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Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiodipyridine
ARCHIVES
OF
BIOCHEMISTRY
AND
Determination
BIOPHYSICS
119,
(1967)
41-49
of Sulfhydryl
Groups
with
2,2’-
or 4,4’-
Dithiodipyridine’ D. R. GRASSETTI,’ Institute
AND
J. F. !MURRAY,
JR.
of Chemical Biology, University of San Francisco, and Arequipa 1645 Russ Building, San Francisco, California 94104 Received
Foundation,
July 5, 19GG
A procedure is described that permits determination of SH grol~ps in simple compounds and in biological materials. The procedure utilizes the reaction of 2,2’-dit,hiodipyridine or of 4,4’-dithiodipyridine with thiols, which gives the corresponding 2- or 4-thiopyridone. The ultraviolet absorption of the thiopyridone formed is measured. The method permits determination of less than 1.5 pg SH when 2,2’-dithiodipyridine is used, and of less than 0.5 pg SH when 4,4’-dithiodipyridine is used. The proposed method is compared with known methods for the determination of snlfhydryl groups.
quite different from those of the corresponding disulfides, which contain the resonating pyridine ring. It is thus possible to follow spectrophotometrically the course of the reaction of 2-PDS and 4-PDS with thiols. Both the disappearance of the disulfide and the formation of the thiopyridone can be followed, and t’hiols can be determined quantitatively. Other disulfides capable of reacting with SH groups with formation of colored or UVabsorbing species are known (4-i).
Grassetti et al. (1) have recently studied the effect of a number of sulfur-containing pyridines on the metabolism of Ehrlich ascites tumor cells. As a consequence of that study it was found that 2-PDS3 and 4PDS react readily and completely with thiols, with the formation of the corresponding thiopyridone, e.g. : 12 s-s
i(’ A,-
)
+
2 RSH-2
c&
+ RSSR S ;
2-Thiopyridone and 4-TP are almost exclusively in the tautomeric thio form, with the mobile hydrogen attached to the nitrogen (3). This causes the ultraviolet absorption spectra of these thiopyridones to be
METHODS 2-Thiopyridone and 4-TP were obtained from Aldrich Chemical Co., Milwaukee, Wisconsin. 2-PDS was prepared by iodine oxidation of 2-TP, according to Marckwald et al. (8), and 4-PDS was prepared by nitric acid oxidation of 4-TP, according to Comrie and Stenlake (9). Homogeneity of 2-PDS and 4-PDS was assayed by thin-layer chromatography on Florisil: one single spot was given with three or more solvent systems. The following systems were used for 2-PDS: tolllenemethanol [80:20], benzene-met,hanol [95:5], benzene-chloroform [l:l), and benzene-acetone [90: 101. The following were used for 4-PIX: tolueneacetone [90:10], toluene-chloroform [l:l], and toluene-methanol [95:5). The spots were vislialized by iodine vapor. The dihydrochloride of 2-PIE and the dinitrate
1 This investigation was supported by Public Health Service grant CA 07296, from the National Cancer Institute. 2 Inquiries concerning this paper should be directed to Arequipa Foundation, Russ Building, San Francisco, California 94104. 3 Abbreviations used: 2-PDS = 2,2’-dithiodipyridine (from the empirical name “pyridine disulfide”); 4-PDS = 4,4’-dithiodipyridine; 2. TP = 2-thiopyridone; 4-TP = 4-thiopyridone; KRP buffer = Krebs-Ringer phosphate buffer, pH 7.2 (2). 41
42
GRASSETTI
AND MURRAY
of 4-PDS were used in our early experiments; we subsequently found that these salts slowly decompose on standing even in the crystalline state. The crystalline free bases, however, are stable for at least several months. When dissolved in KRP buffer, the bases decompose slowly: it is thus desirable to prepare fresh solutions daily and to carry blanks throughout the procedure. Heating of the solution accelerates the decomposition. Bovine serum albumin was the crystalline Pentex Corp. product; rabbit muscle aldolase was A-grade Boehringer preparation; both were obtained through Calbiochem, Los Angeles, California. Mercaptopyruvic acid was prepared according to Schneider and Reinfeld (10). Cysteine and the other thiols used in this work were reagent-grade products. ASSAY PROCEDURES Simple thiols and proteins. A suitable amount of the material to be analyzed (0.0150.5 pmole SH) was dissolved in 1.5 ml of KRP buffer. This solution was mixed with 1.5 ml of PDS solution (2 X 10e3M) in the same buffer. The UV absorbance at the appropriate wavelength could be determined immediately after mixing in the case of simple thiols, and after 15 minutes at room temperature
in the case of bovine serum albumin. Longer delays did not affect the absorbance. Rabbit muscle aldolase was analyzed in the presence of 5 M urea in KRP buffer; the absorbance was determined after 90 minutes at room temperature. Protein determinations were carried out by the 280/260 method, using Layne’s coefficients (11). Tissue homogenates. Organs from healthy female Swiss mice were homogenized in KRP buffer (1:lO) at 24” in a Potter-Elvehjem homogenizer provided with a Teflon pestle. The homogenate was heated in a water bath at 90” for 5 minutes and then cooled rapidly under running water. The equivalent of approximately 10 mg of tissue (0.1 ml homogenate) was incubated with 4.9 ml of PDS solution in the same buffer (lo-* M) for 15 minutes at 37”, with shaking. The suspension was filtered and the absorbance of the filtrate was determined at the appropriate wavelength against a PDS blank. A sample of tissue was carried through the same procedure, in absence of PDS, in order to determine the blank absorbance. The same procedure was used with washed Ehrlich ascites cells. Urine. Freshly collected human urine was heated 5 minutes at 90” in a water bath. One ml of the cooled urine sample was then incubated 15 minutes at 37” with 4.0 ml of PDS (lOya M in KRP
QLS-SQ
IO1 x Id4 M
x = 08x ij 24 Q 0.6-
220
240
260
280 Wavelength
300
320
340
360
380
mp
FIG. 1. UV spectrum of P-PDS in KRP buffer, pH 7.2.
