Since an Important Part Played by Sulphydryl Compound in the Tissue

The Journal of Biochemistry, Vol. XII, No. 2.

POTENTIOMETRIC DETERMINATION OF CYSTINE AND CYSTEINE.

KAGEYU YAMAZAKI.

(From the Institute of Medical Chemistry, Kyushu Imperial University, Fukuota. Director: Prof. Keizo Kodama.)

(Received for publication, June 14, 1930.)

Since an important part played by sulphydryl compound in the tissue respiration has been confirmed by the brilliant work of Hopkins on glutathione, its content in various tissues has been intensely studied by many investigators. It is regrettable, how ever, that the method used for its quantitative determination seems to be not fully satisfactory. Tunnictiffe's method (1925) can be applied only when a large amount of material is available, for the endreaction, using sodium nitroprussid as external indicator, is less sensitive, when the concentration of -SH compound is small, and often escapes our attention, owing to the transitory nature of the colour developed. Okuda (1925) published a valuable method which consists in extracting the sulphydryl compound with sulfosalicylic acid and titrating with iodate solution in the presence of iodide, the endreac lion being read by the appearance of yellow colour of free iodine. Of course this method can not be applied to the coloured solution, such as the hydrolysate of protein. In a later publication, Okuda (1929) recommended to add at first a definite amount of iodine solution to the hydrolysate and to titrate back the excess of iodine with thiosulfate, the endreaction being taken by the disappearance of the colour of iodine absorbed in chloroform, added to the reaction system. It was found previously however, in testing this method, that the titration of iodine absorbed in chloroform cannot be executed

207 208 K. Yamazaki: in a convincible manner. Hence we endeavoured to remove this disadvantage and found that a potentiometric method is more con venient and accurate for the estimation of the minute amount of cystine and eysteine, even in the coloured solution.

PROCEDURE. The titrations-vessel of about 50cc. capacity was fitted with the rubber stopper, which carries 4 bores for the electrode, the agar salt bridge, the titratiog microburette and a tube for bubbling. As the electrode the blank Pt wire of 0.5mm. diameter and of 8cm. length was used. The gold was also tried, but found to be rather unfit, owing, to its slowness to follow the potential change. As the half cell the saturated calomel electrode was used, and it was connected with the reactions-vessel by means of the agar salt bridge. The air was bubbled through the solution at a constant rate for the purpose of stirring. The potential reading was taken one minute after each addition of an aliquot amount of the oxidising solution from the microburette. The endreaction was read by a point where maximal potential spring occurred. In this case it is not necessary to wait until the potential attains to the equilibrium as the estimation of the real potential value is not to our purpose.

EXPERIMENTS. I. The titration of eystine with bromate in the presence of bromide. Cystine is oxidized with bromine as the following equation shows. R-S-S-R+10Br.+6H2O=2R•SO3H+10HBr. When the dilute bromine solution is used, the endreaction of the titration is not clear. In the potentiometric titration , as soon as the titration arrives at the endreaction, the potential of the solu tion gives rise to a marked jump, as is indicated in Table I. It is possible, therefore, to carry out microestimation with fair accuracy. Potentiometric determination of eystine and cysteine . 209

In the following tables the potential was represented in milli volt in reference to the saturated caromel electrode .

TABLE I. The titration of eystine by KBrO4 in the presence of KBr .

The temperature and the acidity of eystine solution to be examined have some influence upon the sharpness of potential break. The stronger the acidity and the higher the temperature, the more distinct is the potential break at the endpoint of the titration. The quantity of bromide solution exerts also a great effect upon the sharpness of the potential break. 210 K. Yamazaki:

II. The titration of cystine with bromate in the absence of bromide. Bromate in the absence of bromide has an oxidizing power, which may be represented by the following equation.

TABLEII. The effect of temperature. Potentiometric determination of cystine and cysteine . 211

TABLE III. The effect of acidity .

BrO3-+6H+6ƒÆ=Br-+3H2O

Therefore, cystine is oxidated by the bromate in absence of bromide as the following equation shows.

3R-S-S-R+5KBrO3+5HCl+3H2O =6R-SO3H+5HCl+5BrH. 212 K. Yamazaki:

TABLE IV. The effect of the concentration of KBr.

As will be seen from Table V the electrometrical titration of cystine with bromate solution gives satisfactory value if the temperature and acidity of the titrated solution are properly adjusted. Since 5 molecules of bromate correspond to 3 molecules of cystine, it can easily be calculated from the result above mentioned,

TABLEV. The titration of cystine with potassium bromate solution. The titrationsvessel contained 10cc. of 2% HCl, 1cc. of 4% HCl, 1cc. of 2% HCl, containing ca. 0.1mg. cystine. Potentiometric determination of eystine and cysteine . 213

that the solution contains 0.108mg of cystine, which stands in close agreement with the theoretical value . The effect of temperature and acidity is fairly pronounced as

will be clearly demonstrated in tables VI and VII .

TABLE VI. The effect of temperature upon the titrationsvalue of

cystine with potassium bromate . The titrations vessel contained 12cc . of 2% HCl and 1cc. of 0.46M/1000 cyctine solution.

