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`
`The Reaction of Combined Cystine of Wool
`with Sodium Bisulfite
`
`G. J. Schuringa, C. Schooneveldt, and T. Konings
`Vezelinstituut T.N.O., Delft, Holland
`
`THE DISULFIDE cross-linkages in wool,~ due
`to the combined cystine, are of fundamental impor-
`tance concerning the physical and mechanical prop-
`erties of the wool. The changes in the elastic prop-
`erties of wool, brought about by different reagents,
`are mainly due to the breaking of these disulfide cross-
`linkages.
`If wool is treated with sodium bisulfite, according
`to Clarke [5] and Speakman [17], the combined
`cystine undergoes the following reaction:
`
`’
`
`In 1946, Carter, Middlebrook, and Phillips [4]
`summarized the results of previous investigations [7,
`8, 12]. They came to the conclusion that not all of
`the combined cystine reacts in the same way with
`sodium bisulfite, but that cystine can be divided
`into different fractions. According to these authors,
`about 25 % of the cystine does not react with sodium
`bisulfite, another 25% gives combined a-amino-
`acrylic acid, and one-half of the cystine is converted
`into cysteine and cysteine sulfonate side-chains.
`From the latter fraction part of the cystine can be
`restored by rinsing with water.
`Moreover, Speakman [18] found that when hu-
`man hair is reduced with sodium bisulfite the cystine
`cross-linkages can be restored by rinsing with a solu-
`tion containing no oxidizing agent.
`In connection with the extensive application of bi-
`sulfite for various treatments of wool, it is very im-
`portant to know which reaction takes place in the wool
`and how this reaction can be influenced.
`Therefore, we investigated the final effect of the
`reaction between wool and sodium bisulfite, and the
`influence of rinsing.
`
`Experimental Procedure and Results
`Samples of 400 mg. of woolen yarn, degreased by
`extraction with a mixture of trichloroethylene and
`
`ethanol, were first soaked for 30 min. at room tem-
`perature in 125 times their weight of 5 % sodium
`bisulfite at pH 5.2,* and then reduced in an identical
`solution which had been preheated in a boiling water
`bath to 92°-95°C and kept at that temperature for
`30 min. Heating the solution in a water bath pre-
`vented it from reaching the boiling point. Boiling had
`to be prevented because dispersion of tiny wool par-
`ticles by the movement of the liquid might cause er-
`rors in the analytical data.
`The bisulfite-treated wool was then subjected to
`the following aftertreatments: (a) no rinsing, im-
`mediate hydrolysis; ( b ) before hydrolysis, rinsing
`for 20 hrs. in 0.50~o sodium acetate adjusted to pH
`5.2 by the addition of acetic acid; (c) before hy-
`drolysis, rinsing for 20 hrs. in 95% ethanol.
`Two additional wool samples were reduced for
`60 min. and two more for 90 min. In each case, one
`sample was hydrolyzed without rinsing and one was
`hydrolyzed after 20 hrs. of rinsing with the sodium
`acetate buffer solution.
`The hydrolyses essential for determining cysteine
`and cystine were carried out in open test tubes with
`10 ml. of 6N sulfuric acid. Hydrolyzing under
`C02 atmosphere did not make any difference.
`Al-
`though after 5 hrs. of boiling the keratin had not
`been completely hydrolyzed to amino acids, hydro-
`lyzing for more than 5 hrs. caused no change in the
`values found for cysteine and cystine.
`The amounts of cysteine and cystine were deter-
`mined according to a method indicated by Shinohara
`[16], which enables cysteine and cystine to be deter-
`mined simultaneously.
`We started with three equal parts of the . hy-
`drolyzate, and adjusted them to pH 5.2 with sodium
`acetate and acetic acid. To the first part an aqueous
`solution of HgCl, and phosphotungstic acid reagent
`(Folin and Marenzi [ 10 ] ~ was added. The color of
`
`* This solution was maintained throughout the investiga-
`tion because of its optimum reducing action.
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`this solution served as a blank. To the second part
`only phosphotungstic acid was added. With the aid
`of a standard calibration curve the cysteine content
`was calculated from the measured color intensity.