400
DETERMINATION
OF SULFHYDRYL
43
GROUPS
14 12 1.0 0.8 8 6 $ 06 3 04 -
02
-
OL -
I
220
240
260
280
300
Wavelength
320
340
360
380
400
mp
FIG. 2. UV spectrum of 2-TP in KRP buffer, pH 7.2.
buffer). Absorbance at 343 rnp (for 2-PDS) or at 324 rnp (for 4-PDS) was read against a blank prepared as above but containing no PDS. A correction was applied for a PDS blank, which was carried through incubation. Urine normally contains substances that absorb at 343 and 324 rnp; therefore, the size of the urine sample must be adjusted to take this into account. Normally it was found feasible to use samples as large as 1 ml. RESULTS
AND DISCUSSION
Figures l-4 show the absorption spectra of 2-PDS, 4-PDS and their corresponding thiopyridones, in KRP buffer. The extinction values of the UV maxima of these compounds are reported in Table I. When 2-PDS reacts with a thiol, the formation of 2-TP can be followed by the change in absorbance at 343 rnp, since the corresponding disulfide, 2PDS, has virtually no absorption at that wavelength. Conversely, the consumption of 2-PDS can be estimated from its absorption at 233 rnp, provided that a correction for the absorbance of 2-TP at this wavelength be made. Analogous considerations apply to 4-PDS, the consumption of 4-PDS being followed through the absorbance at 247 rnp, and the formation of 4-TP at 324 mM. The extinction of 4-TP at 324 rnp is almost three
TABLE I UV ABSORPTION IN KRP BUFFER, PH 7.2 Compound 2,2’-Dithiodipyridine 2-Thiopyridone 4,4’-Dithiodipyridine 4-Thiopyridone
bmx bd
233 281 271 343 247 230 324
Molar
extinction 1.39 9.73 1.04 7.06 1.63 9.6 1.98
x x x x x x X
104 10” 10’ 103 104 lo3 lo*
times as high as the extinction of 2-TP at 343 rnp (Table I) ; thus 4-PDS is the more sensitive reagent. Calibration curves for 2-PDS were obtained (Fig. 5) by adding increasing amounts of cysteine (O-O.9 fimole) to a solution of 2PDS (0.32 pmole). Similarly, Fig. 6 gives calibration curves for 4-PDS disappearance and 4-TP formation. Lower concentrations were used in this case because of the higher extinction of the 4-isomer. Various thiols have been tested for reaction with 2-PDS and with 4-PDS, and were found to form the respective thiopyridones. These compounds include cysteine, reduced glutathione, reduced lipoic acid, mercapto-
44
GRASSETTI
AND MURRAY
pyruvic acid, and coenzyme A. Figure 7 [solid Iine) gives the UV spectrum of a mixture of 2-PDS and cyst.eine (in slight excess) in KRP buffer. This curve is representative of the spectrum obtained from
mixtures of 2-PDS and other thiols as well; it is essentially identical with the spectrum of 2-TP (Fig. 2). The dotted curve in Fig. 7 was obtained by incubating a solution of 2PDS with intact washed Ehrlich ascites
3.1x d
Wavelength
M
m,u
FIG. 3. UV spectrum of 4-PDS in KRP buffer, pH 7.2.
7.0 x 10-5M
0” 0.8 5 i $ 0.6 04-
01
I
1
I
I
I
220
240
260
280
300
Wavelength
I
320
340
360
m/l
FIG. 4. UV spectrum of 4-TP in KRP buffer, pH 7.2.
