THE EFFECT OF PYRUVIC ACID ON THE ESTIMATION
`OF CYSTINE AND CYSTEINE
`BY M . X. SULLIVAN A .ND W. c. HESS
`(From the Chemo-Medical Research Institute, Georgetuwn University,
`Washington)
`
`(Received for publication, Augll8t 24, 1937)
`
`In a study of the reaction of aldehydes and ketones with thiol
`acids Schubert (1, 2) found that cysteine reacts with various
`aldehydes to form condensation products with the loss of water.
`The probable structure of the complex formed he gives as
`
`CH,S"-
`"-cHR
`1
`CHNH/
`I
`COOH
`
`Independently, Ratner and Clarke (3) found that formaldehyde
`reacts with cysteine to form thiazolidinecarboxylic acid and that
`formaldehyde and aminoethyl mercaptan give thiazolidine, and
`described the compounds in detail.
`Since aldehydes such as formaldehyde, glyoxal, and methyl(cid:173)
`glyoxal and keto acids such as pyruvic acid are possible metabolic
`products of proteins, carbohydrates, and fats, and as such might
`occur more or less in biological solutions, blood and urine, con(cid:173)
`siderable study has been made in this laboratory on their effect
`on the determination of cystine and cysteine.
`In previous work (4, 5) it was shown that in cystine or cysteine
`determination the various aldehydes have little effect in low molar
`ratios and dilute solutions. With increasing concentration of
`cysteine and aldehyde and decreasing acidity, on the other hand,
`new compounds no longer reacting like cysteine arc formed.
`These compounds, derivatives of thiazolidinecarboxylic acid,
`described by Schubert and by Ratner and Clarke, do not yield
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`12
`
`Pyruvic Acid, Cystine, and Cysteine
`
`cysteinc readily either by the action of zinc and hydrochloric acid
`or by dilute acid hydrolysis and are negative in the various reac(cid:173)
`tions for cysteine.
`In his early work Schubert (2) considers that cysteine and
`pyruvic acid, on the other hand, make a simple addition compound
`with a probable structure of
`
`CHa"
`
`OH /
`
`/COOH
`C
`"-s-cH2- CHNH2- COOH
`
`but in more recent work (personal communication) he questions
`his early conclusions and considers that the complex between
`cysteine and pyruvic acid may also be a thiazolidine derivative.
`Without prejudice as to the nature of the pyruvic acid-cysteine
`complex, in the present paper we deal with the effect of pyruvic
`acid on cystine and cysteine determinations by various methods
`and also with the possibility of liberating cysteine from the
`complex by simple means.
`
`EXPERIMENTAL
`Pyruvic Acid and Cystine- Solutions of cystine and pyruvic
`acid in 0.1 N HCI were made so that 5 cc. contained 1 mg. of cystinc
`and increasing amounts of pyruvic acid: (A) 0.3 mg., (B) 3.0 mg.,
`and (C) 5.0 mg. of pyruvic acid. Within 30 minutes of mixing,
`these solutions were analyzed for cystine by the Sullivan (6),
`the Okuda (7), and the Folin-Marenzi (8) procedures. The
`cystine was estimated practically quantitatively by all three
`methods, even in Solution C, with a molar ratio of pyruvic acid
`In fact, with 24 hours contact,
`to cystine of approximately 14: 1.
`100 per cent of the cystine was recovered colorimetrically in
`Solution C. Likewise, with relatively concentrated solutions
`of cystine (1 mg. in 1 cc. of 0.1 N HCl and proportions of pyruvic
`acid as given above) there was little if any effect on the deter(cid:173)
`mination of cystine even in 24 hours standing of the mixture.
`Thus with 14 moles of pyruvic acid to I of cystine, the cystine
`findings were 97 per cent of the theoretical.
`Experiments were also carried out at pH 6.8, the pH of the
`distilled water, and in phosphate buffer, pH 7.4, the solutions
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`

