Catalytic Activity of Cysteine and Related Compounds in the Iodine-Azide Reaction D. W. WHlTAIAN AND R . MCL. WHITNEY Department of Food Technology, University of Illinois, Crbana, 111. volume of each JVarburg flask was determined by the mercury displacement method described by Loomis ( 9 ) , and the flask constants were calculated by the method of Umbreit, Burris, and Stouffer ( I S ) .
Interest in a quantitatiie determination of the sulfhjdrjl groups in milk and other biological products resulted in an iniestigation of their cataljsis of the iodine-azide reaction. The catal) tic actiiit? of cjsteine and related compounds in the etolution of nitrogen bj the iodine-azide reaction was measured in Warburg respirometers at pH 4.63. Evidence was obtained as to the mechanism of the reaction. The volume of nitrogen etolted is quantitatively related to the su1fhydr)ls and disulfide present in the sample and is also dependent on the time of reaction, the temperature, the reagent concentrations, and the group attached to the su1fh)drjI group. Although the catal) sis of the iodine-azide reaction under the conditions in\estigated is not suitable for the measurement of mixtures of sulfhjdrjl groups without further studj, its specificit) mal be of c*onsiderable\ alue in following changes in abailahle sulfhj drj 1 groups in complex biological s ) stems.
I
REAGENTS
All water used in this study was deionized by passage through a bed of mixed exchange resins. This water had a cpecific resistance of 1 megohm or more. Hydrochloric acid, 0.01 S. Stock cysteine solution! 2 X 10-3.1f in 0.01 .Y hydrochloric acid. This solution n-as diluted Kith 0.01 .I’hydrochloric acid to the appropriate roncentrations. Preliminary studies indicated that in 0.01 S hydrochloric acid these Polutions were stable for a t leaat 8 hours; thereforr, thry w i ~ epwp:trPd less than 8 hours before use.
400
-.,300 -
R E C E S T years intense interest has been shown in the sulfhydryl groups in milk and other biological liquids as a criterion of such properties as keeping quality ( I d ) , protein denaturation ( I O , I L ) , and suitability for certain uses (4,6-8, I l l . Therefore, their catalytic effect on the liberation of nitrogen by the iodine-azide reaction described by Feigl ( 2 ) has been investigated. He listed the following groups as catalyzing the reaction with an instantaneous evolution of nitrogen: sulfhydryl, thiocyanate, thiosulfate, thioketone, and diacyl disulfide. In addition, he observed that compounds containing a disulfide group (except diacyl disulfide) catalyzed the reaction very little if at all. His observations were ronfirnied in part by Frietimann ( 3 ) ) who quantitatively investigated the reaction and suggested the following mechanism for catalysis Iiy sulfhydryl groups: ?;
R-SH
+ I? R-SH
-
R-SI:
(11)
12
----f
R--S R-4
I
$
n W
3
z
200
z W
W
0
a
r100
/ Cysteine-treated-with-Iodine\, 0
t
2 .z
h
n
1
10
20
30
40
50
60
TIME (rnin.)
+ 2HI
Figure 1.
It-SH
Catalysis of Iodine-Azide Reaction
A t 11.5’ C. by cysteine, cystine, a n d cysteine treated with iodine (1.8 peq. per ml.)
Both the catalytic ( I ) and the oxidation (11)reactions, in his view, continued simultaneouslj until stopped by the depletion of the limiting constituent, which n as the sulfhydryl group. Friedmmn also attempted t o investigate the effect of disulfides upon the reaction, observing little if any catalytic activity. However, he obtained his “cystine” by the iodine ovidation of cysteine a t 17.5’ C., which doesnot actuallj take place, as indicateti by the results reported in this paper. APPARATUS
The evolution of nitrogen by the iodine-azide reaction, due t o the catalytic activity of the compounds investigated, was followed b y means of Warburg respirometers using Clerici’s solution ( I S ) or Brodie’E solution ( 1 3 ) as the retaining liquid. The
Stock cystine solution, 0.25 X 10-3M in 0.01 -Yhvdrochloric acid. The procedure for dilution and storage - was the same aa for cysteine solutions. Iodine-azide solution, 0.05 S iodine, 0.06 aV potassium iodide, 0.01 S sodium azide, and 0.60 ‘V acetate buffer at p H 4.63. G E 3 ERAL PROCEDURE
Before use, the Warburg flasks were washed with detergent solution, immersed in cleaning solution for several hours, rinsed, and immersed for 5 minutes in a n approximately 0.4 N oxalic acid solution t o remove any trace of oxidants. after which they n-ere rinsed and inverted in a rack t o dry. I n making a determination, 2 ml. of catalyst solution was pipetted into the reaction trough and 1 ml. of the iodine-azide solution into the side well of a Warburg flask. The respirometer was assembled, placed in a water bath a t 17.5” C., and tempered for I hour without rocking. Upon equilibration, the contents of the side arm were mixed with the catalyst solution and the respirometer was set in motion in the constant temperature bath.
