Since an Important Part Played by Sulphydryl Compound in the Tissue
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The Journal of Biochemistry, Vol. XII, No. 2. POTENTIOMETRIC DETERMINATION OF CYSTINE AND CYSTEINE. BY 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.