Amplifier Output
-
0
Ground t o
4K
50K
t,,-
II 6000
1 5 v.
3K
L
IO0
Brown 50
1G
3K
Brown Amp.
Span Adj.
Heliput
U
3
1Y 25
type described have been used continuously for several months in laboratory and pilot plant Xork with maintenance required.
15
50 Z e r o Adj. Heiipot
The absorption of beta particles by liquids may be used for very accurate determination of density of binary liquid mixtures or mixtures where t,he content of h y d r o g e n ( c h e m i c a l l y bonded) or heavy elements does not vary significantly. The density of such liquids may be recorded continuously with an accuracy of ~t0.0002 gram per ml. at p = 1.00 gram per ml. I n combination with an independent density measurement, beta particle absorption measurements may be used to determine the weight per cent of rhemically bonded hydrogen in liquid
RECEIVEDfor re,iew August 23, l9:3 Accepted Xovember 20, 1953. Presented before the Dlvlslon of Petroleum Chemlstrv a t the 124th .\feetine of the AMERICASCHEMICAL SOCIETY, Chicago, Ill.
Amperometric Mercurimetric Titration of Sulfhydryl Groups in Biologically Important Substances At the Rotated Platinum Wire Electrode as Indicator Electrode 1. M. KOLTHOFF, WALTER STRICKS, and LOES MORREN School of Chemistry, University o f Minnesota, Minneapolis 14, M i n n .
A
MPEROMETRIC titrations of sulfhydryl groups in amino acids and proteins with silver nitrate have been reported (1, 5, 9). I n recent years the authors have found that the argentimetric amperometric titration of reduced glutathione (GSH) in ammoniacal medium gives low results, the error being a function of the ammonia concentration in the titration mixture. The cause of the deviation appears to be the appreciable dissociation of silver glutathionate near the end point. The argentimetric titration of reduced glutathione in the presence of much alcohol gives satisfactory results (1, 8). However, it was found that the titration of disulfide after addition of sulfite cannot be applied when the reaction mixture contains about 5070 or more alcohol. Thus, the application of the argentimetric method
in the presence of much alcohol is limited to the determination of sulfhydryl in substances which are soluble in the medium. Sulfhydryl in proteins cannot be determined properly in the presence of much alcohol because of denaturation and precipitation of the protein. Polarographic studies ( 7 ) of the reaction of mercury Jvith reduced glutathione and with cysteine indicated that it should be possible to make this reaction the basis of a rapid amperometric titration of sulfhydryl groups in biological materials. It is shown in this paper that with the rotating platinum wire electrode as indicator electrode traces of sulfhydryl in cysteine, reduced glutathione, and native and denatured proteins can be titrated accurately in aqueous media.
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4
367
Argentimetric amperonietric titration of glutathione gives low results in aqueous ammoniacal medium. The argentimetric titration of sulfhydryl groups i n biological materials in the presence of alcohol has limited applicability if denaturation of proteins is to be avoided. From polarographic studies of the reaction of mercuric mercury with reduced glutathione and with cysteine it is found t h a t this reaction can be made the basis of a mercurimetric titration of sulfhydryl i n cysteine and glutathione in aqueous medium, using the rotated platinum wire electrode as indicator electrode. Procedures are given for the accurate and rapid determination of traces of sulfhydryl in amino acids, peptides, and in native and denatured proteins. The method allows the determination of 7 to 507 of sulfhydryl in biological materials in aqueous solutions with a n accuracy of *29". The method can be applied to the determination of sulfhydryl in proteins in normal and pathological sera.
position of the electrolyte. Polarographic measurements with the mercury or platinum electrode do not allow a differentiation between Hg,(GS)* and GS-Hg--r. EXPERIMEWTAL
Materials Used. Glutathione and cysteine hydrochloride were Pfanstiehl products, which were found to be better than 99% pure by titration with cupric copper ( 4 ) . Stock solutions of glutathione and cysteine ( 10-3M) were prepared in air-free conductivity water. Fresh stock solutions were prepared every day. Mercuric acetate was a Powers-Weightman-Rosengarten Co. (P.W.R.) analytical reagent grade product and mercuric chloride (c.P.) was from Mallinckrodt. The stock solutions of these compounds were 0.05M in mercury(I1). Crystallized bovine plasma albumin was an Armour product which was found to contain 7% water upon heating a t 110" C. to constant weight. All the other materials were C.P. reagent grade products.
14
13
Polarographic studies shohved that rnercur!. :idded to a 1educed glutathione solution reacts to give the mercaptide Hg(GS)?, nhich at pH higher thxn 2 5 can react nith more mercury to form the compounds IIgdGS)?and Hg,(GS)? ( 7 ) . The foiniation of the compounds Hg(CS), and Hg,(GS)? is also observed in titrations with the p1,itinurn electrode as indicator electrode, but the further upt'tke of mercury to form Hga(GS)? cannot be demonstrated undei the e\perimental conditions a t this electrode. The basic reaction for the mercurimetric ampeionietric titration of sulfhydryl in amino acids, peptides, and ploteins is the formation of the compound in which one mercury atom is present per one sulfhydryl group. Such a compound may be represented by the formulas Hg2(GS)2or GS-Hg--r, where z m%v be a halogen or an amino group, depending on the com-
le II
10
$
K
w P
50
9 O
a 7
0
I s 8 4
3 12
e
I
't
10
0
1
8
I 0.3 0.2
0
0.1
E m e Y (r
I
u
, VOLT
-0.2 VS.