1523
ANALYTICAL CHEMISTRY
1524
The pressure readings were taken a t 10-minute intervals for 1 hour, thermobarometer corrections were made, and the results were converted to microliters of nitrogen evolved under standard conditions. This procedure was used throughout the work, except as specifically indicated.
Table I. Volume of Nitrogen Evolved by Iodine-Azide Reaction at Various Cysteine Concentrations Cysteine Concn., peq./MI.
RESULTS
Exploratory Results. As Friedmann (3) had performed the only quantitative work on this reaction, his work was employed as a starting point in this investigation. These conditions differed from those of the general procedure in that, when cysteine was investigated, 1.0 ml. of 0.05 S iodine solution, 0.5 ml. of 0.10 N sodium azide solution, and 1.3 ml. of 0.12 N acetate buffer a t p H 4.63 were placed in the reaction trough and 0.2 ml. of cysteine solution (18 peq. per ml.) in acetate buffer in the side well. When “cystine” was investigated it was obtained by placing 1.0 ml. of 0.05 iV iodine and 1.0 ml. of cysteine solution (3.6 peq. per ml.) in acetate buffer in the reaction trough. I n this case 0.5 ml. of 0.10 N sodium azide solution and 0.5 ml. of the acetate buffer were added from the side well. Typical results are shown in Figure 1. However, in order to test the validity of Friedmann’s assumption that cystine could be prepared in this manner, an additional determination was performed in which 2.0 ml. of cystine solution (1.8 peq. per ml.) in 0.06 N buffer and 0.06 S potassium iodide was placed in the reaction trough and 1.0 ml. of an iodine-azide solution, 0.05 N with respect t o each, was added from the side ell. The results shown in Figure 1 indicate that the data for cystine do not agree with those obtained employing Friedmann’s procedure; therefore the product he investigated could not have been pure cystine but rather some othcr oxidation product of the cysteine.
0.20 0.30 0.40 0.50 0.60 0.70
0.80 0 90
Volume of Nitrogen Evolved a t Time Indicated, pl10 min. 20 min. 3 0 min. 40 min. 50 min. 60 min. 105 105 105 99 99 93 99 93 99 93 93 93 209 209 209 197 203 203 215 209 209 203 209 209 304 310 298 304 286 304 261 261 268 255 261 261 435 422 428 428 428 416 422 422 416 410 416 416 568 554 568 561 561 548 514 507 514 514 514 507 439 432 445 420 432 426 547 553 553 534 541 547 642 636 636 630 636 630 672 666 672 660 660 666 767 751 751 762 766 745 784 778 784 767 773 773 Analysis of Variance ( I ) Degrees Mean of Sum of Square Freedom Squares
Source of Variance Cysteine conon. Time Cysteine concn. X time Error Total
7 5
4 , 3 0 0 , 1 0 0 614,300 2,216 443
35 48 95 2 = 34
310
0.08
Significance.
%
>0.1 N.s. N.S.