`To the third part sodium bisulfite and phosphotungstic
`acid were added; then, with the aid of the blank, the
`known cysteine content, and the standard calibration
`curve, the cystine content was calculated from the
`color intensity caused by this reaction.
`The cysteine and cystine percentages were de-
`termined for the wool samples treated with bisulfite
`in the above manner. The results are given in
`Table I.
`Table I shows that after reduction for 30 min.
`about one-half of the cystine was converted. If,
`however, after the reduction the sample is rinsed
`with pH 5.2 buffer, it appears that cysteine is al-
`most completely reconverted into cystine. Rinsing
`with ethanol does not cause reconversion of cysteine
`into cystine; on the contrary, the reduction of cystine
`appears to continue.
`After more intensive reduction-namely, for 60
`or 90 min.-rinsing again caused a considerable re-
`conversion into cystine. However, a small amount
`of cysteine appears to be irreversible. This amount
`increases with a longer reduction time, being greater
`after 90 min. treatment than after 60 min.
`
`* This value must be multiplied by 2 in order to obtain the
`actual cysteine S content (see text).
`
`Additional samples of wool yarn were reduced for
`45 min. Samples that were not rinsed after the re-
`duction were immediately hydrolyzed. It appears
`from Table II that about two-thirds of the cystine
`was reduced, the cysteine S content being 1.91 °,>o.
`By rinsing after reduction in the pH 5.2 buffer the
`cystine percentage increased, but 0.39% cysteine S
`could not be reconverted into cystine. Also, it ap-
`pears that rinsing with ethanol hampers the recon-
`version into cystine.
`A few reduced samples were treated with mono-
`iodoacetic acid of pH 8.3 for 15 min. at 95 ° C. Ac-
`cording to Sanford and Humoller [15] and Mirsky
`and Anson [13] the free thiol groups are blocked by
`this treatment.
`The color intensity measured is due to cysteine
`formed during hydrolysis from the cysteine sulfonate
`groups. As there is an equal amount of cysteine
`blocked by acetic acid groups, the value found has to
`be increased twofold. The result of the measurement
`was 1.04~le ; according to this method, therefore,
`2.08% cysteine S must have been present.
`It is also possible to block the thiol groups with
`the aid of ethyl iodide [ 13, 15 ] . To enable a com-
`parison with the previous treatment, the reduced
`wool was shaken for 18 hrs. in a suspension of ethyl
`iodide in water at room temperature. The values
`found for the cysteine S and cystine S contents
`showed a large divergence. It appeared, however,
`that much more cysteine was reconverted into cystine
`than with the monoiodoacetic acid treatment.
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`Cuthbertson and Phillips [6] observed that in wool
`treated with a potassium cyanide solution the com-
`bined cystine is converted into combined lanthionine :
`
`In order to examine the effect of this reaction we
`treated wool with potassium cyanide, as described
`by Farnworth, Neish, and Speakman [9].
`Wool with a cystine content of 3.07% was treated
`with 30 times its weight of a O.1M potassium cyanide
`solution at 66°C for 22 hrs. After this treatment the
`wool still contained 2.53% sulfur (determined by
`Blackburn’s method [2] ) ; a determination of the
`cystine content showed that 1.50 0~o cystine S was still
`present. This wool was then reduced for 45 min. in
`5% sodium bisulfite. The results are given in Table
`III.
`From Table III it appears that one-third of the
`disulfide S was reduced to cysteine. After prolonged
`rinsing in the buff er solution practically all the
`cysteine had been reconverted into cystine.
`Woolen yarn was treated in the usual method with
`potassium cyanide for various reaction times. As
`the duration of the treatment was prolonged, the
`amount of nonreduced cystine gradually approached
`a certain final value. After a period of 16z hrs. the
`wool still contained 0.91 % cystine S. A few samples
`of this wool were then, as in the previous experiment,
`reduced in 5 % sodium bisulfite for 45 min. The re-
`sults are given in Table IV.
`Table IV shows that in neither case (a) nor (b)
`was cysteine S present, and no change in the cystine
`S content occurred.
