Polarographic and Am perometric M ercurometric Determination of Disulfide Groups In Cystine, Oxidized Glutathione, and Proteins WALTER STRICKS, I. M. KOLTHOFF, and NOBUYUKI TANAKA' School o f Chemistry, University o f Minnesota, Minneapolis 14, M i n n .
MATERIALS
From a polarographic study of the reaction in alkaline medium in the presence of sulfite between mercuric mercury and disulfide groups in amino acids, peptides, denatured proteins, and acid protein hydrolyzates, it was inferred that this reaction can be made the basis of an accurate mercurimetric amperometric titration as well as of a direct polarographic determination of disulfide in biological materials, using the dropping mercury electrode as indicator electrode. Complete hydrolytic disintegration of the protein into amino acids, as practiced in the conventional methods of disulfide determination in biological material can be replaced by a more rapid procedure involving pepsin digestion and subsequent partial hydrolysis in 1.M hydrochloric acid. Procedures are given for the accurate determination of disulfide in cystine, oxidized glutathione, and denatured and partially hydrolyzed protein. An accurate and rapid determination of disulfide is of great significance in investigations of biological material such as normal and pathological blood serum. The new method is more rapid, specific, and precise than methods which have been used previously.
Cystine was a Merck reagent grade product. Directions for the preparation of cystine stock solutions have been iven previously ( 5 ) . Reduced glutathione was a Pfanstiehf product which was found t o be 99% pure by amperometric titration with cupric copper (4). A stock solution of oxidized glutathione was prepared as described in a previous paper (IO). Crystalline bovine plasma albumin was an Armour product which was found to contain 6% water. The albumin stock solution was about 7% in albumin and was kept in a refrigerator. All the other chemicals used were commercial C.P. reagent grade products. EXPERIMENTAL METHODS
Current voltage curves were obtained with a manual apparatus and circuit, described by Lingane and Kolthoff (8). The same apparatus was used in the amperometric titrations. All current voltage curves were taken at 25' i 0.1" C. Potentials are expressed versus the saturated calomel electrode (S.C.E.). Oxygen was removed from solution with a stream of oxygen-free nitrogen. During an experiment an atmosphere of nitrogen was maintained over the solution. A layer of chloroform a t the bottom of the cell was used to prevent the mercury metal from reacting with excess mercury in solution. Corrections were made for the residual current. The volume of the solution in the polarographic cell was about 20 ml. All currents were referred to the same volume (20 ml.). The characteristics of the capillary used were: m = 2.17 mg. see.-]; t = 3.91 sec. (open circuit); m213 t ' ' 6 = 2.104 mg.*l3 see.-'/*; and h = 60 cm. Preparation of Solutions of Pepsin-Digested Albumin. Ten milliliters of a 10-3.W albumin solution were placed in a 50-ml. volumetric flask and diluted to the mark with a .solution which was 0.005% in pepsin (a Pfanstiehl product, indicated atrength 1 to 10,000) and 0.05-lf in hydrochloric acid. This mixture was shaken thoroughly and allowed to stand for 1 hour a t 25' C. Preparation of Solutions. of Hydrolyzed Albumin. AFTER PEPSIN DIGESTIOK. Ten milliliters of the digest were placed in a 50-ml. round-bottomed flask provided with standard taper. To this solution were added 2 ml. of 6M hydrochloric acid or 7.15 ml. of 1 2 X hydrochloric acid if the hydrolysis was to be carried out, in 1M or 5M hydrochloric acid, respectively. The resulting solution was refluxed on a sand bath for various periods of time and then transferred quantitatively to a 25-ml. volumetric flask. Water was added to fill to the mark. The resulting solution was 8 X 10-5M with reference to original albumin and approximately 0.5M or 3.4111in hydrochloric acid. BEFOREPEPSINDIGESTION.Four milliliters of a albumin stock solution were placed in a 20-ml. round-bottomed flask with standard taper and 2.9 ml. of 12M hydrochloric acid was added to make the resulting solution 5 X in hydrochloric acid. The mixture was refluxed and diluted in the same way as described in the previous paragraph. For the measurement of current voltage curves and for the titrations