4,358,090
Both cysteine and cystine were investigated and the volumes of gas liberated in 1 hour were plotted against the concentration of azide in the iodine-azide solution a t each level of iodine concentration. Effect of Cysteine Concentration. Upon completion of the exploratory work and the selection of experimental conditions the effect of cysteine concentration upon the volume of nitrogen evolved by the iodine-azide reaction was investigated over the range 0.1 to 2 Ieq. per ml. From consideration of typical results presented in Table I it can be seen that the catalytic activity of cysteine is not statis-
-
, CATALYST CONC. = 0.10U Cysteine
2ooL
- - - E
-
”,
2 x 17SoC, Cysteine, (1.8~ eqhl) 3 0 37.5’C, Cystine, ( 0 . 5 eq/ml) ~ 4 o I7.5’C, Cystine, ( 0 . 5 , ~e q h l )
9 1,156
____55,464
Variance Ratio 531.6 0.38
Cystine
1
0.1
/ //
400
0
eq/ml
/
/ /
2 ( K 1 3 ) = 0 . 4 5rneq/ml
/
b ( K 1 3 ) : 0 . 2 5 rn ealrnl
/’
/
y -
(KI,I ~ 0 . 4 5m e q h l (K13)= O 25 m eq/ml W 1 3 ) -0.05 meq/ml
0.5 0.9 AZIDE CONCENTRATlON(m /(K13):0.05 eqlml)
rn e
q h
CATALYST CONC. = 0 . 3 5 1 e m / /
TlME(min.) Figure 2. Effect of Temperature on Catalysis Iodine-Azide Reaction by Cysteine and Cystine
of
In the exploratory stages of this study it was thought desirable to investigate briefly the effect of temperature, iodine concentration, and sodium azide concentration. The results of the study of the temperature effects are illustrated in Figure 2, where both 1.8 peq. per ml. of cysteine and 0.5 Ieq. per ml. of cystine were studied under Friedmann’s conditions a t temperatures of 37.5” and 17.5’ C. I n Figure 3 the effect of three different levels of iodine concentration in combination with three different levels of sodium azide concentration for each of two levels of the catalysts is recorded.
0.06 0.08 0.’10 AZIDE CONCENTRATION(meq/ml)
0.02 0.04 Figure 3.
Effect of Iodine and Azide Concentrations on Nitrogen Evolved
In 60 minutes by catalysis of iodine-azide reaction by cysteine and cystine
V O L U M E 2 5 , N O . 10, O C T O B E R 1 9 5 3
1525
tically significant after 10 minutes. Therefore, in considering the effect of concentration upon the volume of nitrogen evolved, the time-mean values may be employed. For low concentrations of cysteine, it may be noted in Figure 4 that a graph of the volume of nitrogen evolved against the square of the cysteine concentration yields a straight line passing through the origin. However, in experiments performed a t moderate concentrations the reaction followed a linear relationship between the volumes of nitrogen evolved and the cysteine concentration.
Table 11. Nitrogen Evolved by Iodine-Azide Reaction a t Various Cystine Concentrations Cystine Concn., peq./hll. 0.15
Volume of Nitrogen Evolved a t Time Indicated, pl. 10 min. 20 min. 30 min. 4 0 & . 50 min. 60 min. 12 12 17 23 29 35 12 17 12 17 23 23 0.30 12 24 24 36 49 49 18 24 24 30 43 49 0.45 12 24 31 43 61 67 18 24 31 37 61 67 Analysis of Variance ( 1 ) Degrees Signifiof Sum of Mean Variance cance, Source of Variancr Freedom Squares Square Ratio % Cystine concn. Linear response 206.8 >0.1 1 2481 2481 Other response 3.58 N.s. 1 43 43 Time 446.4 >0.1 Linear response 1 5357 5357 5.33 5 Quadratic response 1 64 64 4.25 5 Other response 3 152 51 Time X cystine concn. 0.1 465 3 8 75 Linear response 2 930 0.42 N.s. 8 43 5 Other response 18 223 12 Error 35 9293 Total
-
240
This relationship fits the data within the 95% confidence limits.