-0.3 -OA
-05
-0.6
-0.7
S . C. E.
Figure 2. Current-Voltage Curves a t Rotated Platinum Electrode of 30 M1. of 3.12 X 10-6M Glutathione (0.1M CH&OONa, 0.011M CHSCOOH, pH 5.6) after Adding (A) 0.2 Ml., ( B ) 0.45 MI., and (C) 0.6 MI. of Mercuric Acetate Solution
P Y
8
-0.1
4
I 2
Experimental Methods. Current-voltage curves were nieasured a t 25.0" 0.1' C. with a Heyrovskj. self-recording polarograph. Amperometric titrations were carried out as described in previous publications ( 4 , 6). The platinum wire electrode was rotated a t a speed of 900 r.p.m. All potentials are expressed versus the saturated calomel electrode (S.C.E.). Five-, 2-, and 1-ml. semimicroburets with 0.01-ml. divisions were used in the titrations. Oxygen was removed from the solutions with a stream of pure nitrogen which was bubbled through the solution during the entire titration. The pH was measured with a Beckman p H meter, laboratory Model G.
t
I B 0 2
0
-02
-os
-01
E , VOLT Vs.5
-08
-I 0
C E
Figure 1. Current-Voltage Curves a t Rotated Platinum Electrode of Mercuric Mercury Solutions [10-6iM Hg(TI), 0.05M Borax] in Absence and Presence of Sulfite A. B. C.
Residual (nosulfite) 10-6 MHg(I1). no sulfite 10 -5 MHg(II), 0.1M Na2SOs Current-voltage curves are obtained by varying the potential from plus to minus
CURRENT-VOLTAGE CURVES A T THE ROTATED PLATINUM WIRE ELECTRODE
Sulfhydryl groups in amino acids, peptides, or proteins dissolved in buffers of various pH (1 to 13) do not give an anodic current a t the rotating platinum electrode. A mercuric chloride or acetate solution gives a well-defined cathodic wave a t this electrode (Figure 1,B). The diffusion current of the cathodic
360
ANALYTICAL CHEMISTRY __
-
: tI -
JO-’M H91CH3 C 0 0 ) ~ ADDED, ml
Figure 3.
-____.__
o
A
0.1
A . In absence of chloride i o n B . In presence of 0.1M potassium chloride C. Reagent l i n r (in abqenco of G S H )
0.2
03
04
os
a6
07
1
OB
,
os
Figure 4. Amperometric Titration of 30 MI. of 2 X 10-6M Glutathione in 0.05.W Borax, 0.1M Potassium Nitrate, pH 9.2, with lO-31M Mercuric Acetate a t -0.2 Volt us. Saturated Calomel Electrode A. B.
In absence of chloride ion In presence of 0.1M KCl
C. In presence of M KCI
0
lo3
‘VI
w
E ’
,
4
so
f
M Hp Cle ADDED, ml.
f
a 3
c .
- - --
lo-’ M Hg C l z
EwK
ADDED, ml
a 5 a
3
0
0:
0
2
I
I
0 I
1::-
0
02
A 04
lO-’MHg
Figure 5 .
OB
0.0
IO
I2
14
IS
IS
l C y C O O ) 2 ADDED, m l
Titration of 30 Nil. of 4.7 X 10PM Albumin
A In 0.05M borax 0 1M KNOa,pH 9.2 B’ In 0 05M borax’ O k M K C l H 9.2 C: In 0:05M N a p H h , 0 . 0 0 8 d k a O H H 10 77 D. In 0.05M Na,HPO4, 0.008M ’ k a O H 0.033M NH4NOa a t -0.2 volt us. s a t u r a t e d c a d m e 1 electrode B I . 4 X IO-SM pepsin-digested a l b u m i n in 0.05M borax, 0.5M KCl ( t i m e of digestion 85 m i n u t e s a t 40° C.)
I
LO
I U 3 M Hg(CH3 COO12 A D D E D , m l
Amperonietric Titration of 30
MI. of 2 X 10-6M Glutathione in 0.1M Acetic Acid, 0.1M Potassium Nitrate, pH 3, with lO-3M Mercuric Acetate a t -0.2 Volt
J
::-d.
IO-’
Figure 6.
M HgCl2
ADDED, ml.
Titration of 30 MI. of Albumin
X 10-OM a l b u m i n in 0.03M NHt, O.O~MNHINOI “3, 0.3M NHaNOs,pH9.3 C. 1.87 X 1 0 - b M a l b u m i n in0.1MCHaCOONn. 0.01M CHaCOOH, 0.5M KCl, pH 5.6
A.
4.7
B.
