The: J,mrnal of Bio :·ll€•1ni~try, Vol. YIII, No.:?.
`
`STUDIES ON THE SOLUBILITY OF CYSTINE UNDER
`VARIOUS CONDITIONS, AND ON A NEW
`METHOD OF CYSTlNE PREPARATION .
`
`B,
`
`LEN OKABE.
`
`(F,·om the Biochem-ical Laborntory, Kitasato I nsti t ute, Tol.:yo.
`L llboi·atory chief: D1'. r. 1.'ernuc hi.)
`
`(Recei\•ed for publication, December 17, Hl:27.)
`
`L
`
`l~TRODUCTIO~.
`
`I t is a well known fact that the preparation of cystine, one
`of the biologically importa.nt amino acids, from liy<lrolysate of
`h air for the purpose of various bioch emical studies is not easy.
`Its yield is Yery scanty in m::u1 y ca~es and it is Yery difficult
`to eliminate the defield tyrosin comp letely from the cystine
`obtained. In order to find the method, v,hich can increase the
`yield of c·ystine from protein-hytlrolysate, I feel the n ecessity of
`kuo11,·ing the solubility of cystine under Yarious conditions. F or
`this purpose it is necessary to know the quantib.tiYe estimation
`of the whole amo unt of cy~tine in the liq uitl a nd the cystine
`r emaining in tlie media after crystallisation. It has been r egret(cid:173)
`ted, howeYer, that there ,Yas not auy method of easy and accurate
`quantitatiYe estimation. R ecently, Okuda r eported on the iodine
`metho 1 of the quantitative determination of cyst.inc-, and I adnptccl
`I sh a ll briefly describe h ere Ok nda's
`h is method for m y studies.
`iodine m f:thod :
`After t.he hydrolysate of proteins by hydrochloric acid is decolorizecl by the
`addit,ion of animal charcoal, and a small amount of zinc powder is put into
`the liquid, the cystine is reduced t o cysteine as follo,rs:
`CH2·s- s·CH2
`\ CH~ ·s a )
`I
`I
`1
`~H ·NH2 ? H 'NII , + ~H = '.! ·l~H·~H~ 1'·
`COOH
`COOH
`COOH
`Cysteine
`Cystine
`
`4.41
`
`Eton Ex. 1061
`1 of 17
`
`

`

`442
`
`L. Okabe:
`
`Caust,ic soda solution is added to make the acidity of the liquid to 2 .%' hydro(cid:173)
`chloric acid solution. Into a certain amount of that liquid (20.0 cc.) are added
`5.0 cc. of a 5 J6 potassium iodide solution and also 5.0 cc. of a 4.0 JG hydro(cid:173)
`chloric aci9, solution. ,Vhen a potassium iodate solution having a certain fi xed
`value (1/300 mol) is dropp~d into the liquid, iodine will be freed according to
`the following equation:
`5KI + KI0,1 + 6HCI::: 3H 20+6I + 6KCI
`The freed iodine immediately acts upon the cysteine in the solution at room
`t,emperatnre, and cystine and HI are produced according to the following
`reaction:
`
`CH2·SH}
`CH2·s-s·CH2
`I
`I
`I
`\
`2lcH·NH2 + 21 = CHNH2 cH·NH2 + 2Hr
`I
`I
`I
`COOR
`COOH COOR
`iodine
`Cysteine
`Cysteine
`When all the cysteine bas been oxidised completely, the freed iodine from
`more than a drop of potassium iodate solution gives a yellow colour to the
`liquid. If this yellow colour should be left unchanged for one minute the final
`reaction is attained; and the amount of cystine is calculated from the amount
`of consumed iodate solution.
`Experimentally, if 4.65 cc. of a 1/300 mol potassium iodate solution is used
`0.01 gm. of cysteine (0.01 gm. cysteine = 0.0l0l gm. cystine) will be completely
`oxidised:
`
`total amount of hydrolvsnts
`consumed cc. of KI0 3 x
`20·0
`100
`- - -- - - - - - - - -- -- - -- - -- X
`.
`f
`4.65
`amount o protem
`employed in gm.
`= Cystine contents in % in the protein.
`It has been generally known that for the separation of
`I adopted
`cystine, a certain H-ion concentration was necessary.
`the H-ion concentration test by the use of the indicator method
`and know that the pH optimum for the separation was pH=4.S.
`This fact has favoured the preparation of cystine to my satis(cid:173)
`faction . According to the results of my long series of experi(cid:173)
`ments, to make clear the necessary conditions for the separation
`of cystine, I have learnt that the kind of electrolyte which had
`been dissolved in the medium interfered to a considerable extent
`in the separation of cystine. Especially NaCl and Na2SO, aug,
`mentecl the solubility of cystine, while NH4Cl or CH3 COO(NH4)
`had little effect.
`In the routine method of the preparation of
`
`Eton Ex. 1061
`2 of 17
`
`