At high concentration, above 1 peq. per ml., the volume of nitrogen evolved is beginning to approach a maximum as indicated by the curvature toward the sulfhydryl concentration axis. Effect of Cystine Concentration. When cystine is used as the catalyst under the conditions of the general procedure, the results illustrated in Table I1 are obtained. The analysis indicates significant linear responses for cystine concentration, time, and the interaction product. The linear least square equations for the relationship between the volume of nitrogen evolved and the reaction time were calculated for each cystine concentration and the coefficients for the slopes divided by their respective cystine concentrations. The average of the resulting quotients, 1.95, was then used in the equation,
v
Catalyst Concn., peq./Ml. Cysteine Cystine 0.0 0.00
-
0.0
0.30
^ 0 = 4
0.0
0.45
0.3
0.00
0.3
0.15
0.3
0.30
0.3
0.45
0.6
0.00
0.6
0.15
0.6
0.30
0.6
0.45
0.9
0.00
0.9
0.15
0.9
0.30
v
n w
3
P W
160-
z W (3
0
E
80
-
i 0.9
OO
0.04 0.08 0.12 CYSTEINE CONCENTRATION SQUARED (,u eq/ml)*
Representative data were assembled and plotted in Figure 5. For lorn- and medium cysteine concentrations ( 5 1 . 0 peq. per ml.), this relationship can be expressed by the quadratic least squares equation,
V =
- 33
+ 819[SH] + 31.5 [SH]*
V = volume of nitrogen evolved,
pl.
and
[SH] = concentration of cysteine, peq. per ml.
(2)
0.45
Volume of Nitrogen Evolved a t Time Indicated, pl. - .__ Calculated from Observed Values Eq. 3 -__ in en 20 3.0 40 50 60 1.0 ._ min. nun. rnin. min. min. min. min. rnin. 6 6 6 6 1 2 1 2 - 24 - 24 6 6 6 6 6 6 12 12 17 23 29 35 -21 - 6 12 17 12 17 23 23 12 24 24 36 49 49 - 18 11 18 24 24 30 43 49 15 12 24 31 43 61 67 29 18 24 31 37 61 67 196 196 196 189 210 196 225 225 176 183 169 176 189 189 172 178 178 178 197 197 243 228 134 146 140 146 165 165 23 1 222 234 234 240 258 258 260 168 186 186 192 216 216 175 192 197 208 225 230 234 278 230 247 247 258 279 279 366 366 360 372 372 378 478 478 453 459 465 471 471 471 493 493 493 510 505 510 496 481 452 464 470 476 476 487 462 462 462 480 480 486 513 484 420 426 432 444 450 450 465 471 471 490 496 502 487 531 477 477 483 502 508 514 730 737 737 744 744 744 739 739 771 771 771 784 784 777 731 738 738 744 750 750 742 757 700 700 706 719 719 719 745 738 744 744 756 762 762 774 714 726 726 744 744 744 748 740 745 751 762 767 773 792 31Sa 334 340 362 372 378
-
__
-
a Row of d a t a rejected a s more t h a n four times t h e average deviation from its replicate a n d clearly in error.
Figure 4. Evolution of Nitrogen by Iodine-Azide Reaction a t Low Cysteine Concentrations
where
+ 1.95t [-ss-I
Table 111. Volume of Nitrogen Evolved by Iodine-Azide Reaction with Mixed Catalyst Solutions
0.15
3
9
The constant, 9, was obtained as the average intercept of the
0.0
-
=
(1)
Analysis of Variance ( 1 ) ([SHI = 0.90 peq./ml. dropped from this analysis) Degrees of Sum of Mean Variance Source Freedom Squares Square Ratio Cysteine concn. 2 4,636,897 2,318,448 3,078. 19.68 44,460 14,820 Cystine concn. 3 3,093 4.10 Time 5 15,463 Cysteine X cys5,460 7.24 32,762 tine concn. 6 Cysteine concn. X time 10 805 80 0.11 Cystine concn. X 15 3,710 247 0.33 time Cysteine X c y s -
Significance,
%
>O
1 1 1
N.S.
N.s.
1
1526
ANALYTICAL CHEMISTRY
ordinate, T- is the volume of nitrogen evolved in microliters, 1 is the time in minutes, and [-SS-] is the cystine concentration in microequivalents per milliliter. Effect of Mixtures of Cysteine and Cystine. I n order to determine whether the effects of sulfhydryl and disulfide concentrations were additive in mixtures of the two, observations were made on mixed catalj st colutions containing both cysteine and cystine (Table HI).