appropriate volumes of the hydrolyzates were added to a given volume of an air-free sulfite containing buffer solution which had been placed in an electrolysis cell. The acid content of hydrolyzates prepared with 5-11 hydrochloric acid was determined before being analyzed. For this purpose a separate sample of hydrolyzate (usually 1 ml.) was titrated with 1 M sodium hydroxide using methyl red as indica tor The proper volume of sodium hydroxide required for the neutralization of the hydrolyzate was added to the buffer in the electrolysis cell, thus taking care of the excess acid in the hydrolyzate. Mercurimetric Titration of Cystine (RSSR), Reduced (GSH), and Oxidized Glutathione (GSSG). Air-free solutions of cystine, reduced and oxidized glutathione in borax buffer and in ammoniacal medium, both at pH 9.2 in the presence of sulfite,
P
REVIOUS to the preqent work a simple amperometric titration technique with the rotated platinum wire electrode as indicator electrode and with mercuric mercury as reagent had been developed for the determination of sulfhydryl groups in amino acids, peptidet, and pioteins (6). Sodium sulfite interferes because of formation of a complex with excess mercury, which is not reduced at the platinum electrode under the experimental conditions of the titrations. At the dropping mercury electrode sulfite has practically no effect on the reduction xave of the excess mercury under the same experimental conditions. Use of this is made in an amperometric titration of disulfide in the presence of sulfite. .4t a suitable pH disulfide reacts with sulfite according to the equation ( 9 ) RSSR
+ SO;- ~t RS- + RSSO;
(1)
On the addition of increasing amounts of mercury, RSreacts to form a slightly dissociated mercaptide, Hg(RS)2, and the reaction of Equation 1 goes to completion, the final products being the mercaptide and the sulfonate. A polarographic study of solutions containing cystine (RSSR), reduced glutathione (GSH), or oxidized glutathione (GSSG) in the preqence of sodium sulfite and various concentrations of mercuric mercury revealed that disulfide can be titrated accurately with mercuric chloride at the dropping mercury electrode. Thi.: method can be successfully applied to the determination of disulfide in solutions of partially and completely hydrolyzed proteins. Also, the anodic diffusion current of cysteine observed in an alkaline solution of acid-hydrolyzed portein in the presence of sulfite can be used for a simple direct polarographic determination of disulfide in protein. 1
On k a v e from Tokyo University, Tokyo, Japan
299
300
ANALYTICAL CHEMISTRY
were titrated with 0.01M mercuric chloride. Complete polarograms were taken after the addition of various amounts of mercury.
soon becomes straight on further addition of mercury. The intersection of the straight portion of the exceGs reagent line (dotted line in Figure 3) with the line presenting the residual A few PolaWPms are Presented in Figures 1 and 2 of mixtures current corresponds to the mole ratio of mercury to GSSG of of reduced and oxidized glutathoine in a borax buffer in the pres1 to 2. Mercuric chloride in an ammonia buffer gives a polarogram of abnormal appearance. -4 flat maximum is observed between -0.2 and -0.3 volt and a pronounced minimum a t -0.5 volt (see Figure 4). At -0.8 volt the current attains the same value as a t -0.25 volt. The current at -0.25 volt is proportional to 3 the mercury concentration and corresponds to the diffusion current. The appearance of the polarogram resembles that given by Laitinen and Onstott ( 7 ) for plati2 W num(I1) under certain conditions. ApW a 3 parently the mercury ammine is re4 , duced in the adsorbed state at potentials P of about -0.2 to -0.3 volt. At more X negative potentials the complex is apparently desorbed and this is accompanied W W by a decrease of current. At potentials P ' more negative than -0.5 volt (isoelectric 8 I point) reduction of the complex occurs in 0 the unadsorbed state. The polarogram 0 becomes normal on the addition of a trace -1 of cystine (curve C in Figure 4). The small second wave in polarogram C corI Figure 1. Current Voltage Curves of responds to the reduction of mercuric Mixtures of 5 X lO-4M Reduced cysteinate. The effect of the mercuric Glutathione in 0.05M Borax Buffer Figure 2. Current Voltage Curves of &5ixtures of 5 x 10-4M Oxidized cysteinate upon the shape of the mercury with Various Amounts of 0.01M Mercuric Chloride Glutathione in 0.05M Borax Buffer ammine wave is not understood a t preswith Various Amounts of 0.01M ent and will be subject to further study. Borax buffer. 