4.7 X 10-6M a l b u m i n i n 0.3M
369
V O L U M E 2 6 , N O . 2, F E B R U A R Y 1 9 5 4 Table I.
i V o l i i m ~of titration mixture, 30 in1 ) Init. GSH, Concn. of Soln. Titrated (Approx.1,
.ir
1.9
2
2 2
x
lo-:
x 10-5 x 10-5 x 10-5
. C oiiiposition
Conrn of A I e ~ ~ i Soln , If 10-7, HgCli 10-3, Hg(dc)e 10-3 H ~ ( A ~ ) ~ IO-?' HgiAc)?
~ i ~
Buffer Phosphate Phosphate Phosphate Phoyiliate
of Electrolyte OSII Chloride Taken concn , .If pH AIg 7 . z 0.174 h-one 7 . , 0.185 None 7 . 7 0.185 0.1 7 . 7 0.185 0 5
10-1
7.7
0.910
2
x
10-4
7.7
1.820
2
x
10-5
9.0
0.18.5
2
x
10
0.0
0.189
9.2 9.2 9.2 9.2 0.2 9.2
0.0923_ 0.0923 0.1840.184 0.184 0.184
9.2
n.2 9.2
0.923 0.923 0.923 i.623 .4.625 5.550
None
0.8
0.185
0.5
9.8
0.185
~i
10-5 10-5 2 x lo--; 2 x 10-5 2 x 10-5 2 x 10-5
H ~ -I . 10-3, Hg(Ac)n 10-8 Hg(Ac)e 10-3' Hg(Ac)n to- FIg(Ac)a
10-4 10-4 10-4 5 X 10-1 S X 10-1 6 x 10-1
5 5 8 2 2 2
fi
x
s u l f h y d r y l . Currentvoltage rurves obtained in mercuryglutathione mixtures at varqing pH were found to be similar to those illustrated in Figure 2. USIT Titration of Reduced GlutaI. ounil, JIg. thione. I n m e r c u r i m e t r i c +1.1 0 176 titrations the platinum elec-0.1) 0.184 + l .I trode becomes coated with 0.187 .. Curved titrat. . .. mercury after an excess of line, no rnrl point mercury is added. It then + 3 . 2 Titrated at 0.9351 behaves like a mercury elec-0.1 volt trode. On a clean electrode 4 2 . 0 Titrated a t 1.856 hydrogen discharge starts in -0.1 volt a borax buffer which is free 0.181 -2.2 of mercury a t -0.7 volt and t2hecurrent rises sharply from 0.174 - 6 . 0 Eiid point n o t sharp then on (curve .4 in Figure 1j, 0 0953 - 3 . 2 while in the same buffer con-0.4 O.OQl!I -1.6 0.181 t a i n i n g 1 0-5M m e r c u r i c 0 .0 0.184 rhloride the .sh:mp rise is not -1.6 0.181 0.178 - 3 . 3 Exccss reagent found until about - 1.0 volt us. line flat, rnrl point n n t S.C.E. (curve B , Figurr: 1): sharp the increase in overvoltage be0.OO.i -1.9 -1.6 0.908 ing due to the thin coat of 0.932 T I .o -1.6 mercury. 4.550 4.550 - 1 . 6 I n t,itrations of glutat,hione -2.8 Reaction linp T,.XRT, has break at : i d proteins new indicator elecfirst end point t,rodes which had been cleaned - 2 . 3 After wiping with nitric acid and washed electrode ivitlc filter paper. with water were found t.o bc reaction l i n r Bat Nomewhat insensitive in the first two or three tit,rations - J . 8 Reaction line very steep, but responded normally t.herc:end point not sharp aft,er. Perfectly reprotlucibk: -1 . I 0.183 results were obtained in some -:3.2 0.179 hundred titrations of gluta-1.1 0.162 t,hione and proteins a t the same -0.5 0.183 electrode without removing the 0180 -2.7 mercury deposit from the elec0.184 -0.5 tiode. Titrations of gl u tm2t thione \WY carried out under varyina conditions. The effect.s of plI and of chloride arid glut,:i.fhione concentration Miere studied. Titration curves obtained :tt, -0.2 volt us. S.C.E. with retiucetl glutathione solutions a t p H 3 :m(I cl.2 are illustrat,ed in Figures 3 and 1. Two end points are observed :tt pfl 3, thr first one corresponding to the formation of Hg(GS), and the second t o Hg2(GS),. A small fraction of the mercury in Ilg2(GS), is reduced a t -0.2 volt (compare Figure 2 ) and the sccond end point is not sharp. The line after the second end point is parallel to the reagent line (graph C in Figure 3). Chloride suppresses ( 6 ) the formation of Hgz(GS)z but two end point,s :&restill observed in 0.1M chloride (graph B in Figure 3). When the chloride concentration is made 0.531 the entirc titrat'ion linc is curved and no end points cttn be detected. Apparently the mercury in €Igr(OKj, is bound to sulfhydrjd :tnd the carboxyl group. Thus it is expected that a high pH is Eavor&le for the formation of Hg2(GR)2. This is evident, from the tit,ration lines in t: I.)or:is medium :it p € f 9.2 where only one eritl point is observed, corresponding to 1 he formation of Hgz(GS)2. The end point is sharp, even in tlw presence of 0.1 M chloride (Figure 4). I n the presence of 1.11 chloride two breaks are observed, corresponding to Hg(GS)s and Hg,(GS)p, but the end point is not sharp. From t,hese observations it is evident tha,t reduced glutathione is best titrat,ctd in :iIk:tlirir~ medium.