380
400
DETERMINATION
01
02
03
OF SULFHYDRYL
0.4
05
Mcromoles
0.6
07
45
GROUPS
0%
09
IO
cysteine
FIG. 5. Calibration curves of 2-PDS. 0 Absorbance at 343 ml; A absorbance at 233 rnp. Zero t,o 0.9 pmole of cysteine was added to 0.318 pmole of 2-PDS (in 0.3 ml KRP buffer) in the spectrophotometer cuvette. Volume was brought to 3.0 ml with KRP buffer, pH 7.2, after mixing. and absorbance was measured immediately
tumor cells, in amounts sufficient to give full reaction. The similarity in the two curves of Fig. 7 shows that the method is directly applicable to biological materials. Analogous results were obtained with mixtures of 4PDS and t’hiols, and of 4-PDS and intact Ehrlich ascites cells, as shown in Fig. 8. In this case, although the UV spectrum of the reaction mixture is very similar to that of 4TP, the end-absorption (below 230 rnb) is higher. Table II presents t’he results obtained when 2-PDS and 4-PDS were used for SH determination of two pure proteins, various tissue homogenates, and human urine. Literature values for t’he same tissues are included in Table II to allow comparison. The values found by Flesch and Kun (17), who used l- (4 - chloromercuriphenylazo) naphthol-2 and an extraction procedure, and those found by Merville et al. (18), who used o-iodosobenzoate, do not differ too
much from the values found with our reagents in various tissue homogenates. However, these methods are much more laborious. Amperometric titration of bovine serum albumin (12, 13), and the use of pchloromercuribenzoate with aldolase (15), gave results in full agreement with those report,ed here. The value for the SH groups of aldolase (Table II) was obtained in the presence of 5 M urea at room temperature: under these conditions 2-PDS and 4-PDS react rapidly with the SH groups of the protein (about 90% reaction in 15 minutes). In the absence of urea the reaction is much slower and incomplete, as evidenced by one experiment in which aldolase was treated with 1O-3 M 2PDS or 10d3M 4-PDS in KRP buffer at room temperature. In this case 2-PDS reacted with 7-S SH groups in 30 minutes, with 10 SH groups in 2 hours, and with 13 SH groups after 20 hours; no further reaction
16
I. 4
1.0 z 5 0.8 (0 4” 0.6
Micromoles
cysteine
FIG. 6. Calibration curves of 4-PDS. 0 Absorbance at 324 rnp; a absorbance at 247 ma. Zero to 0.28 rmole of cysteine was added to 0.038 pmole of 4-PDS (in 0.1 ml KRP buffer) in the spectrophotometer cuvette. Volume was brought to 3.0 ml with KRP buffer, pH 7.2, and absorbance was measured immediately after mixing.
Wavelength
mp
FIG. 7. Solid line: UV spectrum of a mixture of 151 mpmoles of 2-PDS and 404 mpmoles of cysteine in 3.0 ml of KRP buffer, pH 7.2. Broken line: UV spectrum of the supernatant solution obtained by incubating 4.4 pmoles of 2-PDS with 275 X 10” Ehrlich ascites cells (buffer-washed) for one hour at 37” in 5 ml of KRP buffer, pH 7.2. This suspension was centrifuged 10 minutes at 3506 9, and then 0.15 ml of the supernatant solution was diluted to 3.0 ml with the same buffer. 46
02 0 Wavelength
m,u
FIG. 8. Solid line: UV spectrum of a mixture of 88 mpmoles of 4-PDS and 214 mpmoles of cysteine in 3 ml of KRP buffer, pH 7.2. Broken line: UV spectrum of the supernatant solution obtained by incubating 4.8 pmoles of 4-PDS with 387 X lo6 intact Ehrlich ascites cells (buffer-washed) for one hour at 37” in 5 ml of KRP buffer, pH 7.2. This suspension was centrifuged for 10 minutes at 3500 g; 0.1 ml of the supernatant solution was then diluted to 3 ml with the same buffer. TABLE SH CONTENT No. ietns.
Material
OF SOME
II
PROTEINS,
TISSUES,
AND
OF
Jaluesa obtained by present method using Units
2-PDS
Bovine serum albumin Mw = 66,OOOb
5
SH/mole
Aldolasec (MW = 147,000b Ehrlich ascites cells (homogenized)
3 5
SH/mole pmoles SH/mg N mmoles SH/ 100 gm dry mmoles SH/ 100 gm fresh mmoles SH/ 100 gm fresh mmoles SH/ 100 gm fresh mmoles/liter mmoles/liter
5 Mouse
liver
Mouse kidney Mouse Human Human
heart urine urine
homogenate homogenate homogenate (fasted) (fed)
5 5 5 4 4
URINE
3.672 f
T
Values from literature
4-PDS
0.010
-I-0.687
28.1 f 1.0 0.554d
27.4 zk 1.4 0.54Bd
0.67 (12); 0.68 (13) ; 0.60 (14); 0.36 (5) 28 (15) 1.206 (la)
7.84 + 0.14
7.72 zt 0.14
-
2.79 f
0.07
2.71 f
1.79 f
0.08
1.78 zk 0.08
1.27 =t 0.04 0.161 f 0.278 f
0.020 0.020
z!c 0.002
0.05
1.23 rt O.OG 0.169 & 0.021 0.271 f 0.024 _-
1.08-1.80 (17) 3.1 (18) 1.65 (17); 2.6 (18) 1.27 (17)
0.038 (5)
= Figures are given f standard error. b Values from “The Proteins,” Hans Neurath, ed., Vol. I, p. 388, Academic Press, New York (1963). Further references given there. c Determined in the presence of 5 M urea. See text. d Calculated from values based on dry weight. mg dry weight = mg N X 7.07 X 1OW. Thus 7.84X 7.07 x 10-Z = 0.554. 47
48
GRASSETTI