0.5Jf KC1, 0.2.M Na2SOI Mercuric Chloride A . Residual current The polarogram of mercuric chloride in B . 5 X lO-4M GSH (no mercury added) Borax buffer. 0.5M KCl, 0.2M NanSOa borax in the presence of sulfite has a C. After addition of 0.4 ml. mercuric chloride A . After adding 0.4 ml. mercuric chloride D. After addition of 0.8 ml. mercuric chloride B. After adding 0.8 ml. mercuric chloride normal appearance. E . After addition of 1.2 ml mercuric chloride
'I
1
ence of sulfite. Titration curves constructed from Figures 1 and 2 by plotting the current measured at -0.35 volt versus the volume of mercuric solution added are illustrated in Figure 3. A large excess of sulfite was used, so that practically all of the disulfide reacted according to Equation 1 to give sulfhydryl. The anodic current measured before addition of mercury corresponds to a molar sulfhydryl concentration which is practically equal to that of the disulfide originally present. Thus the anodic currents of a 5 X lO-4M GSH and 5 x lO-4M GSSG solution in the presence of 0.2M sodium sulfite were found to be 1.6 and 1.45 pa, respectively. The polarography of cysteine, cystine (2, S), and of reduced and oxidized glutathione (IO) has been studied previously. Neither the anodic waves of RSH and GSH nor the cathodic waves of Hg(RS)*and Hg(GSh are affected by the presence of sulfite or the sulfonate (GSSO,-). This is evidenced by the fact that the polarograms obtained with reduced and with osidized glutathione are practically identical (compare Figures 1 and 2). As increasing amounts of mercuric chloride are added to RSSR or GSSG solutions in the presence of sulfite, the anodic wave decreases in height and disappears when the mole ratio of mercury to RSSR(GSSG) is 1 to 2. The height of the cathodic wave of Hg(RS)* IHg(GS)2]reaches a limiting value at this ratio and remains constant on further addition of mercury. The cathodic wave of the excess of mercury starts a t a much more positive potential than that of Hg(RS)* [Hg(GS)*]. A detailed study of reactions between mercuric mercury and reduced glutathione (cysteine) has been reported (11). It is seen from Figure 3 that with cystine the excess reagent line and the reaction line are straight and intersect when the mole ratio of mercury to RSSR is 1 to 2, where the current is equal to the residual current. In titrations of glutathione (both GSH and GSSG), the iines are slightly curved close to the end point. The excess reagent line
Figure 3. Amperometric Titration with 0.01M Mercuric Chloride at Dropping Mercury Electrode as Indicator Electrode at -0.35 Volt vs. S.C.E. 20-ml. solutions used 0.05M borax buffer. 0.5M KC1. 0.2M N&~SOI A . 5 x 1 0 - 4 ~GBH B. 5 x 10-4MGSSG C. 5 X 10-4M RSSR D . Blank (mercuric chloride added t o buffer) -411 titration lines start a t 0 ml. of 0.01M mercuric chloride solution added.
V O L U M E 2 6 , N O . 2, F E B R U A R Y 1 9 5 4
301
PROCEDURE
Place 10 ml. of 0.1M borax solution and 2.5 ml. of 4 M potassium chloride in a polarographic cell. Add 10 ml. of pure chloroform to cover the bottom of the cell. Add enough disulfide (RSSR, GSSG) to make its final concentration 2.5 X 10-4 to 3 X 10-aM. Add 4 ml. of 1M sodium sulfite solution and water to bring the total volume to 20 ml. Introduce the salt bridge and capillary into the mixture and titrate with 10-2Mmercuric chloride solution a t a potential of -0.35 volt. An atmosphere of nitrogen should be maintained over the mixture during the titration. Determine the residual current a t -0.35 volt in a separate experiment. (The titration can be carried out quickly, since the current need not be measured until it has become cathodic.) The intersection of the straight part of the excess reagent line with the residual current is the end point. One milliliter of 0 01M mercuric solution corresponds to 12.24 mg. of oxidized glutathione or 4.806 mg. of cystine. (The borax buffer can be substituted by an ammonia buffer 0.1M in ammonia, 0.1M in ammonium chloride, and 0.2M in sodium sulfite. If an ammonia buffer ip used, the nitrogen must first be passed through a solution of the same composition as that of the buffw in the titration mixture.'