`
`Discussion
`
`If wool is treated with sodium bisulfite, the disulfide
`cross-linkages are affected. In the opinion of Clarke
`
`[5] and Speakman [17] the combined cystine is de-
`composed into cysteine and cysteine sulfonate :
`
`Hydrolysis of this bisulfite-treated wool causes a
`decomposition of the cysteine sulf onate into cysteine
`and sodium bisulfate:
`
`Reactions (1) and (2) occur when bisulfite-treated
`wool is hydrolyzed without previous rinsing (see
`Table I (a) ). About one-half of the cystine is con-
`verted into cysteine.
`However, if before hydrolyzing the reduced wool
`is rinsed in a buffer solution, cysteine is reconverted
`into cystine (see Table I ( b ) ) . This phenomenon
`can be explained by assuming that reaction (1) is
`an equilibrium:
`
`By rinsing in water, bisulfite is withdrawn from the
`reaction, thus shifting the equilibrium to the side of
`cystine.
`Rinsing in ethanol does not shift the equilibrium
`in favor of cystine (see Table I ( c ) ) , the solubility
`of sodium bisulfite in ethanol being very low.
`The assumption of the existence of an equilibrum
`is in close agreement with the result of Katz and
`Tobolsky [ 11 ] . These authors studied the relaxation
`of wool fibers in water, bisulfite, etc. For fibers
`treated with bisulfite, the rate of relaxation appears
`to be much greater than that for fibers immersed in
`water. If the bisulfite-treated fibers are rinsed before
`stretching, the rate of relaxation is equal to that of
`the untreated fibers. In the case of the increased rate
`of relaxation, a great part of the disulfide cross-link-
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`ages are broken. These linkages are recovered by
`rinsing, and the rate of relaxation then decreases to
`that of untreated wool.
`Table II shows that a higher cysteine percentage
`was found after treatment with monoiodoacetic acid.
`This phenomenon can he explained by assuming that
`at the beginning of the reaction with monoiodoacetic
`acid the concentration of bisulfite in the fiber is still
`high. The rinsing effect during this short reaction
`time (15 min.), however, is small. This results in
`a continuing reduction, causing a somewhat higher
`cysteine content.
`By assuming an equilibrium, it can also be seen
`why so much cysteine is reconverted into cystine after
`the ethyl iodide treatment, for in this case the’ wool
`has been exposed to a certain rinsing eff ect for 18 hrs.
`After an intensive reduction and subsequent rins-
`ing in pH 5.2 buffer a small part of the cysteine and
`cysteine sulfonate is not reconverted into cystine
`(see Table II ( b ) ) , but remains in the form of cys-
`teine. This phenomenon led to the conclusion that
`the wool disintegrated to such an extent that some
`cysteine groups were not able to react with cysteine
`sulfonate groups in order to form combined cystine
`and sodium bisulfite. This may be explained by as-
`suming displacement of the corresponding cysteine
`and cysteine sulfonate groups so that they are beyond
`the reach of each other.
`This conception is supported by the results of the
`reduction of KCN-treated wool. Here, all disulfide
`cross-linkages are re-formed because the more stable
`lanthionine groups prevent the wool from disinte-
`grating.
`Brown and Harris [3] ] observed a similar phe-
`nomenon when wool was reduced with sodium hy-
`drosulfite and new cross-linkages were subsequently
`formed on treatment with alkyl halides. If there was
`some lapse of time between these treatments, the
`wool appeared to be badly damaged, as the newly
`formed cross-linkages were insufficient in number.
`However, if the treatments were carried out almost
`simultaneously, the combined cysteine groups had
`no opportunity to shift, and damage was avoided.
`From Table IV it is apparent that the cystine frac-
`tion remaining after prolonged treatment with potas-
`sium cyanide, being about 30% of the original cystine
`content, cannot be reduced by sodium bisulfite.
`This means that the cystine groups which could
`not be changed by potassium cyanide were also un-
`affected by sodium bisulfite. This cannot be a matter
`
`of equilibrium but must be due to the structure of
`keratin.
`Thus, we conclude that the reaction of the combined
`cystine with sodium bisulfite must be considered to
`be an equilibrium reaction; also, it is probable that
`not all cystine groups are equally reactive. This as-
`sumption holds for the reaction with sodium bisulfite
`as well as for that with potassium cyanide and prob-
`ably also for the reaction with thioglycolic acid [14].
`This difference in reactivity must be attributed to a
`difference in accessibility of the keratin. For various
`reagents the accessibility has not the same magnitude.