was observed up to 70 hours. 4-PDS reacted with 15 SH groups of aldolase after 20 hours. The behavior of these new reagents is similar to that observed by Swenson and Boyer (15) for p-chloromercuribenzoate with aldolase; in the absence of urea, these authors found S-11 SH groups in aldolase after 90 minutes of reaction. When aldolase (1 mg in 3 ml) was treated with 4-PDS (lop3 M in KRP buffer), the solution became cloudy after l-2 hours. The absorbance of the clear solution obtained after centrifugation was used to estimate the SH content. No cloudiness was caused by 2-PDS under the same conditions. Srere (19) has observed that when the SH content of crystalline citrate condensing enzyme is determined with p-chloromercuribenzoate (pCMB), a precipitate of pCMB-enzyme is formed that does not form when the analysis is carried out in 4 M urea. We have observed that citrate con-
3
AND
MURRAY
obtained densing enzyme (Boehringer, through Calbiochem, Los Angeles, California) and 2-PDS in the absence of urea do not give a precipitate. Preliminary experiments with 2-PDS indicate that the values found for the SH content of citrate-condensing enzyme are the same as Srere found with pCMB, in the absence as well as presence of 4 M urea. During the course of this investigation, we were impressed by the findings of Jocelyn (14), who showed the importance of pH in the determination of SH groups by 5,5’ - dithiobis - (2 - nitrobenzoic acid) (DTNB). Whereas Ellman (5) found only 0.36 SH per mole of bovine serum albumin at pH 8 with DTNB, Joceyln reported a value of 0.6 at pH 7.6 and 0 at pH 6.8 (14). Values obtained with PDS were 0.674.69 SH per mole when the pH of the medium was varied from pH 6.2 to 8.4. Thus, in the range of physiological pH, the theoretical
I
I
I
I
I
I
I
I
4
5
6
7
8
9
IO
II
PH
FIG. 9. Values for SH determined with 8-PDS (0). Each cuvette contained 1.5 ml of 2 X 1W3 M 2-PDS in water, 1.5 ml of 0.11 M universal buffer (26) of varying pH, and 0.01 ml of 1.1 X 1OW M cysteine in water. Absorbance (343 rnp) was measured against a blank without cysteine. Values for SH determined with DTNB (A). Each cuvette contained 0.03 ml of 1 X 10e2 M DTNB (in 0.1 M phosphate buffer of pH 7.2), 3.0 ml of 0.11 M universal buffer (20) of varying pH, and 0.01 ml of 1.1 X 1OW M cysteine in water. Absorbance (412 ma) was measured against a blank without cysteine. SH content was calculated from molar extinction coefficients of 7.06 X 103 for 2.PDS and 1.36 X lo4 for DTNB (5).
DETERMINATION
OF SULFHYDRYL
SK content of bovine serum albumin can be obtained with these new reagents. At pH 3.3, no reaction occurred between DTNB and cysteine; however, when the pH of the medium was increased, theoretical SH values were obtained in the range of pH between 7.5 and 10.4 (Fig. 9). When 2PDS was used as the reagent, correct SH values were obtained in the pH range of 3.4S.1 (Fig. 9). Thus, the range of pH at which 2-PDS can be effectively used CLYan SH reagent is broader and includes those pH values within the physiological range. The dependence of the DTNB assay on the pH of the medium can be ascribed to the nature of the product formed. Smith et al. (6) showed that reactions of mercaptans with disulfides in which an anion is produced are dependent on pH. 2,2’-dithiodipyridine does not react with either ascorbic acid or reduced diphosphopyridine nucleotide at pH 7.2, under the conditions of these assays. In addit,ion, Fe++ ions do not react with 2-PDS in aqueous solution, in the presence or absence of EDTA. ACKNOWLEDGMENTS The authors wish to thank Dr. S. Abraham for helpful discussions and advice, and Dr. M. E. Brokke and A. D. Gutman for preparing the compounds used in this work. REFERENCES 1. GRBSSETTI, I). R., BHOKKE, M. E., AND MURRAY, J. F., JR., J. Med. Chem. 8, 753 (1965). 2. UMBREIT, W. W., BURRIS, R. H., AXD ST.~UF-
3.
4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. Id. 19. 20.
GROUPS
49
Techniques,” p. FER, J. F., “Manometric 149. Burgess, Minneapolis (1957). ALBERT, A., AND BARLIN, G. B., in “Current Trends in Heterocyclic Chemistry” (A. Albert, G. M. Badger, and C. W. Shoppee, eds.), p. 51. Butterworths, London (1958). ELLMAN, G. L., Arch. Biochem. Biophys. 74, 443 (1958). ELLMAN, G. L., dxh. Biochem. Biophys. 82, 70 (1959). SXITH, H. A., DOUGHTY, G., :INU GORIN, G., J. Org. Chem. 29, 1484 (1964). BITNY-SZLECHTO, S., in. “Progress in Biochemical Pharmacology” (It. Paoletti and R. Vertua, eds.), Vol. 1, p. 112. Butterworths, Washington (1965). M~RCKWALD, W., KLEMM, W., .INU TR~BERT, H., Chem. Ber. 33, 1556 (1900). COMRIE, A. M., ;\ND STENL~~KE, J. B., J. Chem. Sot. 1968, 1853. SCHNEIDER, F., .\ND REINFELD, E., Biochem. 2. 318, 507 (1948). L.IYXE, E., in “Methods ill Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 454, second equation. Academic Press, New York (1957). BENESCH, R.E., L.~RDY, II. A., AND BENESCH, R., J. Biol. Chem. 216, G63 (1955). KOLTHOFF, I. M., AND AN.IST,\SI, 9., J. Am. Chem. Sot. 80, 4248 (1958). JOCELYN, I’. C., Biochem. J. 86, 480 (1962). SWENSON, A. D., AND BOYER, P. D., J. Sm. Chem. Sot. 79, 2174 (1957). DIP~OLO, J. A., J. Cell Camp. Physiol. 65, 57 (1965). FLESCH, P., AND KUN, E., F’roc. Sot. Exptl. Biol. Med. 74, 249 (1950). MERVILLE, R., DEQUIDT, J., BND CORTEEL, M. L., Ann. Pharm. Franc. 18, 625 (1960). SRERE, P. A., Biochem. Biophys. Res. Commun. 18, 87 (1965). BRITTON, H. T. S., ND WELFORD, G., J. Chem. Sot. 1937, 1848.