`

`Studies on the solubility of cystine.
`
`443
`
`cystine, NaOH or Na 2CO~ had been em ployed for the lowering
`of the II-ion concentration of the hydrochloric ncicl b ydrolysate
`I n this case, however, the NaCl, which had been
`of hair.
`produced by the neutralization process, woula augment the solu(cid:173)
`bility of cystine, and interf~re with its crystallization. For the
`lowering of tLe acidity of the their hydrolyf:ate, therefore am(cid:173)
`I also studied the influence
`monium water shou1J be employed.
`I have devised an
`of ethyl-alcohol on the solubility of cysline.
`easy and fruitful method for the preparation of cystine, as
`compared with the hitherto known methods.
`
`II.
`
`I ~FJ,UESCE OF THE H-I0:-1 CO~CESTRATIO~ OF THE
`MEDIA OX THE SOLUBILITY OF CYSTI~E.
`The crystallization optimum or solubility minimum of the
`hardly soluble electrolytes which having acid n.nd alkaline reac(cid:173)
`tion, amino acids etc., conforms with the isoelcctric poiuts, which
`fact h as been pointed out by Mich ae lis and Davi<lsohn.
`Recently Sano carried out an inYestigation on the solubility of
`leucin, tyrosin and cystine, which arc horJ1y soluble amino
`acids, at various H-ion concentrations and supported the theory
`of ~lichaelis and othen. H aving been assured of the fact that
`the solubility of cystine oscillated according to the concentration
`nud kinds of salts dissolve:1 in thfl me:lia (as it is sta.teu in
`Section III), I testecl the solubility of cystine at various H-ion
`concentration s of m e;clia, the salt concentration of which had
`been put at a certain fixed value. From the results, I have
`learnt the fact that the solving curves of cystine at the certain
`fixed concentrations of salts obtained by employing dh·ergent
`H-ion concentration were almost indifferent to the kinds and
`concentration of salts; and they w~re all similar. Tlie amoun~
`of residue cystine dissolved in the medium after the scparn,tion
`of cystine oscill:J.tecl only according to the variations of the H(cid:173)
`ion concentration of tbe medium.
`
`Eton Ex. 1061
`3 of 17
`
`