`Alexander, Hudson, and Fox [ 1 ] studied the reaction
`of oxidizing reagents with combined cystine of wool
`and observed that different amounts of cystine were
`oxidized by KMnO~, peracetic acid, and chlorine.
`There is a great similarity between these results and
`the estimations of the amount of crystalline matter
`in cellulose. Using chemical methods, the degree of
`crystallinity observed depends upon the method.
`Hence, in the case of cellulose it is also better to
`speak about accessibility.
`The accessibility of wool for sodium bisulfite and
`for potassium cyanide is about the same. With both
`reagents, about two-thirds of the combined cystine
`can be converted, whereas one-third is not affected.
`It seems possible that, under certain conditions,
`the combined cystine forms combined a-aminoacrylic
`acid by splitting off hydrogen sulfide. However, we
`did not find any indication of this reaction taking
`place. In a few cases the sum of the cysteine S and
`cystine S contents was, after the treatments of reduc-
`ing and hydrolyzing, somewhat lower than the
`original cystine S content of wool, but the difference
`never exceeded 10 % . This difference need not be
`caused by the formation of a-aminoacrylic acid, but
`may be due to other causes-for instance, the forma-
`tion of lanthionine.
`
`Conclusions
`
`1. The effect of sodium bisulfite on wool can be
`represented by the reaction
`
`2. At 95°C about two-thirds of the disulfide cross-
`linkages of the wool react according to this equation.
`By rinsing in water, all of these cross-linkages are
`re-formed. The remaining one-third does not react
`with sodium bisulfite at all.
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`3. After a prolonged reduction the rinsing does
`not reconvert all of the cysteine and cysteine sulfonate
`into combined cystine. This phenomenon is due to
`molecular shiftings in the fiber, which prevent the re-
`formation of some of the disulfide cross-linkages.
`4. The cystine groups which cannot react with po-
`tassium cyanide to form lanthionine do not react
`with sodium bisulfite either. This may be explained
`by assuming different accessibilities for different parts
`of the keratin fiber.
`
`Literature Cited
`
`1. Alexander, P. A., Hudson, R. F., and Fox, M.,
`Biochem. J. 46, 27 (1950).
`2. Blackburn, S., Tech. Comm. Proc. 2, P72 (1948).
`3. Brown, A. E., and Harris, M., Ind. Eng. Chem. 40,
`316 (1948).
`4. Carter, E. G. H., Middlebrook, W. R., and Phil-
`lips, H., J. Soc. Dyers and Colourists 62, 203
`(1946).
`5. Clarke, J., J. Biol. Chem. 97, 235 (1932).
`6. Cuthbertson, W. R., and Phillips, H., Biochem. J.
`39, 7 (1945).
`
`7. Elsworth. F. F., and Phillips, H., Biochem. J. 32,
`837 (1938).
`8. Elsworth, F. F., and Phillips, H., Biochem. J. 35,
`135 (1941).
`9. Farnworth, A. J., Neish, W. J. P., and Speakman,
`J. B., J. Soc. Dyers and Colourists 65, 447 (1949).
`10. Folin, O., and Marenzi, A. D. J., J. Biol. Chem. 83,
`109 (1929).
`11. Katz, S. M., and Tobolsky, A. V., TEXTILE RE-
`SEARCH JOURNAL 20, 87 (1950).
`12. Middlebrook, W. R., and Phillips, H., Biochem. J.
`36, 428 (1942).
`13. Mirsky, A. E., and Anson, J. Z., J. Gen. Physiol. 18,
`307 (1935).
`14. Patterson, W. J., Geiger, W. B., Mizell, L. R., and
`Harris, M., J. Research Natl. Bur. Standards 27,
`89 (1941).
`15. Sanford, D., and Humoller, F. L., Ind. Eng. Chem.,
`Anal. Ed. 19, 404 (1947).
`16. Shinohara, K., J. Biol. Chem. 112, 683 (1935).
`17. Speakman, J. B., J. Soc. Dyers and Colourists 52,
`335 (1936).
`18. Speakman, J. B., U. S. Patent 2,410,248 (1946) ;
`2,351,718 (1944).
`
`(Manuscript received December 12, 1950.)
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