OF
BIOCHEMISTRY
AND
Determination
BIOPHYSICS
119,
(1967)
41-49
of Sulfhydryl
Groups
with
2,2’-
or 4,4’-
Dithiodipyridine’ D. R. GRASSETTI,’ Institute
AND
J. F. !MURRAY,
JR.
of Chemical Biology, University of San Francisco, and Arequipa 1645 Russ Building, San Francisco, California 94104 Received
Foundation,
July 5, 19GG
A procedure is described that permits determination of SH grol~ps in simple compounds and in biological materials. The procedure utilizes the reaction of 2,2’-dit,hiodipyridine or of 4,4’-dithiodipyridine with thiols, which gives the corresponding 2- or 4-thiopyridone. The ultraviolet absorption of the thiopyridone formed is measured. The method permits determination of less than 1.5 pg SH when 2,2’-dithiodipyridine is used, and of less than 0.5 pg SH when 4,4’-dithiodipyridine is used. The proposed method is compared with known methods for the determination of snlfhydryl groups.
quite different from those of the corresponding disulfides, which contain the resonating pyridine ring. It is thus possible to follow spectrophotometrically the course of the reaction of 2-PDS and 4-PDS with thiols. Both the disappearance of the disulfide and the formation of the thiopyridone can be followed, and t’hiols can be determined quantitatively. Other disulfides capable of reacting with SH groups with formation of colored or UVabsorbing species are known (4-i).
Grassetti et al. (1) have recently studied the effect of a number of sulfur-containing pyridines on the metabolism of Ehrlich ascites tumor cells. As a consequence of that study it was found that 2-PDS3 and 4PDS react readily and completely with thiols, with the formation of the corresponding thiopyridone, e.g. : 12 s-s
i(’ A,-
)
+
2 RSH-2
c&
+ RSSR S ;
2-Thiopyridone and 4-TP are almost exclusively in the tautomeric thio form, with the mobile hydrogen attached to the nitrogen (3). This causes the ultraviolet absorption spectra of these thiopyridones to be
METHODS 2-Thiopyridone and 4-TP were obtained from Aldrich Chemical Co., Milwaukee, Wisconsin. 2-PDS was prepared by iodine oxidation of 2-TP, according to Marckwald et al. (8), and 4-PDS was prepared by nitric acid oxidation of 4-TP, according to Comrie and Stenlake (9). Homogeneity of 2-PDS and 4-PDS was assayed by thin-layer chromatography on Florisil: one single spot was given with three or more solvent systems. The following systems were used for 2-PDS: tolllenemethanol [80:20], benzene-met,hanol [95:5], benzene-chloroform [l:l), and benzene-acetone [90: 101. The following were used for 4-PIX: tolueneacetone [90:10], toluene-chloroform [l:l], and toluene-methanol [95:5). The spots were vislialized by iodine vapor. The dihydrochloride of 2-PIE and the dinitrate
1 This investigation was supported by Public Health Service grant CA 07296, from the National Cancer Institute. 2 Inquiries concerning this paper should be directed to Arequipa Foundation, Russ Building, San Francisco, California 94104. 3 Abbreviations used: 2-PDS = 2,2’-dithiodipyridine (from the empirical name “pyridine disulfide”); 4-PDS = 4,4’-dithiodipyridine; 2. TP = 2-thiopyridone; 4-TP = 4-thiopyridone; KRP buffer = Krebs-Ringer phosphate buffer, pH 7.2 (2). 41
42
GRASSETTI
AND MURRAY
of 4-PDS were used in our early experiments; we subsequently found that these salts slowly decompose on standing even in the crystalline state. The crystalline free bases, however, are stable for at least several months. When dissolved in KRP buffer, the bases decompose slowly: it is thus desirable to prepare fresh solutions daily and to carry blanks throughout the procedure. Heating of the solution accelerates the decomposition. Bovine serum albumin was the crystalline Pentex Corp. product; rabbit muscle aldolase was A-grade Boehringer preparation; both were obtained through Calbiochem, Los Angeles, California. Mercaptopyruvic acid was prepared according to Schneider and Reinfeld (10). Cysteine and the other thiols used in this work were reagent-grade products. ASSAY PROCEDURES Simple thiols and proteins. A suitable amount of the material to be analyzed (0.0150.5 pmole SH) was dissolved in 1.5 ml of KRP buffer. This solution was mixed with 1.5 ml of PDS solution (2 X 10e3M) in the same buffer. The UV absorbance at the appropriate wavelength could be determined immediately after mixing in the case of simple thiols, and after 15 minutes at room temperature
in the case of bovine serum albumin. Longer delays did not affect the absorbance. Rabbit muscle aldolase was analyzed in the presence of 5 M urea in KRP buffer; the absorbance was determined after 90 minutes at room temperature. Protein determinations were carried out by the 280/260 method, using Layne’s coefficients (11). Tissue homogenates. Organs from healthy female Swiss mice were homogenized in KRP buffer (1:lO) at 24” in a Potter-Elvehjem homogenizer provided with a Teflon pestle. The homogenate was heated in a water bath at 90” for 5 minutes and then cooled rapidly under running water. The equivalent of approximately 10 mg of tissue (0.1 ml homogenate) was incubated with 4.9 ml of PDS solution in the same buffer (lo-* M) for 15 minutes at 37”, with shaking. The suspension was filtered and the absorbance of the filtrate was determined at the appropriate wavelength against a PDS blank. A sample of tissue was carried through the same procedure, in absence of PDS, in order to determine the blank absorbance. The same procedure was used with washed Ehrlich ascites cells. Urine. Freshly collected human urine was heated 5 minutes at 90” in a water bath. One ml of the cooled urine sample was then incubated 15 minutes at 37” with 4.0 ml of PDS (lOya M in KRP
QLS-SQ
IO1 x Id4 M
x = 08x ij 24 Q 0.6-
220
240
260
280 Wavelength
300
320
340
360
380
mp
FIG. 1. UV spectrum of P-PDS in KRP buffer, pH 7.2.