TABLE VII. The effect of the acidity upon the titrationsvalue of cystine with potassium bromate. The titrationsvessel contained 11cc . of HCl of various concentration and 1cc. of 0.46M/1000 cystine solution.

III. The titration of cysteine with bromate in the presence of bromide.

Cysteine is oxidised to cysteinie acid by the bromine, which is produced by the action of bromate upon bromide in the presence of a sufficient amount of acid. The reaction may be represented as follows.

KBrO3+5KBr+6HCl•¨3Br2+6KCl+3H2O

3Br2+3H2+R-SH•¨RSO3H+6BrH 214 K. Yamazaki:

Hence, one molecule of potassium bromate corresponds to one molecule of cysteine. In carrying out the titration it was found that this was really the case as shown in Table VIII, and the potential jump at the endreaction was also sharp enough.

TABLE VIII. The titration of cysteine with bromate in the

presence of bromide.

The titrations-vessel contains:

- 0.84M/ 1000 cysteine 1.0cc. 2% HCl 10.0 •V

4% HCl 1.0 •V

M/10 KBr 1 .0 •V

The effect of the acidity in this case was not so marked as in

the foregoing experiment, being only perceptible when the tempera

ture of the titrated solution was lowerd. As will be seen from

Table IX, at the temperature of 18•Ž no difference in the endreac

tion was observed between 1% and 4% hydrochloric acid. But when the temperature was lowered to 4.5•Ž, more than the

theoretical amount was required. Potentiometric determination of cystine and eysteine. 215

TABLE IX. The effect of temperature and acidity upon the titrationsvalue of cysteine with bromate in the presence of bromide.

IV. Poteartiometric titration of cysteine with potassuium bromate.

Cysteine can be titrated with bromate in absence of bromide.

The reaction here involved may be written as follows.

R-SH+KBrO3+HCl•¨RSO3H+KCl+HBr.

Hence, one molecule of KBrO3 corresponds to one molecule of

eysteine. But this is satisfied when the temperature and the acidity

are well controlled. As will be seen from Table IX and X respec

tively the lower tmperature and the lower acidity lead to a higher

value than the theoretical. 216 K. Yamazaki:

TABLE X. Influence of temperature.

TABLE XI. Influence of acidity.

V. Potentiometric titration of cysteine with iodate in the presence of iodide. The iodemetric titration of cysteine or the compounds has been extensively used by many investigators of biological field. Prof. Okuda also has given very valuable method, which is now widely used in our country. According to him the titration of cysteine with iodate in the presence of iodide is more complicated than with bromate owing to the fact that cysteine can be oxidised to the stage of cystine or cysteinic acid. The ratio of cystine and cysteinic acid formed varies with the temperature and the acidity at which the reaction is carried out. When cystine is formed, one molecule of cystine cor responds to one atom of iodine, while in the case of cysteinic acid, 6 atoms. The intermediate value of iodine, therefore, shows that cystine and cysteinic acid are produced at variable ratios. Potentiometric determination of cystine and cysteine. 217

The whole problem was studied once more, using the potentio metric titration. The experimental procedure is the same as in the previous experiment. The results of the experiment on the effect of the temperature and the acidity are summarised in Tables XII and XIII.

TABLE XII. The effect of the temperature. The reactions-vessel contained 1.0cc. 0.85M/1000 cysteine, 10cc. of 2% HCl 1cc. of 4% HCl and 1cc. of M/10 KJ.

Remark: At the point marked* faint yellow colour was produced.

TABLE XIII. Influence of the acidity. 218 K. Yamazaki:

As will be seen from Table XII the potential spring is not so apparent as in the case of bromate. Nevertheless the end reaction is precise enough and is obtained just before the yellow colour due to free iodine appears. The theoretical value of the titration of 1cc. of 0.00085 mol of cysteine with 0.001 mol iodate solution should be 0.141cc., when the oxidation stops at the stage of eystine, while when all of the molecules are oxidised to cysteinic acid, 0.85cc. is required. All the experimental results gave the intermediate values. Moreover, the higher the temperature and the lower the acidity, the larger the titrations value. This means that here more cysteinic acid was produced. In the course of the above experiment it was also noticed that when the oxidation of eysteine with iodine proceeds more slowly, the more eystine is formed and in the reverse case the more cysteinic acid. Therefore, the rate of titration exerts here also a great influence. The following experiments gives the evidence. Into 3cc. of the solution of the acidity equivalent to 2% HCl, which contained 1.0cc. of 0.00084 mol cysteine and 0.1 mol KJ, 2.0cc. of 0.001 mot KJO3 was added in one experiment as quickly as possible, and in another slowly drop by drop. The excess of iodine was titrated back with thiosulfate. In the first case 0.55cc. of 0.005N iodine solution was used, while in the second, 0.46cc. It should be concluded, therefore, that the titration of cysteine with iodine should be carried out under a strictly controlled condition in respect of temperature, acidity and the rate of addition of iodine, and the value should be calculated from the experimental value obtained on pure cysteine titrated exactly under the same condition. This is the serious drawback in the iodine method. This potentiometrical method can be satisfactorily applied to the colored hydrolysate, as well as to the extract obtained from tissues with sulfosalicylic acid. The cleavage products of protein, aceton-bodies, creatine and creatinine do not interfere. To deter mine eysteine in the proteinhydrolysate, Okuda first decolorized Potentiometric determination of cystine and cysteine. 219 with charcoal and then titrated with iodate as mentioned above. According to him, cysteine was absorbed by charcoal to a great extent, but this could be removed by washing thoroughly. But it was found that this recovery was not so complete when a small amount of cysteine was present. It seems, therefore, more pre ferable to avoid the use of charcoal and to titrate directly, this being only possible by the application of the potentiometric method. Okuda described that the reduction of cystine with zinc dust in coloured hydrolysate is incomplete. The author's experiments agreed with his observation.