Titration of Heduced Glutathione in Alkaline 3Iediiim (pH > 7) in Abseikce and Presence of Chloride at -0.2 Volt us. Saturated Calomel Electrode
(0-8.
I
I~
0.05.W borax 0.03M borax 0 . 0 5 M borax 0.05Mborax 0 0.5Jiborax
X 10-3, llg(Ac)z 0 05.16 borax X lo--, Hg(Ao)l 0,0g5.11borax
X 10-3, X 10-2, X 10-2,
x
10-2.
Hg(Ac)i 0 . 0 5 M horax Hg(Ac)z 0.05.M borax Hg(Ac)z 0.08.11 borax IIa(Ao)r 0 03.11 borax
None Sonr 0.1
0.5
1.0
iuonc 0.1 0.5 h-oni. 0.1 None
9.2 9.2 9.2
lo-'
7 x 1 0 ,
2
x
10-6
in-:, IIgciz
2
x
10-6
10-2, HgCli
2 2 2
x x x
10-5 lo-; 10
IO-a, HgCh 1 0 - 8 , IIgClz 10-3, 1 1 ~ ( . 4 ~ ) .
2
x
10-5
__
.
Carbonate-bicarbonate Carbonate-bicarbonate Phosphate Phosphate 0.026.11 -1)orax
None 0.5 0.5
10.8 0.184 1 0 . 8 0.184 1 1 . 2 0.18.; 12.9
0.183
Y
mercury wave is found to be proportional to the meicury concentration in the entire p l l range (1 to 9 ) investigated. The mercury n a v e is completely eliminated in the presence of sulfite (Figure 1,C). Mercury forms a strong complex with sulfite which is not reduced a t the p1:ttinum electrode. It is therefore not possible to apply mercurinietric sulfhydryl titrations at thc platinum electrode to thr determination of disulfide groups in the presence of sulfite. Figure 2 exemplifies polnrograrns a t the rotating electrode of mixtures a t p H 5.6 containing mercury and glutathione in the mole ratios 0.855 to 2 and 1.92 to 2. If the mole ratio of mercury to glutathione is 1 to 2 or smaller no appreciable current is ohserved between +0.2 and -0.2 volt. The cathodic wave starting a t -0.2 volt is due to reduction of mercury in the compound Hg(GS)z. Upon addition of mercury to Hg(GS)2 this WRW becomes more drawn out and starts a t a more positive potential Its height increases as more mercury is added and finally :Lttains a constant value when the mole ratio of mercury to glut;LthioncL becomes greater than 1 to 1. Apparently this wave is due to the reduction of a compound which may be presented by the formula Hg,(GS)z in which one mercury is more loosely bound than in the mercaptide, Hg(GS)z When meicury is in excess of the formation of HgZ(GS)z the "free" mercury wave is observed. Use of these observations is inadr in the amperometric titration of
ANALYTICAL CHEMISTRY
370 Table 11.
Titration of Reduced Glutathione in Acid iMedium (pH < 7) in Absence and Presence of Chloride at -0.1 Volt vs Saturated Calomel Electrode (Volume of titration mixture, 30 ml.)
GSH
Init. GSH.
Found from Second
,trolyte do-
10-5
10-3, Hg(Ac)z
3.0
0.186
0.182
10-3. Hg(Ac)z
0 . 1 M CHaCOOH, 0.11M Kx03 0 . 1 M CHaCOOH
...
2 X 10-5
0.1
3.0
0.185
0.187
+1.1
2 X 10-5
10-3, Hg(.4c)z
0 . 1 4 1 CH3COOH
0.2
3.0
0.186
0.202
i8.6
3 x 10-5 1 . 9 X 10-5
10-3, Hg(Ar)z 10-3, HgClt
Phosphate Acetate
...
5 , 2 0.285 5 . 6 0.174
0.281 0.175
+O.G
2 x 10-5 2 X 10-5 2 x 10-5 2 x 10-5
10-8, 10-3, 10-3, 10-3,
2 X 10-5
10-3, Hg(bc)z
2
x
2 X 10-5
x
... ...
-2.1
-1.4
Acetate Acetate Acetate Acetate
10-4 10-3 10-2
5,6 5.6 5.6 5.6
0.1875 0.187 0.1875 0.187 0.1875 0.183 0.1875 0.177
-0 2 -0.2 -2.3 -5.7
Acetate
...
5.6
0.185
0.186
+0.5
10-3, Hg(Ac)z
A4cetate
...
5.G
0.185
0.187
10-3 H ~ ( A C ) ~ 10-3: H ~ ( A c ) ~
...
5.6 5.6
0.283 0.283
0,283 0.283
HgClz HgC1, HgClz HgCle
3 X 10-5
10-3, Hg(Ac)z
Acetate Acetate, 10 -2.M KNOi Acetate, 0.1.11
...
5.6
0.283
0.283
3 X 10-5
10-3, Hg(Ac)z
Acetate
0.1
5.6
0.283
0.265
-G.4
3 X 10-6 1 . 5 x 10-5
10-3 HgClz 10-3: H ~ ( A C ) ~
Acetate Acetate
...