Table I.
Mercurimetric Titrations of Cystine and Oxidized and Reduced Glutathione
(20-ml. solutions a t -0.35 volt Concn. of Substance Added X Substance Titrated 10-3~ 0.260 RSSR (borax buffer) 0.500 1 .oo 1.00 RSSR (ammonia buffer) 0.250 0.500 1.00 3.00 0,250 GSSG (borax buffer) 0.500
Error,
R
t2.0 A1.0 -1.0 -1.0 -2 0 -2.0 -1.5 -0.6
0,505
0,250
0.500 1.00
0.503 1.00;
3.00 0.50 1 .oo
GSH (borax buffer)
Concn. of Substance Found X 10-351 0.25s
0,990 0,990 0.245 0,490 0.985 2.98 0 254 0 510 0 990 3.01 0 498 (3.992 0.242
1 .oo
GSSG (ammonia buffer)
us. S.C.E.)
+;.5
-- . o -1
+o
(J
J
-0.5 -0.8 -3.0 +0.5 +0.3
RESULTS
Results of mercurimetric titrations of cystine and oxidized and reduced glutathione are listed in Table I. 8
1 8
5 4
1
w
a
g P
0
=
2
I t
o
"I
-2
The height of the anodic wave is found to increase with progressing hydrolysis. It is seen from Figure 5 , curve A , that the rate of increase of the diffusion current of sulfhydryl is large in the initial period of the acid hydrolysis and becomes smaller as the hydrolysis proceeds further. Titrations of pepsin-digested albumin before and after acid hydrolysis for 1, 4, 6, 10, and 18 hours were carried out in a borax buffer in the presence of sulfite. Some complete polarograms of titration mixtures after addition of varying amounts of mercuric chloride are illustrated in Figures 6 and 7 These figures demonstrate that the appearance of the cathodic mercaptide waves in albumin hydrolyzate is similar to the corresponding waves in oxidized glutathione and cystine. The height of the mercaptide and of the excess of mercury waves in solutions of constant hydrolyzate and mercury concentration increases with time of acid hydrolysis in a way similar to that of the anodic sulfhydryl wave, as demonstrated by the dotted curve, B , in Figure 5 .
3 -4
-5 -8
Figure 4. Current Voltage Curves of Mercury(I1) Solutions in Ammonia Buffer
0
2
4
e
I
IO
I2
I4
1%
IS
TIME of HYDROLYSIS (HOURS)
Ammonia buffer. 0.ldf "3, 0.l.U NHICI, 0.2M NaZSOa A . 5 X 10-4-M mercury(I1) B . 10-a.W mercury(I1) C. 10-3M mercnry(I1) in presence of 8 X 10-6M cystine
The accuracy and precision of mercurimetric titrations of
GSSG, RSSR, and GSH at the dropping mercury electrode is i l % at concentrations of 2.5 x 10-4 to 10-aM in borax or ammonia buffers. Determination of DisuEde in Bovine Plasma Albumin after Pepsin Digestion and Acid Hydrolysis. The mercurhnetric sulfhydryl titration at the dropping mercury electrode can be applied to the determination of disulfide in proteins. Upon the addition of a protein hydrolyzate to a borax buffer containing sulfite, disulfide reacts to form sulfhydryl compounds which give an anodic wave at the dropping mercury electrode. A complete polarogram of an albumin hydrolyzate (10 hours' boiling in 1M hydrochloric acid after pepsin treatment) in boras in the presence of sulfite is given in Figure 7 (curve A ) .