400
DETERMINATION
OF SULFHYDRYL
43
GROUPS
14 12 1.0 0.8 8 6 $ 06 3 04 -
02
-
OL -
I
220
240
260
280
300
Wavelength
320
340
360
380
400
mp
FIG. 2. UV spectrum of 2-TP in KRP buffer, pH 7.2.
buffer). Absorbance at 343 rnp (for 2-PDS) or at 324 rnp (for 4-PDS) was read against a blank prepared as above but containing no PDS. A correction was applied for a PDS blank, which was carried through incubation. Urine normally contains substances that absorb at 343 and 324 rnp; therefore, the size of the urine sample must be adjusted to take this into account. Normally it was found feasible to use samples as large as 1 ml. RESULTS
AND DISCUSSION
Figures l-4 show the absorption spectra of 2-PDS, 4-PDS and their corresponding thiopyridones, in KRP buffer. The extinction values of the UV maxima of these compounds are reported in Table I. When 2-PDS reacts with a thiol, the formation of 2-TP can be followed by the change in absorbance at 343 rnp, since the corresponding disulfide, 2PDS, has virtually no absorption at that wavelength. Conversely, the consumption of 2-PDS can be estimated from its absorption at 233 rnp, provided that a correction for the absorbance of 2-TP at this wavelength be made. Analogous considerations apply to 4-PDS, the consumption of 4-PDS being followed through the absorbance at 247 rnp, and the formation of 4-TP at 324 mM. The extinction of 4-TP at 324 rnp is almost three
TABLE I UV ABSORPTION IN KRP BUFFER, PH 7.2 Compound 2,2’-Dithiodipyridine 2-Thiopyridone 4,4’-Dithiodipyridine 4-Thiopyridone
bmx bd
233 281 271 343 247 230 324
Molar
extinction 1.39 9.73 1.04 7.06 1.63 9.6 1.98
x x x x x x X
104 10” 10’ 103 104 lo3 lo*
times as high as the extinction of 2-TP at 343 rnp (Table I) ; thus 4-PDS is the more sensitive reagent. Calibration curves for 2-PDS were obtained (Fig. 5) by adding increasing amounts of cysteine (O-O.9 fimole) to a solution of 2PDS (0.32 pmole). Similarly, Fig. 6 gives calibration curves for 4-PDS disappearance and 4-TP formation. Lower concentrations were used in this case because of the higher extinction of the 4-isomer. Various thiols have been tested for reaction with 2-PDS and with 4-PDS, and were found to form the respective thiopyridones. These compounds include cysteine, reduced glutathione, reduced lipoic acid, mercapto-
44
GRASSETTI
AND MURRAY
pyruvic acid, and coenzyme A. Figure 7 [solid Iine) gives the UV spectrum of a mixture of 2-PDS and cyst.eine (in slight excess) in KRP buffer. This curve is representative of the spectrum obtained from
mixtures of 2-PDS and other thiols as well; it is essentially identical with the spectrum of 2-TP (Fig. 2). The dotted curve in Fig. 7 was obtained by incubating a solution of 2PDS with intact washed Ehrlich ascites
3.1x d
Wavelength
M
m,u
FIG. 3. UV spectrum of 4-PDS in KRP buffer, pH 7.2.
7.0 x 10-5M
0” 0.8 5 i $ 0.6 04-
01
I
1
I
I
I
220
240
260
280
300
Wavelength
I
320
340
360
m/l
FIG. 4. UV spectrum of 4-TP in KRP buffer, pH 7.2.
380
400
DETERMINATION
01
02
03
OF SULFHYDRYL
0.4
05
Mcromoles
0.6
07
45
GROUPS
0%
09
IO
cysteine
FIG. 5. Calibration curves of 2-PDS. 0 Absorbance at 343 ml; A absorbance at 233 rnp. Zero t,o 0.9 pmole of cysteine was added to 0.318 pmole of 2-PDS (in 0.3 ml KRP buffer) in the spectrophotometer cuvette. Volume was brought to 3.0 ml with KRP buffer, pH 7.2, after mixing. and absorbance was measured immediately
tumor cells, in amounts sufficient to give full reaction. The similarity in the two curves of Fig. 7 shows that the method is directly applicable to biological materials. Analogous results were obtained with mixtures of 4PDS and t’hiols, and of 4-PDS and intact Ehrlich ascites cells, as shown in Fig. 8. In this case, although the UV spectrum of the reaction mixture is very similar to that of 4TP, the end-absorption (below 230 rnb) is higher. Table II presents t’he results obtained when 2-PDS and 4-PDS were used for SH determination of two pure proteins, various tissue homogenates, and human urine. Literature values for t’he same tissues are included in Table II to allow comparison. The values found by Flesch and Kun (17), who used l- (4 - chloromercuriphenylazo) naphthol-2 and an extraction procedure, and those found by Merville et al. (18), who used o-iodosobenzoate, do not differ too
much from the values found with our reagents in various tissue homogenates. However, these methods are much more laborious. Amperometric titration of bovine serum albumin (12, 13), and the use of pchloromercuribenzoate with aldolase (15), gave results in full agreement with those report,ed here. The value for the SH groups of aldolase (Table II) was obtained in the presence of 5 M urea at room temperature: under these conditions 2-PDS and 4-PDS react rapidly with the SH groups of the protein (about 90% reaction in 15 minutes). In the absence of urea the reaction is much slower and incomplete, as evidenced by one experiment in which aldolase was treated with 1O-3 M 2PDS or 10d3M 4-PDS in KRP buffer at room temperature. In this case 2-PDS reacted with 7-S SH groups in 30 minutes, with 10 SH groups in 2 hours, and with 13 SH groups after 20 hours; no further reaction
16
I. 4
1.0 z 5 0.8 (0 4” 0.6
Micromoles
cysteine
FIG. 6. Calibration curves of 4-PDS. 0 Absorbance at 324 rnp; a absorbance at 247 ma. Zero to 0.28 rmole of cysteine was added to 0.038 pmole of 4-PDS (in 0.1 ml KRP buffer) in the spectrophotometer cuvette. Volume was brought to 3.0 ml with KRP buffer, pH 7.2, and absorbance was measured immediately after mixing.