5.6
0.288
0.289 0.281
+0.3 -0.7 -1.7 -0.5 -0.8
-0.8 -1.2
3 3
x
10-5
10-5
KSOI
...
...
5 , 6 0.283
2 X 10-3, Hg(Ac)z .4cetate 6 X 10-5 Acetate 9 . 9 X 10-5 5 X 10-3, HgClz 9 . 9 x 10-5 5 X 10-3, Hg(Ac)z Acetate 10 - 4 10-4 10-4 2 x 10-4 3 X 10-5
5 X 10-3 H-(Ac)z Acetate 5 X 10-3: H;(Ac)t Acetate 5 X 10-3, Hg(Ac)2 Acetate 5 x 10-3, Hg(Ac)p Acetate 10-3, Hg(Ac)z Phosphate
10'4
10-3
... ...
5.6 5.6 5.6
0.576 0.910 0.910
0.566 0.905 0.917
5.6 5.6 5.6 5.6 6.7
0.918 0.918 0.918 1.820 0,285
0.911 0,907 0.903 1.796 0.282
End point not sharp End point not sharp, current unstable after end point
E n d point sharp
not
E n d point sharp
not
0.0
Volume of solution, GO ml.
-1.6
-1.3 -1.1
Table 111. Titration of Cysteine in Absence and Presence of Chloride at -0.2 Volt us. Saturated Calomel Electrode (Volume of titration mixture, 30 ml.) Init RSH, Concn. of Solution Titrated (Approx.),
M .
2 2 3:2
Y 10-5 io-5
1.5
x
2
Concn of hfercury Soh, M 10-3
io-3
6 . 4 X 10-5 10-3 6 . 0 X 10-5 2 X 10-3 10-5
10-3
Composition of Electrolyte Chloride concn., Buffer M pH Acetate 5.6 ... 5.6 Acetate . .. 5.6 Acetate 0.2 7.53 Phosphate 0.2 9.20 Borax
4
x x x x x
10-5 10-5 10-5 10-5 10-5
Borax Borax 2 X 10-3 Borax 2 X 10-8 Borax 2 X 10-3 Borax
6
x
10-6
2 X 10-8
6 X 10-5
2 X 10-8
2 2 3 3
...
RSH RSH Taken, Found, Mg. Mg.
Error,
0.151 0.150 0.151 0.155 0.302 0,305 0 . 2 8 4 0,278 0.071 0.0725
-0.7 f2.6 +l.O -2.1 +2.0 +2.8 +2.4 +1.4 0.0 +3.6
%
0.142
...
9.20 9.20 9.20 9.20 9.20
0,142 0.192
0.0987_ 0.0984 0.1440.142 0.199
Borax
0.2
9.20
0.284
0.284
0.0
Borax
0.2
9.20
0,284
0.289
+ 1, 8
10-3 10-3
... 0.2 0.2
0.096 0.096
Remarks End noint not sharo E n d 'point not sharp E n d point not sharp End point not sharp Sharpendpoint
Electrode not cleaned. large anod. current Freshly cleaned electrode, sharp end point . Clean electro4e
6 X 10-6
2 X 10-8 Borax
0.2
9.20
0.284
6 X 10-5
2 X 10-3
Borax
0.5
9.20
0.284
10-5
2 X 10-3
Borax
0.2
9.20
0.284
6 X 10-5
2 X 10-3
Borax
0.2
9.20
0.284
9 X 10-5
2 X 10-3
Borax
B,2
9.20
0.427
0.431
9 X 10-5
2 X 10-3
Borax
0.2
9.20
0,427
0,433
1 . 5 x 10-4 3 X 10-4
5 X 10-8 5 X 10-3
Borax Borax
0.2 0.2
9.20 9.20
0.713 1.425
0,718 1.448
4th Titrat. after cleanina , anodic current, -3,6 microamp. + O , 9 Freshly cleaned electrode (2nd Titrat. after cleananodic current, +1.4 Y . 7 microamp. +0.7 Cleanelectrode +1.6 Cleanelectrode
6 X 10-5 6 X 10-6 6 x 10-6
2 X 10-8 2 X 10-3 2 X 10-3
Borax-NaOH Phosphate-NaOH 0.1M NaOH
0.2 0.2 0.2
10.62 10.61 13.0
0.284 0.284 0.284
0.276 0,293 0.266
-2.8 +3.2 -6.3
6
x
E
3rd Titrat. after clean-
anodic current, -3.6 microamp.