VI. Potentiometric titration of cysteine with iodate. The titration of cysteine with iodate in absence of iodide was studied potentiometrically. The experiments showed that the influence of temperature is here also fairly significant, but that of acidity is inconsiderable.

TABLEXIV. The effect of the temperature on the titration with iodate. Each titrations-vessel contained 1.0cc. of 0.00084mol cysteine, 10cc. of 2% HCl.

TABLE XV. The effect of the acidity.

Each titrations-vessel contained 1.0cc. of 0.00084 mol cysteine and 12cc. of HCl of the concentration given in the table.

Temperature 25•Ž.

No aminoacid save cysteine is oxidised by iodate in an acid solution, containing 2% hydrochloric acid. But if the cysteine solution contained tryptophan, it required more iodate solution 220 K. Yamazaki:

and at the same time ammonia was found to develop as is shown in Tables XVI and XVII. Since typtophan alone is not oxidised by iodate at all, certain catalytic process of cysteine might intervene in this oxidizing process.

TABLE XX. Influence of tryptophan upon the titration value of cysteine with KJO3 alone.

TABLE XXI. The ammonia production due to the oxidation of tryptophan with iodate in the presence of cysteine.

DISCUSSION. The titration of cystine or cysteine with bromate was first introduced by Prof. Okuda and also was discussed by him in con nection with the effect of the temperature and the acidity of the titrated solution. In this method the endreaction is recognized by the appearance of brown color due to the free bromine. But this Potentiometric determination of cystine and cysteine. 221 appeals to our eye when the amount of free bromine attains to a certain amount in the titrated solution. Hence in a very diluted solution, as is usually the case in the biological micro estimation of SH-compound contained in the biological fluid, the percentage of error in the titration value becomes relatively large. This can be avoided to a certain extent by the application of potentiometric titration. The potential jump always occurs prior to the first appearance of yellow colour and in a distinct manner so that no individual error is allowed to come in. There exists no alternative whether we titrate with bromate solution in the presence of bromide or in its absence, if proper precautions are paid for the temperature and the acidity of the reaction system. One of the drawbacks in the titration with bromate is the fact that certain amino acids such as tyrosine and tryptophan are oxidised. Therefore, for the solution containing these amino acids with cysteine as protein hydrolysate the bromate cannot be used. In this case the iodometric titration is rather preferable. In the presence of iodine the iodate only oxidises cysteine, though in its absence tryptophan may be simultaneously oxidised to a certain extent. But owing to the lower oxidationspotential exhibited by iodate, the oxidation of cysteine to cysteinic acid does not run completely. Some portion of the cysteine remains at the stage of cystine. Moreover, the proportion of cystine and cysteinic acid formed is affected seriously by the temperature, the acidity, the concentration of cysteine present and the rate at which the oxidising agent is added to. It is obvious, therefore, that the iodometric titration is also far from satisfactory. Unfortunately there is no other good method at present. We are obliged to use this method under utmost precautions.

SUMMARY. It is recommended to apply the potentiometric titration for the determination of cystine and cysteine, the details of which are reported in this paper. Here the following summary is given. 222 K. Yamazaki.

1. Pure cystine and cysteine solution can be accurately titrated with bromate either in the presence of bromide or in its absence. 2. If the solution contains other aminoacids besides cystine, such as protein hydrolysate the titration with iodate in the presence of iodine is more accurate. 3. As is already depicted by Okuda, the temperature and the acidity of the solution have the marked effects upon the the titrat ing value, because cysteine can be oxidised to cystine or crysteinic acid in varying ratio as these factors change. 4. It was found, moreover, that the rate of titration with iodine has also some influence. When it is carried out more slowly the more cystine is formed and in the reverse case the more cysteinic acid. 5. Among various aminoacids only tryptophan exerts a definite influence upon the titrating value of cysteine by iodate. Perhaps the oxidation of tryptophan may be induced by the presence of cysteine. The author is deeply indebted to Prof. Keizo Kodama for his kind advice throughout this investigation.

REFERENCES. Okuda, Y. (1925): Journ. of Biochemistry, 5, 201. Okuda, Y. (1929): Nihon Nogeikagakukai Zasshi, 5, 549. Tunnicliffe (1925): Bioch. Journ., 19, 194.