Figure 5. Height of Anodic Sulfhydryl Wave, A (Measured at -0.35 Volt); Cathodic Mercaptide Wave, B (Alb.: Hy = 0.16:l) (Measured at - 0.8 Volt) in Protein Hydrolyzate after Pepsin Digestion us. Time of Hydrolysis in 1M Hydrochloric Acid Electrolyte: 0.05M borax, .0.5M KCI. 0.2.V SaxSOa, 1.6 X 10-6.U albumin
Titration curves a t -0.35 volt in Figure 8 show that the slope of the excess reagent line is flat in a titration of pepqin-digested albumin without acid hydrolysis but increases markedly upon boiling with hydrochloric acid. As the time of boiling is extended over 4 hours no further increase in the elope of the excess reagent line is observed. The end point in these titrations was obtained in the same way as described for cystine and oxidized glutathione. According to Equation 1, 1 mole of disulfide yields 1 mole of sulfhydryl after reaction with sulfite. The molar
A N A L Y T I C A L CHEMISTRY
302 concentration of sulfhydryl found, therefore, is equal to the molar concentration of disulfide originally in the protein. Titration results are listed in Table 11. A value of 18.7 moles of disulfide per mole of albumin is obtained after 1 hour or longer boiling with 1M hydrochloric acid, in good agreement with the value 18.66 (taking in account the sulfhydryl content of albumin) reported in the literature ( 1 ) . Prolonged boiling does not change the results of the titration. Thus all the disulfide in albumin can be made available for reaction with sulfite after pepsin digestion and subsequent boiling with 1Ai' hydrochloric acid for 1 hour. The data in the third column of Table I1 indicate that not until after 10 hours of boiling with 111.1hydrochloric acid is an i d / c value of the anodic wave a t the start of the titration reached m-hich is approximately equal to that of an equimolar solution of reduced glutathione ( i d l e = 2.6) (IO). In order to ascertain whether the hydrolyzate after 18 hours of boiling with acid has a suppressing effect on the anodic diffusion current of cysteine, a known amount of cystine was added to a hydrolyzate (after 18 hours boiling with 1M hydrochloric acid) in a borax buffer, 0.2M in sodium sulfite and 0.5M in potassium chloride, and the increase in height of the anodic wave was measured after correction for change in volume. In one experiment the increase of the anodic diffusion current in a hydrolyzate, originally 1.6 X
25
20
I5
w
IC
I I : W L
4
g
b!
0
= t
P
-O!
-I
c
-02
-04
E d e 3 VOLT
-06 VI.
-08
-1 0
SCE
Figure 7 . Current Voltage Curves of 20-M1. Mixtures of Acid Albumin Hydrolyzates in 0.05M Borax Buffer with Various Amounts of 0.01M Mercuric Chloride Added Albumin was pepsin-digested and boiled 10 hours in 1M HCl Original albumin concentration was 1.6 X 10-6M Borax buffer: 0.5M KC1, 0.2M Na?SOa A . 1.6 X lO-SrM hydrolyzed albumin (no mercury added) B. A after adding 0.2 ml. mercuric chloride C. A after adding 0.3 ml. mercuric chloride D. A after adding 0.4 ml. mercuric chloride E . A after adding 0.8 ml. mercuric chloride
Table 11. Amperometric Titrations and Anodic Waves of 1.6 X 10-5M Pepsin-Digested and Hydrolyzed Albumin (1M HCl)
0
-02
-04
E de,
-06
VOLT
VI.
-08
-10
S C E
Figure 6. Current Voltage Curves of 20-M1. Mixtures of 2 X 10-6M PepsinDigested Albumin (without Acid Hydrolysis) in 0.05M Borax Buffer with Various Amounts of 0.01M Mercuric Chloride Added Borax buffer: 0.5M KCl, 0.2M NazSOt A . Residual current B. 2 X 10-6M digested albumin (no mercury added) C. Biter adding 0.4 ml. mercuric chloride D. After adding 0.8 ml. mercuric chloride
in added cystine, was 1.71 lO-SM in albumin and 5 X pa while the corresponding value for the anodic current in an electrolyte of the same composition and originally 5 X 10-4M in cystine in the absence of hydrolyzate was found to be 1.76 pa. Thus the suppressive effect of the hydrolyzate on the diffusion current of cysteine is practically negligible. The increase in the anodic i d / c values with time of hydrolysis is indicative of the decrease in particle size of the disulfide-containing constituents in the solution and is a measure of the average diffusion coefficient of these constituents. From the results in Table I1 it follows that disulfide groups in high molecular weight constitu-
(Titrating agent. 0.OliN mercuric chloride. supporting electrolyte. 0.05M borax, 0.5M KC1, 0.2M NaiSOs; 20-ml. 'solutions of albumin used) Time of Boiling Anodic Current', with 1M HC1, Moles Disulfide/ id/c a t -0.35 Volt Hr. N o l e Albumin pa./lO-aM Sulfhydryl 0.21 17.5 1.37 18.7 I 2.34 18.8 4 2.40 6 18.7 2.70 10 18.7 2.94 18.8 18 i d / c is t h e diffusion current, cprrespondinq t o ! O - ' M sulfhydryl. T h e molar Concentration of sulfhydryl is found by titration.