0,308
+8.5
\
E n d point not sharp E n d point not sharp Titrat. line curved a t end point
T i t r a t i o n of S u l f h y d r y l Groups in Protein. For a study of the applicability of amperometric mercurimetric titrations to the determination of sulfhydryl groups in proteins, commercial crystallized bovine plasma albumin has been chosen as a typical example. It has been shown by Hughes ( 2 , 3 ) that crystallized albumin consists of two fractions. One of these fractions, which makes up two thirds of the total albumin, is called mercaptoalbumin and is found to contain one sulfhydryl group per mole. Thus, c o m m e r cia1 crystallized albumin should contain 0.66 mole of sulfhydryl groups per mole of protein. Air-free albumin solutions of various c o n c e n t r a t i o n s i n buffers of various composition a t pH 1 O . i i t o 1.13 were titrated with mercuric chloride or mercuric acetate solutions at a potential of -0.2 volt us. S.C.E. Figurep. 5 and 6 illustrate examples of these titrations. In borate (pH 9.2), phosphate (pH 10.8 to i . 3 ) , or acetate (pH 5.6 to 4.0) buffers no distinct end point is observed in the absence of chloride, the slope of the excess of reagent line being extremely flat (Figure 5 , A and C). I n the presence of chloride the excess reagent line becomes markedly steeper and a well-defined end point is observed (Figures 5 3 , 6,D, and 6,C). I n ammoniaammonium ion buffers, even when chloride-free, a sharp end point is observed which becomes better defined as the buffer concentration is i n creased (Figures 6 , A and B ) . Taking a molecular weight of albumin of iO,OOO, the end point in these titrations corresponde to a mole ratio of mercury to albumin of 0.65 f 0.02, which is in good agreement with Hughes's results. Under the experimental conditions one mercury atom combines with one mole of mercapto albumin by reaction XTith the sulfhydryl group. I n the presence of chloride ion or ammonia the excess reagent line apparently corresponds to the reduction of mercury in the mercuric chloride or ammine complex. I n the absence of
V O L U M E 2 6 , N O . 2, F E B R U A R Y 1 9 5 4
371
these complexing agents mercury can be bound by albumin by groups other than sulfhyConcn. of Mole Ratio, dryl groups. It appears that Albumin in Mercury t o Titration Albumin a t mercury bound to albnmin is Mixture. .I1 Supporting Electrolyte pH E n d Point Remarks not or only partially reduc4 . 7 X 10-5 Phosphate 10.77 End point not detectable Sharp end point 10.77 0 . 6 4 ' ' 4 . 7 X 10-5 Phosphate, 0.033.1.1 NHaNOi ible a t the platinum elec9 . 6 4 0.65 Sharp end point 6 X 10-5 0.05 21 borax, 0 . l M KHa trode, as indicated by the ex9.64 0.64 Sharp end point 4 . 7 X 10-5 0.05.W borax, 0 . 1 M NH3 9 . 6 4 0 . 6 4 , O . 68 Sharp end point 4 . 7 X 10-5 0.05M borax, 0.1.11 NH3 ceedingly small slope of the 9.10 E n d point not detectable 4 7 X 10-5 005,W borax, 0 1.11 KNOJ excess reagent line in titra9.10 0 . 6 i " Sharp end point 4 7 X 10-5 0 0 5 M borax, 0 1.11 KCI 8 . 9 4 0.63 Slope of excess reagent line small tions of albumin solutions in 8 X 10-3 0 0534 borax, 0 5.11 KCI 8 . 9 4 0.81 Sharp end point 4 7 X 10-5 0 05.W borax, 0 5.W KC1 the absence of chloride or 8 . 9 4 0.61,O. 62 Sharp end point 2 X 10-5 0 05.W borax, 0 5.11 KC1 ammonia. Sharp end point 0.05M borax 0.OB.li KHaNOa 8.90 0.66 4 X 10-5 Sharp end point 0.05M borax' 0.05-11 KHdNOa 8.90 0.66 2 x 10-6 The best reproducible re8.71 0.66 Sharp end point 2 x 10-6 0.05.W borax' 0.05-WKHdNOa 0,531KC1 Sharp end point, titration carried 8.71 0 . 6 3 1 . 5 x 10-5 0.0534 borax: 0.05.11 hT&NOa: 0.5.11 KC1 sults were obtained with proout with 5 X 10-4M HgCh i. 7 X 10-5 0.3.21 N H I , 0.351 XHah'Oa 9.3 0.63 Sharp end point tein solutions in borax buffers 9 . 3 0.64 Sharp end point 4 7 X 10-5 0 li21 "3, 0 1 2 1 SHaNOs (pH 8.8) in the presence of 9.3 0.64 Slope of excess reagent line small 4 7 X 1 0 - 6 0 0 3 M NHs, 0 03.11 N H ~ N O J chloride (0.5M) and a small 7 . 3 1 0.68 Sharp end point 10-4 Phosphate, 0 5-V KC1 7.31 0.68 Sharp end point 4 X 10-5 Phosphate, 0 5 M KC1 amount of ammonium nitrate 7.31 0.65 Titration carried out with 5 X 1 j X 10-5 Phosphate, 05-W KC1 10-4,M HgCln (O.O5M), in phosphate buffers 4 X 10-6 Acetate, 0.5.M KC1 5 . 5 9 0.65,O. 68 Sharp end point (pH 7 . 3 ) in the presence of 2 X 10-5 Acetate, 0.5M KC1 5 . 5 9 0.67 Sharp end point 0.5M potassium chloride, and 1 . 5 x 10-5 Acetate, 0 . 5 N KC1 5 . 5 9 0.68 Titration carried out with 5 X l O - a M HgCb in acetate buffers (pH 5.6) in 4 x 10-5 Acetate, 0.3.21 KC1 5 . 5 9 0.67 Excess reagent line straight, only close after end point the presenre of 0.5M potassium 4 X 10-6 Acetate, 0.l.M KC1 B.59 0 . 6 7 Excess reagent line straight, only chloride. close after end point 4 X 10-5 Acetate, 0.5.W KCI 3.97 0.68 Titration of Cysteine. The 4 X 10-5 0.2.M CHaCOOH, 0.5X KC1 2.93 0.49 4 x 10-5 0.1M HC1. 