Wavelength
mp
FIG. 7. Solid line: UV spectrum of a mixture of 151 mpmoles of 2-PDS and 404 mpmoles of cysteine in 3.0 ml of KRP buffer, pH 7.2. Broken line: UV spectrum of the supernatant solution obtained by incubating 4.4 pmoles of 2-PDS with 275 X 10” Ehrlich ascites cells (buffer-washed) for one hour at 37” in 5 ml of KRP buffer, pH 7.2. This suspension was centrifuged 10 minutes at 3506 9, and then 0.15 ml of the supernatant solution was diluted to 3.0 ml with the same buffer. 46
02 0 Wavelength
m,u
FIG. 8. Solid line: UV spectrum of a mixture of 88 mpmoles of 4-PDS and 214 mpmoles of cysteine in 3 ml of KRP buffer, pH 7.2. Broken line: UV spectrum of the supernatant solution obtained by incubating 4.8 pmoles of 4-PDS with 387 X lo6 intact Ehrlich ascites cells (buffer-washed) for one hour at 37” in 5 ml of KRP buffer, pH 7.2. This suspension was centrifuged for 10 minutes at 3500 g; 0.1 ml of the supernatant solution was then diluted to 3 ml with the same buffer. TABLE SH CONTENT No. ietns.
Material
OF SOME
II
PROTEINS,
TISSUES,
AND
OF
Jaluesa obtained by present method using Units
2-PDS
Bovine serum albumin Mw = 66,OOOb
5
SH/mole
Aldolasec (MW = 147,000b Ehrlich ascites cells (homogenized)
3 5
SH/mole pmoles SH/mg N mmoles SH/ 100 gm dry mmoles SH/ 100 gm fresh mmoles SH/ 100 gm fresh mmoles SH/ 100 gm fresh mmoles/liter mmoles/liter
5 Mouse
liver
Mouse kidney Mouse Human Human
heart urine urine
homogenate homogenate homogenate (fasted) (fed)
5 5 5 4 4
URINE
3.672 f
T
Values from literature
4-PDS
0.010
-I-0.687
28.1 f 1.0 0.554d
27.4 zk 1.4 0.54Bd
0.67 (12); 0.68 (13) ; 0.60 (14); 0.36 (5) 28 (15) 1.206 (la)
7.84 + 0.14
7.72 zt 0.14
-
2.79 f
0.07
2.71 f
1.79 f
0.08
1.78 zk 0.08
1.27 =t 0.04 0.161 f 0.278 f
0.020 0.020
z!c 0.002
0.05
1.23 rt O.OG 0.169 & 0.021 0.271 f 0.024 _-
1.08-1.80 (17) 3.1 (18) 1.65 (17); 2.6 (18) 1.27 (17)
0.038 (5)
= Figures are given f standard error. b Values from “The Proteins,” Hans Neurath, ed., Vol. I, p. 388, Academic Press, New York (1963). Further references given there. c Determined in the presence of 5 M urea. See text. d Calculated from values based on dry weight. mg dry weight = mg N X 7.07 X 1OW. Thus 7.84X 7.07 x 10-Z = 0.554. 47
48
GRASSETTI
was observed up to 70 hours. 4-PDS reacted with 15 SH groups of aldolase after 20 hours. The behavior of these new reagents is similar to that observed by Swenson and Boyer (15) for p-chloromercuribenzoate with aldolase; in the absence of urea, these authors found S-11 SH groups in aldolase after 90 minutes of reaction. When aldolase (1 mg in 3 ml) was treated with 4-PDS (lop3 M in KRP buffer), the solution became cloudy after l-2 hours. The absorbance of the clear solution obtained after centrifugation was used to estimate the SH content. No cloudiness was caused by 2-PDS under the same conditions. Srere (19) has observed that when the SH content of crystalline citrate condensing enzyme is determined with p-chloromercuribenzoate (pCMB), a precipitate of pCMB-enzyme is formed that does not form when the analysis is carried out in 4 M urea. We have observed that citrate con-
3
AND
MURRAY
obtained densing enzyme (Boehringer, through Calbiochem, Los Angeles, California) and 2-PDS in the absence of urea do not give a precipitate. Preliminary experiments with 2-PDS indicate that the values found for the SH content of citrate-condensing enzyme are the same as Srere found with pCMB, in the absence as well as presence of 4 M urea. During the course of this investigation, we were impressed by the findings of Jocelyn (14), who showed the importance of pH in the determination of SH groups by 5,5’ - dithiobis - (2 - nitrobenzoic acid) (DTNB). Whereas Ellman (5) found only 0.36 SH per mole of bovine serum albumin at pH 8 with DTNB, Joceyln reported a value of 0.6 at pH 7.6 and 0 at pH 6.8 (14). Values obtained with PDS were 0.674.69 SH per mole when the pH of the medium was varied from pH 6.2 to 8.4. Thus, in the range of physiological pH, the theoretical