`

`444
`
`L. -Okaibe :
`
`A. Solubility of cyst,ine a{ the varioiis H-ion concen(cid:173)
`trations (in a 0.046 mol NaCl m.edia.)
`A serie3 of te3t tubes are filled each with s·.o cc. of a 1.0 %
`cystine hydrochloric acid solution (1.0 % HCl) and then A- few
`drops of indicator added. They were then warmed in the water
`bath (which process had be~n applied to prevent immediate
`crystallizution).
`Into the liquid is .then dropped enough of a
`0.1 mol caustic soda solution to attain the lowering of the· H-ion
`concentration to the desired H-ion concentration. Then distilled ·
`water and a 0.5 mol NaCl solution are added in adequate pro(cid:173)
`portions to make-the NaCl concentration of each test tube to be
`0.0-!6 mol, and the total amount of each test tube to be 30.0 cc.
`by adding of 0.046 mol NaCl solution. The tubes are then
`cooled for one hour in an ice and NaCl mixture (at 0°C), and
`the cysline is crystallized. Tho content8 of the tubes are then
`filtered through a small piece of filter paper and then a certain
`amount of the filtrate (vi7.. 10.0 cc.) is withdrawn into a test tube
`and the amount of cystine is estimated according to the method
`of Okuda, as described above.
`
`TABLE I.
`
`Solnbilit.y of cystine at the Yarious H-ion concentrations
`(in a 0.046 mol NaCl solution).
`
`No. of test-tube
`
`pH
`
`Residue cystine J~
`in the filtrate
`
`-
`
`1
`
`8
`7
`6
`2
`4
`5
`3
`- - - - - -
`- - -- --
`-
`8.9
`6.7
`7.8
`5.6
`3.6
`4.6
`2.6
`1.6
`-
`- - -
`- - - - --
`-
`- - - -
`-
`0.168 0.070 0.062 0.049 0.059 0.045 0.064 0.195
`
`9
`- -
`9.1
`- -
`0.427
`
`Eton Ex. 1061
`4 of 17
`
`

`

`Studies on tho solubility of cystine.
`
`445
`
`Fig. 1.
`Solubilit-y-cm·,·e of cyst.iue at the various H-ion concentrations
`(in a 0.0-16 mol NaCl solution).
`
`·- ~,.,
`~o--'\~ - - - -- - -- --
`:§ I
`~ r-::;:_ - -+--\c-
`~
`
`"O ,-:, ~?l
`
`'-::;~---;;:::---
`pH
`2 .0
`
`-;:'-::-----:-:---
`.3.0
`--1-.U
`
`--,:-'-,------
`~. 0
`Q.V
`
`-
`
`-,,
`
`B . Solubility of cy.sti/lc at d-i1.·crgcnt plI in a 0.0.~13 m0l
`a1m11onitPn chloride solution.
`
`The follo,rin g Table II shows the results of the test carrieu
`out after the same rnetho::l
`:=ts in Experiment A . the only
`difference being that a 0.04G mol of a11Jmonium chloriuc solution
`is employed as the medium insteat1 of the same mol solution of
`NaCl at the tiine of crystallisation.
`
`TABLE II.
`
`Solubility of cyst,ine at the nnions H-ion conce11tration
`(in a 0.046 mol );If.Cl solution).
`
`5
`
`6
`
`7 I 8
`
`~o. of test-tube I 1 I 2 I :J I 4
`I 1.6 1 ~-6 1 3.6
`R~~<lt~e c:f::~:c J~ 1~1~r~~1~ ~1~1~
`
`p H
`
`4.8
`
`5.6
`
`6.8
`
`7.6 j 8.9
`
`Eton Ex. 1061
`5 of 17
`
`

`

`446
`
`. L. Okabe:
`
`Fig. 2.
`Solubilit.y-curvc of cystine at the various H-ion conce:1tration
`{in a 0.046.mol NH.Cl solution).
`
`~ '.:2
`coi-----r-- --+----+---+---~ - - .
`
`pH
`
`zo
`
`J .0
`
`4.0
`
`5.0
`
`6.0
`
`7.0
`
`9.0·
`
`III.
`
`l~FLUE::SCE OF THE VARIOUS KINDS OF SALTS DISSOLVED
`
`rn TilE )lEDIA AGAL,s·r TilE SOLUBILITY OF CYSTINE.
`
`I haYe dealt so far with the relation of the Hsion concen(cid:173)
`tration of the medium and the- solubility of cystine. On the
`influence of rnlts upon the solubility of cyst.inc or other amino(cid:173)
`acids, however, there is no literature.
`f have been convinced
`that in the preparation of cystine, there occurred oscillation
`regarding the yield of cystine by employing caustic soda or
`ammonium, which is employed for the lowering of the H -ion
`concentration of the HCI hydrolysate of hair. By my investiga(cid:173)
`tion the fact was confirmed that NaCl and NH,Cl were the
`factors causing these oscillations. Therefore, I carriel out ex(cid:173)
`periments with a number of salts i n special reference to their
`influence on the solubility of cy3tine at divergcut concentrations
`of salts.
`I prepared media of diYergent salt concentrations in which
`a certain amount of pure cystine was dissolved and the pH was
`fixed at 4.S and cooled at 0°0. for one hour, during which period
`the contents were every now and then stirred an::l thus the
`cystine was made to crystallize. The results are shown in the
`following rrable III, from which it will be seen that the higher
`
`Eton Ex. 1061
`6 of 17
`
`