0.5.11 KC1 1.13 0.40 surface conditions of the electrode are critical in mercurimetric titrations of cys7 represents teine. Figure titrations of a 5 X 10-6M cysteine solution a t an electrode which had been freshly cleaned with nitric acid (diagram il) and a t electrodes which had been used for more than one titration after experiment A (diagrams B and C). After each experiment the electrode was rinsed with distilled water. It is seen that no appreciable current is detectable before the second end point in the titration with the clean electrode while with the mercurycoated electrode anodic currents are observed which increase with the amount of mercury deposited. Thus starting with a clean electrode the anodic current a t the beginning of the second, third, and fourth consecutive titration of a 5 X lO-5.W cysteine solution was found to be 0.5, 3.6, and 5.6 pa., respectively. On the addition of mercury the anodic current decreases and disappears in the vicinity of the first end point. Further addition of mer2a cury gives an increasing cathodic current a t the mercury-covered k!4 electrode. Many experiments have been carried out with used z a electrodes and with electrodes which had been cleaned in various 0 cr ways (hot concentrated nitric acid, cleaning solution, anodic uI 2 polarization in potassium nitrate, borax buffer, and sodium hydroxide). It appears that electrodes which are covered with a slight amount of mercury or whirh are not perfectly clean are extremely sensitive to the loosely bound mercury in the cysteine mercury complex, Hg,(RS)?, and give appreciable cathodic currents after the first end point. Since the excess reagent line is practically the same a t clean and mercury-coated electrodes, -2 the intersection of this line with an abnormal reaction line yields kt high results. Thus the errors of the titrations presented in Figure 7 are -0.1, + O S , $5.3, and +8.3% for the first, second, third, -4 and fourth titration, respectively. Similar results were obtainel i n titrations of various cysteine concentrations in phosphate and ammonia buffers in the absence and presence of chloride. These observations show that freshly cleaned electrodes must be used 0.2 0.4 os 0.8 10 1.2 for mercurimetric titrations of cysteine. 2 X IO-' M HgClp ADDED mL The mercury deposit can be best removed with warm nitric Figure 7. Titration of 30 M1. of 6 X 10-5M Cysteine i n acid (1 to 1 by volume) or by polarizing the electrode a t a poten0.05M Borax, 0.2M Potassium Chloride with 2 X 10-3M tial of +0.5 to f0.2 volt us. S.C.E. in a 0.1M potassium nitrate Mercuric Chloride a t -0.2 Volt solution for 5 to 10 minutes, After thorough rinsing with disA . First titration C. Fourth titration after cleaning of tilled water the electrode is ready for use. B . Second titration platinum electrode with nitric acid
Table IV.
I
1
Titration of 30-M1. Albumin Solutions i n Various Buffers a t -0.2 Volt vs. Saturated Calomel Electrode with 10-3M Mercuric Chloride
372
ANALYTICAL CHEMISTRY
Mercurimetric titrations of cysteine were carried out under various conditions in the same way as described for glutathione. The best medium for the titration of cysteine was found to be a borax solution. RECOMMEYDED PROCEDURES
Glutathione and Cysteine. Introduct. 30 nil of a solution whirh is 0.05111 in borax and 0.1M in potassium chloride into a 120-nil. beaker which is provided with a rubber stopper nith holes for electrode, salt bridge, buret, and inlet tube for nitrogen. Immerse a platinum wire electrode in the solution. Remove air with purified nitrogen and pass nitrogen through the mixture during the entire titration. To the air-free solution add enough sample so that the glutathione or cysteine concentration in the mixture is between and 6 X 10-4M. Immerse the Qalt bridge and tip of the buret in solution and titrate with mercuric acetate or mercuric chloride of suitable concentration (10-3 to 2 X 10-SM) a t an applied potential of -0.2 volt IS. S.C.E One milliliter of 10-SM mercuric chloride corresponds to 0 307 mg. of reduced glutathione, 0.121 mg. of cysteine, or 0.033 mg of sulfhydryl. Titrations of glutathione should be carried out with an electrode precoated with mercury, the electrode being rinsed with distilled water after each titration. Cysteine must be titrated a t an uncoated platinum c~lectrotlt~ Preceding the cysteine titration place the electrode into warm nitric acid (1 to 1 by volume) for about 5 minutes and waqh with distilled water, or place the electlode in 0.1M potassium iiitiatt. and apply a potential of +0.5 to +0.2 volt us. S.C.E. for 5 to 10 minutes. After rinsing with water the electrode is ready for UQP If not in use keep the electrode in distilled water Proteins. Into a 120-ml. beaker intioduce 30 nil. of a mlution \\hich is 0.01M in monosodium phosphate (SaH,POI), 0.08211 in tlisodium phosphate (NhHPO,), and 0.5M in potassium chloride To the air-free solution add enough of the protein solution to make the mixture 1 to 7 X 10-5JI in sulfhydryl. While passing a gentle stream of nitrogen through the solution titrate with a mercuric chloride or acetate solution at an applied potential of -0.2 volt us. S.C.E. The treatment of the electrode is the same as for the titration of glutathione. (Mixtures of reduced glutathione and protein can be titrated by the procedure given for glutathione. Cysteine cannot be titrated mercurimetrically in mixture. with glutathione or protein.)