?
ents of denatured or partially hydrolyzed albumin react rapidly with sulfite. The complete disintegration of the protein into amino acids, as practiced in the conventional methods of disulfide determinations in biological materials, can thus be eliminated and replaced by denaturation and partial hydrolysis. Titration results obtained with hydrolyzates of albumin in 5M hydrochloric acid are listed in Table 111. Correct results with these hydrolyzates are obtained after refluxing for 1 to 2 hours. Pepsin digestion has little effect on the results if 5M hydrochloric acid is used. Values of the anodic diffusion currents were found equal to those of equimolar solutions of cysteine in the same supporting electrolyte. Experiments with mixtures of hydrolyzates (from 5M hydrochloric acid) and known amounts of cystine indicated that the hydrolyzate has no suppressive effect on the anodic wave of cysteine. The anodic diffusion current meas-
303
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 Table 111. Amperometric Titration and -4nodic Waves of 1.6 x 10-6M Albumin, Hydrolyzed in 5 M Hydrochloric Acid (Titrating agent: 0 01 M mercuric chloride. supporting electrolyte. 0 0 5 M borax, 0 5-X KCI, 0 23f xa%SOa;i o - m l solutions used) Moles Anodic Wavea, Time of Disulfide/ id/c a t - 0 . 3 5 Boiling with 5 M Mole Volt, fia./lO-3 M Sulfhydryl HC1, Hr. Albumin 1 1
Native albumin
2 2 18
Pepsin-digested albumin
1
1 a
17.2 17.8 1?.8 18.2 18.4
3.53 3.54 3.54 3.43 3.57
18.3 17.5
3.50 3.70
c, molar concentration of sulfhydryl a s found b y titration.
I
-
A
1
0 01 M HP G I rADDED,ml
Figure 8. Mercurimetric Amperometric Titration at Dropping Mercury Electrode as Indicator Electrode at -0.35 Volt us. S.C.E. of 20 M1. of Pepsin-Digested Albumin after Various Times of Boiling with 1M Hydrochloric Acid Hours boiled. A .
B.
c.
0 1
4
D. 6 E . 10 F . 18 0.05M borax, 0.5.M KC1, 0.2M Na&& All curves s t a r t a t 0 ml. 0.01M mercuric chloride added
ured in a hydrolyzate prepared with 5M hydrochloric acid which is added to a borax buffer containing sulfite can thus be used for a direct polarographic determination of disulfide in protein. RECOMMENDED PROCEDURES
Titration. PEPSIN DIGESTIONAND HYDROLYSIS IN liM HYDROCHLORIC ACID. Place 5 ml. of a 7 % protein solution in a 25-ml. volumetric flask and fill up to the mark with a solution
which is 0.005% in pepsin and 0.05M in hydrochloric acid. Shake thoroughly and allow to stand for 1 hour a t 25” C. Place 10 ml. of the digest solution in a 50-ml. round-bottomed flask with standard taper to fit a reflux condenser. Add 2 ml. of 6M hydrochloric acid, put a capillary with sealed end on top into the solution, and reflux on a sand bath for 2 hours. Transfer the solution quantitatively into a 25-ml. volumetric flask and fill up to the mark with distilled water. HYDROLYSIS IS 5M HYDROCHLORIC ACID. Place 2 ml. of a 7% protein solution and 8 ml. of distilled water in a 50-ml. round-bottomed flask with standard taper to fit a reflux condenser. Add 7.2 ml. of 12151 hydrochloric acid and reflux on a sand bath for 2 hours. Transfer quantitatively t o a 25-ml. valumetric flask and fill up to the mark with distilled water. Pipet
