form in 3 months) a t room temis slox (l5yO perature, I n preliminary experiments the yellow rhodium chloride Lvas prepared by redissolving the freshly precipitated hydroxide in hydrochloric acid. The yellow form behaved as a cation and the pink form as a n anion toward ion exchange resins. These and other reactions have been used in developing a series of separations of rhodium from several mixtures with platinum, palladium, and iridium.