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`M. X. Sullivan and W. C. Hess
`
`13
`
`being so made that each 5 cc. contained 1 mg. of cystine and 0. 73
`mg. of pyruvic acid (2 moles). After 18 hours standing of the
`reactants, with the controls respectively in distilled water and
`buffer at pH 7.4, colorimetric estimation on 5 cc. samples indi(cid:173)
`cated 92 per cent of the theoretical cystine at pH 6.8 and 89 per
`cent at pH 7.4.
`At pH 6.8 and 7.4 the solubility of cystine in the presence of
`pyruvic acid is questionable, so the results were interpreted to
`indicate little if any effect of pyruvic acid on the estimation of
`cystine; in short, no combination such as occurs between cysteine
`and pyruvic acid, presently detailed.
`Pyruvic Acid and Cysteine, Dilute Solution- Mixtures were
`made of cysteine hydrochloride and pyruvic acid so that each 5 cc.
`contained 1.0 mg. of cysteine and 0.73 mg. of pyruvic acid, that is
`mole for mole, with the solutions adjusted to pH 1, 2, and 3 re(cid:173)
`spectively. Analyzed by the Sullivan cysteine (9) procedure, at
`intervals up to 24 hours, the cysteine was recovered quantitatively
`within the limits of error, at all three pH ranges. The minimum
`finding of cysteine even after 24 hours standing of the pyruvic
`acid and cysteine at pH 1, 2, and 3 respectively was 95 per cent
`of the theoretical. The Folin-Marenzi method run as for cystine
`showed marked falling off at the end of 6 hours at pH 2.0 (84
`per cent return) and at pH 3.0 at the end of 6 hours (80 per cent
`return).
`Pyruvic Acid and Cysteine, Concentrated Solutions-Mixture-s
`were made of cysteine hydrochloride and pyruvic acid so that 2.5
`cc. of solution contained 20 mg. of cysteine weighed as the hydro(cid:173)
`chloride and 15 mg. of pyruvic acid, adjusted respectively to pH
`I, 2, and 3. Analyzed by the Sullivan method at the end of 6
`hours, 70 per cent, 63 per cent, and 59 per cent of the cysteinc
`were recovered at pH 1, 2, and 3 respectively and at 24 hours the
`results were practically the same. Less rcacti ve cysteine was
`found by the Folin-Marenzi procedure without sulfite than by the
`Sullivan procedure. At the end of 2 hours standing the respective
`percentage recoveries of cysteine at pH 1, 2, and 3 were, Sullivan,
`84, 80, and 76; Folin-Marenzi, 75, 60, and 53. The Okuda method
`without reduction at the end of 24 hours showed 60, 50, and 50
`per cent recovery at pH I, 2, and 3 respectively. However, when
`the Okuda cystine procedure, reduction with zinc and hydro-
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`

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`14
`
`Pyruvic Acid, Cystine, and Cysteine
`
`chloric acid, was employed, 100 per cent return of the cysteinc was
`obtained even at the end of 24 hours contact of the reactants at
`each pH. The Okuda values indicated several possibilities:
`either the phenomenon was due to greater oxidation of cysteine
`in the presence of pyruvic acid or the pyruvic acid-cysteine com(cid:173)
`plex was split by the action of zinc and hydrochloric acid or by the
`acid alone.
`In order to determine just which of these possibili(cid:173)
`ties was correct the cysteine-pyruvic acid complex was prepared
`according to the method of Schubert (2).
`Cysteine-Pyruvic Acid Complex-The crystalline compound
`melted at 149-151° uncorrected and gave a negative nitroprusside
`reaction with dilute ammonia, while the addition of aqueous
`sodium cyanide slowly produced a faint color, as did the addition
`of dilute sodium hydroxide. For quantitative work 17.2 mg. 1 of
`the compound were dissolved in 50 cc. of 0.1 N HCl, making a
`solution equivalent to 200 parts per million of cysteine. The
`Sullivan cysteine reaction on this solution was very faintly posi(cid:173)
`If the cystine reaction was used (2.0 cc. of 5 per cent NaCN
`tive.
`in N NaOH, 10 minutes contact) and the solution compared with a
`200 parts per million cysteine solution similarly treated, a color
`was obtained indicating a liberation of 44 per cent of the cysteine.
`This experiment was repeated but with an increase of the contact
`time of the sodium cyanide from 10 to 30 minutes. Complete
`splitting of the complex resulted and a colorimetric estimation of
`100 per cent of the cysteine. Substituting 2.0 cc. of N NaOH for
`the sodium cyanide and giving 30 minutes contact gave a return of
`77 per cent of the cysteine. Heating 5 cc. of the.pyruvic acid(cid:173)
`cysteine complex (equivalent to 1 mg. of cysteine) at 60° for 10
`minutes with 2 cc. of 5 per cent NaCN in N NaOH completely
`liberated the cysteine and gave 100 per cent matching of 5 cc of
`cysteine (1 mg.) similarly heated, both being cooled to room
`temperature before
`the colorimetric determination. Heating
`with 2 cc. of 4 N NaOH similarly gave 100 per cent return of the
`cysteine by the Sullivan cysteine procedure when matched against
`a cysteine standard similarly treated with both standard and
`pyruvic acid complex run without any sodium cyanide.
`
`1 Later work showed the presence of 8.15 per cent potassium chloride,
`so the actual amount of ash-free material weighed out was 15.8 mg . See
`"Addendum."
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`