off 1 or 2 ml. of this solution and determine the volume of 1M sodium hydroxide required to neutralize the hydrolyzate, using . . methyl red as indicatbr. Place 10 ml. of 0.1M borax solution. 2.5 ml. of TITRATION. 4M potassium chloride, and 10 ml. of chloroform in a polarographic cell. If the hydrolyzate was prepared with 5M hydrochloric acid add the appropriate volume of 1M sodium hydroxide required to neutralize the hydrolyzate. Add 4 ml. of 1M sodium sulfite solution and enough hydrolyzate solution to make the titration mixture 1 to 8 X l O - * M in total disulfide. Sulfhydryl groups contained in the undenatured protein are oxidized to disulfide during hydrolysis. Introduce salt bridge and capillary into the mixture and titrate with an 0.01M mercuric chloride solution at an applied potential of -0.35 volt versus saturated calomel electrode. The further procedure is the same as that in the titration of cystine or GSSG. The end point corresponds to the sum of half the sulfhydryl and tho total disulfide content of the native protein. The sulfhydryl content of the undenatured protein is determined by mercurimetric titration a t the rotating platinum electrode (6). One milliliter of 0.01M mercuric chloride corresponds to 1.283 mg. of disulfide or 4.805 mg. of cystine.
Polarographic Procedure. Place 10 ml. of 0.1M borax solution, 2.5 ml. of 4M potassium chloride, 10 ml. of chloroform, and enough 1M sodium hydroxide to neutralize the volume of hydrolyzate (after refluxing in 5M hydrochloric acid) to be added, in a polarographic cell provided with gas inlet and outlet tube. Cover the cell with a rubber stopper with holes for the salt bridge, dropping mercury electrode, and a pipet. Introduce the salt bridge and place the cell in a thermostat a t 25 C. Add 4 nil. of 1M sodium sulfite solution and enough hydrolyzate to make the to 10-3 in disulfide. Stopper the hole for the solution 5 X pipet. While passing nitrogen over the surface of the solution introduce the dropping mercury electrode and run a polarogram between -0.2 and -0.8 volt. Read the current a t -0.35 volt and refer it to a volume of 20 ml. In a separate experiment measure the diffusion current obtained in a 10-sM cystine solution and the residual current in the same supporting electrolyte with sulfite as used for the sample. From the i d / c value of cysteine and from the current measured in the sample, the sum of RSSR RSH in the protein is calculated. The disulfide content of the protein is calculated in the same way as given for the titration method. The accuracy of the method is of the order of 2%, Thich is of the same order of magnitude as that of the titration. Mercurimetric titrations a t the dropping mercury electrode have been successfully applied to the determination of disulfide in normal and pathological blood sera and their albumin and globulin fractions.
+
ACKNOWLEDGMENT
This investigation was supported by a research grant from the National Cancer Institute, U. S. Public Health Service. LITERATURE CITED
Haurowitz, F., “Chemistry and Biology of Proteins,” New York, Academic Press, Inc., 1950. Kolthoff, I. M., and Barnum, C., J . Am. Chem. SOC.,62, 306 (1940). Ibid., 63, 520 (1941).
Kolthoff, I. M., and Stricks, W., ANAL.CHEM.,23, 763 (1951). Kolthoff, I. M., and Stricks, W., J . Am. C h m . SOC.,72, 1952 (1950).
Kolthoff, I. XI., Stricks, W., and Morren, L., AXAL.CHEM., in press. Laitinen, H. A,, and Onstott, E. I., J . Am. Chem. SOC., 72,4565 (1950).
Lingane, J. J., and Kolthoff, I. M., Ibid., 61, 825 (1939). Stricks, W., and Kolthoff, I. M., Ibid., 73,4569 (1951). Ibid., 74,4646 (1952). Ibid., 75, 5673 (1953). RECEIVEDfor review August 10, 1953.
Accepted October 31, 1953.