I
I
I
I
I
I
I
I
4
5
6
7
8
9
IO
II
PH
FIG. 9. Values for SH determined with 8-PDS (0). Each cuvette contained 1.5 ml of 2 X 1W3 M 2-PDS in water, 1.5 ml of 0.11 M universal buffer (26) of varying pH, and 0.01 ml of 1.1 X 1OW M cysteine in water. Absorbance (343 rnp) was measured against a blank without cysteine. Values for SH determined with DTNB (A). Each cuvette contained 0.03 ml of 1 X 10e2 M DTNB (in 0.1 M phosphate buffer of pH 7.2), 3.0 ml of 0.11 M universal buffer (20) of varying pH, and 0.01 ml of 1.1 X 1OW M cysteine in water. Absorbance (412 ma) was measured against a blank without cysteine. SH content was calculated from molar extinction coefficients of 7.06 X 103 for 2.PDS and 1.36 X lo4 for DTNB (5).
DETERMINATION
OF SULFHYDRYL
SK content of bovine serum albumin can be obtained with these new reagents. At pH 3.3, no reaction occurred between DTNB and cysteine; however, when the pH of the medium was increased, theoretical SH values were obtained in the range of pH between 7.5 and 10.4 (Fig. 9). When 2PDS was used as the reagent, correct SH values were obtained in the pH range of 3.4S.1 (Fig. 9). Thus, the range of pH at which 2-PDS can be effectively used CLYan SH reagent is broader and includes those pH values within the physiological range. The dependence of the DTNB assay on the pH of the medium can be ascribed to the nature of the product formed. Smith et al. (6) showed that reactions of mercaptans with disulfides in which an anion is produced are dependent on pH. 2,2’-dithiodipyridine does not react with either ascorbic acid or reduced diphosphopyridine nucleotide at pH 7.2, under the conditions of these assays. In addit,ion, Fe++ ions do not react with 2-PDS in aqueous solution, in the presence or absence of EDTA. ACKNOWLEDGMENTS The authors wish to thank Dr. S. Abraham for helpful discussions and advice, and Dr. M. E. Brokke and A. D. Gutman for preparing the compounds used in this work. REFERENCES 1. GRBSSETTI, I). R., BHOKKE, M. E., AND MURRAY, J. F., JR., J. Med. Chem. 8, 753 (1965). 2. UMBREIT, W. W., BURRIS, R. H., AXD ST.~UF-
3.
4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. Id. 19. 20.
GROUPS
49
Techniques,” p. FER, J. F., “Manometric 149. Burgess, Minneapolis (1957). ALBERT, A., AND BARLIN, G. B., in “Current Trends in Heterocyclic Chemistry” (A. Albert, G. M. Badger, and C. W. Shoppee, eds.), p. 51. Butterworths, London (1958). ELLMAN, G. L., Arch. Biochem. Biophys. 74, 443 (1958). ELLMAN, G. L., dxh. Biochem. Biophys. 82, 70 (1959). SXITH, H. A., DOUGHTY, G., :INU GORIN, G., J. Org. Chem. 29, 1484 (1964). BITNY-SZLECHTO, S., in. “Progress in Biochemical Pharmacology” (It. Paoletti and R. Vertua, eds.), Vol. 1, p. 112. Butterworths, Washington (1965). M~RCKWALD, W., KLEMM, W., .INU TR~BERT, H., Chem. Ber. 33, 1556 (1900). COMRIE, A. M., ;\ND STENL~~KE, J. B., J. Chem. Sot. 1968, 1853. SCHNEIDER, F., .\ND REINFELD, E., Biochem. 2. 318, 507 (1948). L.IYXE, E., in “Methods ill Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 454, second equation. Academic Press, New York (1957). BENESCH, R.E., L.~RDY, II. A., AND BENESCH, R., J. Biol. Chem. 216, G63 (1955). KOLTHOFF, I. M., AND AN.IST,\SI, 9., J. Am. Chem. Sot. 80, 4248 (1958). JOCELYN, I’. C., Biochem. J. 86, 480 (1962). SWENSON, A. D., AND BOYER, P. D., J. Sm. Chem. Sot. 79, 2174 (1957). DIP~OLO, J. A., J. Cell Camp. Physiol. 65, 57 (1965). FLESCH, P., AND KUN, E., F’roc. Sot. Exptl. Biol. Med. 74, 249 (1950). MERVILLE, R., DEQUIDT, J., BND CORTEEL, M. L., Ann. Pharm. Franc. 18, 625 (1960). SRERE, P. A., Biochem. Biophys. Res. Commun. 18, 87 (1965). BRITTON, H. T. S., ND WELFORD, G., J. Chem. Sot. 1937, 1848.