`

`M. X. Sullivan and W. C. Hess
`
`15
`
`The Okuda method without reduction gave a negative reaction
`with the cysteine-pyruvic acid product. Application of the
`Okuda method after reduction with zinc and HCl or simply
`boiling for 15 minutes with 2 per cent HCI .gave 100 per cent
`return of the cysteine.
`The complex can thus be split either by relatively long contact
`with NaCN and NaOH or by heating with weak acid or relatively
`strong alkali. By use of 5 per cent NaCN in alkali, cystine, if
`present, would be estimated and such estimation for the present
`purpose was undesirable. Accordingly, a more satisfactory
`procedure for splitting the complex into cysteine and pyruvic acid
`was used; namely, boiling the material with dilute hydrochloric
`acid, 2 per cent, and estimating the cysteine by the Sullivan
`cysteine procedure without cyanide. The procedure used is as
`follows: 25.9 mg. of cysteine-pyruvic acid complex were dissolved
`in 75 cc. of distilled water. To 10 cc. was added 0.5 cc. of concen(cid:173)
`t rated HCl and the mixture boiled 10 minutes on the hot-plate,
`cooled, and neutralized by adding 5 N Na.OH dropwise with
`stirring to pH 3.5 and made to 10 cc. with water. 5 cc. were used
`for colorimetric work as follows: To 5 cc. add 1 cc. of a 1 per cent
`aqueous solution of 1,2-naphthoquinone-4-sodium sulfonate,
`shake for 10 seconds, and add 5 cc. of 10 per cent N&2SO3 in 0.5 N
`NaOH, mix, and wait 30 minutes. Then add 1 cc. of 2 per cent
`N&2f¾O, in 0.5 N NaOH. Match against a cysteine standard
`(1 mg. in 5 cc. of 0.1 N HCl) similarly treated. By this procedure
`cysteine only is estimated. A cystine solution containing 1 mg.
`in 5 cc. of 0.1 N HCl was negative in this procedure. The recovery
`of cysteine was 95.4 per cent of the theoretical.
`Complete Combining of Pyruvic Acid and Cysteine-As previ(cid:173)
`ously mentioned, cysteine and pyruvic acid, mixed mole for mole
`and kept for 24 hours at pH 1, 2, and 3, did not become negative
`in the Sullivan reaction for cysteine. Accordingly the effect of
`higher pH and higher amounts of pyruvic acid was tried. Thus
`20 mg. of cysteine and 15 mg. of pyruvic acid were dissolved in 2.5
`cc. of phosphate buff er at pH 6. After 3 hours contact at room
`temperature, 28- 30°, and suitable dilution with 0.1 N HCI for
`colorimetric work, 59 per cent of the cysteine was found by the
`Sullivan cysteine method and the same finding obtained after 24
`hours contact of the reactants, Solution A. When the pyruvic
`
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`16
`
`Pyruvic Acid, Cystine, and Cysteine
`
`acid, however, was increased 10-fold (150 mg.), that is approxi(cid:173)
`mately 10 moles of pyruvic acid to 1 of cysteine, practically no
`cysteine reactive in the Sullivan procedure was left after 1 hour,
`Solution B.
`Complete Dissociation- In both cases the cysteine can be readily
`and completely liberated. Thus 0.3 cc. of each solution was
`diluted to 10.3 cc. with water and 0.5 cc. of 35 per cent HCI was
`added and the solutions were boiled for 10 minutes, cooled, neu(cid:173)
`tralized carefully, and diluted to 15 cc. with 0.1 N HCI. When 5
`cc. of diluted Solution A were matched colorimctrically against a
`fresh cysteine standard (1 mg. of cysteine) without use of sodium
`cyanide, 95 per cent of the theoretical cysteine was found.
`In the case of Solution B similarly treated, 96 per cent of the
`throretical cysteine was found colorimetrically when matched
`against a fresh cysteine standard. On the other hand, when the
`eysteine standard (1 mg. in 5 cc. of buffer at pH 6) was set side by
`side with the pyruvic acid-cysteine mixture, 101 per cent of the
`theoretical cysteinc was recovered.
`For complete combination of cysteinc and pyruvic a<·iJ, till'
`pyruvic acid must be in considerable cxc<·ss. Tiu~ combining
`reaction is favored by low acidity.
`Thiazolidiuecarboxylic acid is not dissociated by lioili ng with
`dilute acid but can be dissociated by other means.
`Preliminary studies of a part day sample of urine tested by the
`Sullivan and Okuda cysteinc methods showed no rysteine in the
`urine as voided and little if any of a material comparable to the
`cysteine-pyruvic acid complex. With the cysteine-pyruvic acid
`compound added to the urine and put through a short heating
`period with 2 per cent HCI, 94 per cent of the added cysteine in
`the complex was found by the Sullivan cysteine procedure.
`
`SUMMARY
`Pyruvic acid added to cystine has little if any effect on the
`colorimetric estimation of cystine either in dilute or in relatively
`concentrated solutions and with long contact.
`In dilute solutions of cysteine, molar quantities of pyruvic acid
`have little effect at pH 1, 2, and 3 on cysteine estimation.
`In concentrated solutions the amount of reactive cysteine
`decreases more rapidly the higher the pH and the greater the
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`