`

`Studies on the solubility of cystine.
`
`447
`
`the concentration of NaCl was the higher the solubility of cystine,
`i. e. the separation is interrupte:::l. by the presence of NaCl. (see
`Table III).
`Then I employed NH~Cl and NHiCiH~o~ instead of NaCl at
`diYergent concentrations with the pH=4.S after t.he sn.me method
`as in the former test with NaCl. The results were that with
`all the dirnrgent concentrations of these salts, the amount of
`the dissol"rnd cystine iu those filtrates was practically the same,
`or in other words, the presence of ammonium <.:hlori<le or am(cid:173)
`monium acetate bQd no affect on the soluuility d cystine (See
`T able l V and Y).
`I11 the llext I tested the influence of ammonium sulphate
`and ~ollium sulpliate upon the solubility of cystine. The re3u}ts
`were , ns a1·e sh own in the Table.<; VI and V II, th:1t the higher
`the concentration of these s::tlts wa<s, the higher was the solubility.
`It m ay be. coududec1 that both ammonium sulphate and sodium
`s ulphate interrupted the separation. of cystine (T a bles VI nnd
`VII).
`Fig. 3. shows the results of the t.ests gi':en in Tables III(cid:173)
`YII, in one chart, from 1,hic1l the influences of various ;-inlts
`contained in the medium upon the solubility of cystine will be
`conYeniently seen for comparison's sake.
`
`T.\.BLE Ill.
`
`Influence of NaCl on the Solul>ility of Cystiue.
`
`Xo . of test-tube I
`NaCl concentra- I 0 .05
`J~ in the filtrate I 0.050
`
`tiou in mol
`
`Dissolved cystine
`
`l
`
`I
`
`'.!
`
`0.,5,5
`
`0.052
`
`\
`
`3
`
`1.05
`
`0.C61
`
`I
`
`4
`
`~.05
`
`I 0.070
`
`Eton Ex. 1061
`7 of 17
`
`

`

`·443
`
`L. Okabe :
`
`TABLE IV.
`Influence of NH.Cl against the Solnbility of Cystine.
`
`No. of test-tube
`
`1
`
`NH.Cl concen•
`tration in mol
`
`0.05
`
`2
`
`0.55
`
`3
`
`1.05
`
`4
`
`2.05
`
`Dissolved cystine
`% in the filtrate
`
`0.047
`
`0 050
`
`0.051
`
`0.049
`
`TABLE V .
`Influence of CH3 C00(!\H,) on the Solubility of Cystine.
`
`No. of test-tube
`
`C.l:i 3 C00<NH 4 1
`concentration
`in mol
`Dissolved c:,stine
`~~ in the filtrate I
`
`1
`
`0.1
`
`2
`
`0.6
`
`0 <>
`
`1.1
`
`-
`
`4
`
`2.1
`
`0.041
`
`I
`
`0.046
`
`O.OH
`
`I 0.045
`
`TABLE VI.
`Influence of (NH.),S0~ on the Solubility of Cystine.
`
`I
`'
`I
`
`No. of test-tube
`
`(NH.,)?SO. concen-
`t.ration in m ol
`
`Dissolved cystine
`% in the filtrate
`
`1
`
`0.05
`
`0.047
`
`I
`I
`I
`
`2
`
`3
`
`4
`
`I
`
`I
`
`0.0~ ~ 2.05
`
`O.OGO
`
`-
`
`0.098
`
`TABLE VII.
`Influence of Na,RO. against the Solubility of Cystine.
`
`No. of test-tube
`
`1
`
`~a"so. concen•
`tration in mol
`
`Dissolved cystine
`% in the tiltrate
`
`0.05
`
`0.050
`
`2
`
`0.05
`
`3
`
`1.05
`
`4
`
`2.05
`
`0.060
`
`0.082
`
`0.125
`
`Eton Ex. 1061
`8 of 17
`
`