1400
% E >
1200 1000
A 0
w'
800
z W
(3
0
k
600 400
teine concentration against the volume of nitrogen evolved gave a straight line. .4t higher concentrations, a statistical equilibrium b e t w e n the pairs and single molecules of sulfhydryl would be maintained by the more frequent collisions, such that the reaction becomes independent of the number of collisions and therefore first order. Some support for this is found in Figure 5 , where a significant curvature occurring a t low concentrations changes to a linear relationship a t moderate concent,rations. At high levels, the iodine and azide concentrations rather than the sLlfhydryl concentration become the limiting factors and hence affect t'he curvature a t the high sulfhydryl concentrations. -1fading of the iodine color was observed in some experiments at this level. The observations of Friedmann that treatment of cysteine ivith iodine practically abolishes its catalytic effect was confirmed; however, the iodine must oxidize the cysteine to some product other than cystine, as the latter exhibits marked catalytic power. The results obtained with cj.stine as a catalyst and with large excesses of iodine and azide (Table 11) indicate a linear dependence of the volume of evolved nitrogen upon both time and cystine concentration; in other words, the cystine under these conditions is not osidized further by iodine within the confidence limits indicated in the analysis of variance. Therefore, a possible mechanism for the reaction might be the following:
R-S,
R-S 200
~ + I ? + 0
R-S 0.4
0.8
1.2
1.6
The analysis of variance indicates a significant interaction between the cyptine and cysteine concentrations and therefore t h e r do not appear to be strictly additive. However, if the least squares equations i l and 2 ) obtained from previous data on the pure compounde are added as indicated, the equation becomes
[-33
+ 819 [SH] + 31.5 [SH]'] (1 - A-') +9 + 1.95 t[-SS-]
(3)
The factor (1 - *-l-') is introduced to provide for the rapid evolution of nitrogen in the first 10 minutes by cysteine. I\.hile A cannot be evaluated from the data available, it must be of such a size as to make the term d-$about equal t o zero a t 10 minutes. The calculated values are also included in Table 111 for comparlson purposes. Effect of Groups Attached t o Sulfhydryl Group. In order t o investigate the effect of the group t o which the sulfhj-dry1 was attached, glutathio~ieand crystalline ovalbumin were rompnred with cysteine. The solutions were prepared on a w i g h t basis. The weight of ovalbumin necessary was calculated according to its labile sulfur content as determined by Kassel and Brand ( 5 ) . The o-iodosohenzoate test as used by Larsen, Jenness, and Geddes ( 7 ) i m p en:plo>-ed as an independent check on the sulfhydryl Concentration. The results are presented in Figure 6. The o-iodosobenzoate test indicated that the glutathione was 100% in the reduced form but, in the case of ovalbumin, only 48% of the equivalents of labile sulfur were present as sulfhydryl.
\
The catalytic mechanism suggested by Friedmann would require, in the presence of large excesses of iodine and sodium azide, that the rate would be controlled b y the number of pairs of sulfhydryl molecules or, in other uvords, the reaction should be second order with respect t o the concentration of sulfhydryl groups, This has been observed at low cysteine concentrations where, as illustrated in Figure 4,a plot of the square of the cys-
2XaK3
\
RAS ~
R-S
,
+ 2xa1 +
3S2t
This rectilinear relationship for the catalytic activity of cystine, combined with the observation in Table I that the evolution of nitrogen by the catalytic activity of cysteine is practically complete in 10 minutes, supplies further evidence that the oxidation reaction for cysteine at 17.5' C. suggested by Friedmann is not satisfactory. Therefore, an independent and parallel reaction is suggested for the oxidation of rysteine at 17.5' C. t o some other oxidation product than cystine. Further evidence that this mechanism is consistent with observations can be obtained from consideration of the theory of rate equations. According t o the implied assumptions of Friedmann, the following equations T o d d represent the catalytic and oxidation reactions a t low concentrations of cysteine. Second-order catalysis.
dT' dt
Second-order oxidation.