`

`M. X. Sullivan and W. C. Hess
`
`17
`
`pyruvic acid content, owing to the formation more or less of a
`non-reactive complex of cysteine and pyruvic acid. At pH 6
`the reaction is very rapid.
`Unlike the complex formed between cysteine and formaldehyde
`the pyruvic acid compound is readily dissociated into its compo(cid:173)
`nents by contact with alkali or acids.
`The most satisfactory procedure for dissociating the cysteine(cid:173)
`pyruvic acid compound, with practically quantitative yields of
`cysteine, is by boiling for 10 minutes with 2 per cent HCl.
`
`Addendum-Prompted by correspondence with Dr. Schubert, we made
`an analysis of the cysteine-pyruvic acid complex which by virtue of its
`and S content seemed to be an addition product. However, t he com(cid:173)
`plex contained 8.15 per cent potassium chloride. Correction for the mois(cid:173)
`ture and ash indicates that the complex is a thiazolidine derivative, tts
`finally concluded by Schubert. The cysteine found colorimctriral!y is
`then the cysteine delivered by a substance of the formula
`CH3
`I
`CH,8 ,
`I
`'c
`CHNH/ I
`I
`( 'OOH
`('OOH
`
`BIBLIOGRAPHY
`1. Schubert, M. P., J . Biul. Chem., 111, 671 (1935).
`2. Schubert, M. P ., J. Biul. Chem., 114, 3-H (1936).
`3. Ratner, S., an<l Clarke, A. T., J . Am. Che1n. Soc., 69, 200 (1937) .
`4. Sullivan, M. X., and Hess, W. C., J. Biol. Chem., 120,537 (1937).
`5. Hess, W. C., and Sullivan, M. X ., J. Biol. Chem., 121, 323 (1937).
`6. Sullivan, M. X., Pub. Health Rep., U.S . P . H. S ., suppl. 78 (1929) .
`7. Okuda, Y., J. Biochem., Japan, ~. 217 (1925).
`8. Folin, 0., and Marenzi , A. D., J. Biol. Chem., 83, 103 (1929).
`9. Sullivan, M. X ., P'Ub. Health Rep., U.S. P . H. S., 44, 1-121 (1929).
`
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`

`THE EFFECT OF PYRUVIC ACID ON
`THE ESTIMATION OF CYSTINE AND
`CYSTEINE
`M. X. Sullivan and W. C. Hess
`
`1937, 122:11-17.J. Biol. Chem.
`
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