to 10-4Jf. Solutions a t pH markedly lower than 4 give low results. With albumin concentrations larger than lO-4M no sharp end point is obtained, the slope of the excesa reagent line being small. Titrations were also carried out with albumin which had been subjected to pepsin digestion for various periods of time in the absence of air. Thus 25 ml. of an air-free solution which waa 6 X 10 -4M in albumin, 0.005~oin pepsin, and 0.05M in hydrochloric acid was kept under nitrogen a t 40" ('. for various periods of time. TWOmilliliters of this digest were titrated in a borax buffer. The mole ratio of sulfhydryl to albumin was found to be 0.65, 0.69, and 0.68 after 85, 105, and 120 minutes, respectively. Hence the sulfhydryl content of albumin as determined by mercurinietric titration hardly rhanges on pepsin digestion. From a romparison of Figure 5,B and 5,B1, nhich present titrations of native and pepsin-digested albumin in the same buffers, it is interesting to note that the slope of the excess reagent line in the titration of pepsin-digested albumin is much smaller than that of the native protein. Mercurimetric titrations a t the rotated platinum electrode have been applied to routine determinations of the sulfhydryl content of the albumin and globulin fractions of normal and pathological blood sera. COlCLUSlONb
For the determination of cysteine the aniperometric titration with mercury has no distinct advantages over that nith silver However, for the determination of glutathione and sulfhydryl in proteins the mercurimetric titration is much more accurate than the argentimetric one. Mercurimetric determinations of sulfhydryl in proteins can be carried out in the presence or absence of ammonia and over a wider pH range (4 to 13) than is possible in argentimetric titrations. The titration of disulfide in the presence of sulfite cannot be carried out nith mercur? ut the rotated wire electrode as the indic:ttor elrctrode. This titration can be carried out a t the dropping mercury electrode as indicator electrode (8).
RESULTS
Tables I and I1 give the results of amperometric titrations of glutathione in alkaline and acid media, respectively An in, spection of these tables show s that mercurimetric titrations of reduced glutathione can be carried out accurately both in alkalilie and in acid media, provided that the chloride concentration in acid medium does not exceed 10-31M. Larger chloride concentrations make the end point less distinct in arid medium. Thus in an acetate buffer a t p H 5.6 the presence of 0.1M potassium (hloride results in an error of -6% whereas titrations in a borax buffer containing 1M chloride gave an error of only -3.3%, w e n though the slope of the excess reagent line is rather small under these conditions. I t wafi found that results of an accuracy and precision of &2% ctin be obtained in borax, phosphate, carbonate, and ammonia 1,uffrr.s in the pH range 9 to 13 a t chloride concentrations smaller than 0.5.V. Satisfactory results are obtained with mixtures containing 10-j to 6 X 10-4M glutathione. A t concentrations larger than 6 X 1O-"lI a sharp end point is not obtained. The results of cysteine titrations are summarized in Table 111. I t is seen that the best results are obtained in a 0.05M borax solution a t pH 9.2. In solutions a t p H markedly higher than 9 2 the end point is not sharp. Chloride ion a t concentrations up to 0.5Bf doe4 not affect the titration results. In a 0.05M borax solution a t cysteine concentrations of 10-5 to 4 X 10-4~11the accuracy and precision of the results me about f1%. Table IV gives the results of titrations of crystallized albumin a t various concentrations and in various buffers. Good results :LI obtained in chloride or ammonia containing solutions a t pH 0.8 to 4 with albumin concentrations between 1.5 X (A
ACKNOWLEUCMER'I'
This investigation was supported by a reseitrch grunt from the National Cancer Institute, LT. S. Public Health Service. LITERATURE CITED
(1) Benesch, R., and Benesch. R. E., Arch. Biochem., 19, 35-45
(1948). (2) Hughes, W. L., Jr., Cold Sprirrg Harho, Symposia Q u a d . B i d , 14, 79 (1949). (3) Hughes, W.L., Jr., J . Am. C h e m Soc., 69, 1836 (1947). (4) Kolthoff, I. ll.,and Stricks, W., ANAL.CHEM.,23, 763 (1951). (5) Kolthoff, I. M., and Stricks, W. J . A m . Chem. SOC.,72, 1952 (1950). (6) Lingane. J. J., and Kolthoff, I. Sf.,Ihid., 61, 826 (1939). (7) Stricks, W., and Kolthoff, I. M., Ibid., 75, 5673 (1953). ( 8 ) Stricks. W., Kolthoff, I. hf.. and Tanaka. N., A N A L . CEEM.,26, 299 (1951). (9) Weissman, N., Schoenhach. E. B., and Arniistead, E. B., J. Bid. Chem., 187, 153 (1950). R F C Y I V fEoDr review July 20 19.53
Accepted December 3, 1953.