.st, this relationship is reversed, with cystine liberating more nitrogen than cysteine, even though the gas volumes evolved by both still increased with increased concentrations of iodine and azide. The analysis of variance of the data on mixtures of cysteine and cystine indicates an interaction between t,he two catalysts, but if this interaction is ignored, as was done In Equation 3, the resulting expression predicts the observed values xithin the 95% confidence limits in most cases. It is readily apparent that the group attached t o the sulfhydryl has a marked influence on the volumes of nitrogen evolved. I t is conceivable that this influence might be due t o a change in the rate of catalysis of the iodine-azide reaction, in the oxidation rate of the sulfhydryl group, or in the diffusion rates of the catalysts. If this phenomenon were considered from the viewpoint of only the relative diffusion rates of the various compounds, one would expect the catalytic activity for glutathione and ovalbumin t o proceed a t I o i ~ erates r than cysteine, and therefore, lower volumes of nitrogen would be evolved before the oxidation of the sulfhydryl groups was complete. This is qualitatively but not quantitatively in agreement with observations. The slope of the curves obtained in Figure 6 for ovalbumin substantiates the results of the o-iodosobenzoate test, in that it suggests the possible presence of disulfides. A calculation of the evolution of nitrogen with time for this situation approsimates the observed curve when based upon the assumptions t h a t : (1) the labile sulfur not detected by the o-iodosobenzoate test was in the form of the disulfides, (2) the volume of gas evolved in the first 10 minutes was due t o the sulfhydryl groups only, and (3) the subsequent evolution was due only t o disulfide, with the nitrogen being evolved according to the cystine equation. As an analytical tool this reaction is appropriate for the measurement of sulfhydryl and disulfides groups attached t o known radicals a t the low concentration encountered in biological liquids. However, the error variance of the results obtained is rather high for some purposes and, in its present form, it is not suitable for mixed sulfhydryls or disulfides, which are common i n biological liquids. LITERATURE CITED
I I I
700
-
600
% Y
8 3
9W
z W
a
$ t z
II I/ 400
I
II I/ 300 II
Key:
A
D
b
500
A
A
conc. by weight Cy eq/ml)
---Cysteine -Glutathione -Ovalbumin
A
0.9 0.6
o
0.3
(1) Cochran, IV. D., and Cox, G. AI., "Experimental Design," Chap. 3. pp. 39-85, Sew York. John Wley 8; Sons, 1950. ( 2 ) Feigl, F., "Qualitative dnalysis by Spot Tests" (trans.), pp. 195-7, 291-4, New York, Elsevier Publishing Co., 1939. (3) Friedmann, E., J . prakt. Chem., 146, 179 (1936). (4) Harland, H. A,, Ashworth, L7. S., and Golding. S . S., Cereal Chem., 20, 535 (1943) (5) Kassel, B., and Brand, E., J . B i d . Chem., 125, 145 (1938). (6) Larsen, R. A , , Jenness, R., and Geddes, IT. F., Cereal Chem., 26, 189 (1949). (7) Ihid., p. 287. (8) Larson, B. L., a n d Jenness, R., J . Duiry Sci., 33, 896 (1950). (9) Loomis, W.F., Science, 109, 491 (1949). (10) Stein, W. H.. Archihald, R. >I,,Brand, E., Cannan, R. K.,
Clarke, H. T., Edsall, J. T., Foster, G. L., AIoore, S.,Shemin, D., Snell, E. E., and Tickery. H. B., Ann. S . Y.A c n d . Sci., 47, 59 (1946).
200
(11) Stewart, A. P., J r . , J . Dairy Sci., 34, 743 (1951). (12) Townley, R. C., and Gould, I. .I.,Ibid., 26, 853 (1943). ,,
IO0
(13) Umbreit, W. R., Burris, R. H., and Stouffer, J. F., Manometric Techniques and Related Methods for the Study of Tissue hIetaholism," pp. 2-5, 41, hlinneapolis, Burgess Publishing Co., 1947. (14) Wald, G., and Brown, P. K., J . Gen. PhysioZ., 35, 797 (1952).
0
Figure 6 .
Y
IO
20
40 TIME (rnin.)
30
50
60
Effect of Group Attached to Sulfhydryl on Catalysis of Iodine- Azide Reaction
RECEIVED for review March 9, 1953. Accepted J u l y 17, 1953. Presented before t h e Biochemistry Section of t h e Eighth Southwest Regional Meeting, AMERICAPCHEMICALSOCIETY,Little Rock, Ark., December 5, 1952. Taken from the thesis of D . W. Whitman, presented in October 1962 t o the L-niversity of Illinois in partial fulfillment of the requirements for the degree of doctor of philosophy.