`

`Studies on the solubility of cystine.
`
`449
`
`Fig. 3.
`Solub ility-curves of cystine at the various concentrati'ons of
`di,·ergent salts (at pH 4.8 and 0°C 1 hour).
`
`,'
`• Na!SO+
`
`I
`
`/
`
`#> <NHilzSO+,
`
`;
`
`, , ,
`..,
`
`/
`
`,
`,
`,
`,
`,
`.,,..
`.,,..
`,
`
`~· -JO
`., /
`,.J,,
`_, .. V ___.....-
`r:---- p -
`
`.. NaCl
`~
`~
`---- (NH1)CI -
`
`(NH1)C2tb02
`
`---
`
`c
`
`I
`
`~.o
`J.o
`2.0
`1.,5
`05
`Salts-concentrnt ion in mol
`From tho abo,·e tests, H will be seen that though the H-ion
`~oncentration of the media should be kept at a certain value
`there occur.s an oscillation in the solubility of cystine due to
`the influence of the two kinds and the concentrations of the salts
`dissolved. From the re.sult, of the experiments described in tho
`previous articles, it can be stabd that NaCl augmented the
`solubility of cystine specially in its concentration, but ammonium
`chloride or ammonium acetate h ad no nffect. Ammonium sul(cid:173)
`phate and sodium sulphate, mark~dly augmented the rnlution
`I n other words, it may be said that Na- and /3O1-
`•of cystine.
`ions augmented the solubility of cystine especially when highly
`concentrated and interrupted the cystine separation, while NIL(cid:173)
`Cl-and acet-ions had no affect on the solubility of cystinc, even
`.at a considerable high concentration.
`
`Eton Ex. 1061
`9 of 17
`
`

`

`450
`
`L. Okabe :
`
`IV.
`
`JNFLUE:-(CE OF· ETHYL ALCOHOL UPON THE
`
`SOLUBILITY OF CYSTINE.
`
`I experimented to see h ow ethyl alcohol in the medium
`iufluences the solubility of cystine. The results Rre shown in
`the following Table VIII. It will be seen from the table, that
`the higher the concentration of the alcohol of the meJimn was,
`the lower was the w l ubility of cystine. Besides, it was noticed
`that by adding alcohol to the medium, the separation of cystinc
`,Yas aecelerated.
`
`TABLE VJII.
`Influence o[ ethyl-alcohol upon the solubility of cystine.
`
`No, of
`test
`tube
`
`l
`2
`:)
`4
`
`Cont.rot
`
`1 ~/dl
`cvstine Abso-
`solut,ion
`lnt,e
`(in
`:t alcohol
`0.:! mol
`cc.
`HCl)
`cc.
`-
`ij .0
`"
`"
`"
`.,
`
`I
`
`I 0
`
`-
`
`10.0
`5.0
`'.l .. ')
`1.25
`
`~
`
`Aqua 0.2 mol
`distil· NH~
`s:,lution
`lat:1
`cc.
`cc.
`
`pH
`
`NH.Cl
`concen- Alcohol
`tration
`0 /
`, u
`mol
`
`Residue
`cystine
`%
`
`0
`!i.0
`7.ii
`8.i:5
`
`5.0
`,.
`"
`"
`
`l --- - - -
`
`10.0
`
`-
`-
`0 .05 c.t.,'iO
`2,i
`"
`] '.l,fi
`"
`,.
`6.'.l->
`
`"
`
`0
`
`0.034
`0.0'.!5
`0.032
`0.038
`
`0.047
`
`4.8
`"
`.,
`"
`-
`"
`
`"
`:Fig. 4.
`Ct:n-e showing the influence of ethyl alcohJ t upon the
`solubilit.y of the cystine.
`
`l
`1~
`
`'
`
`!
`
`I
`I I
`
`10
`
`!
`
`\
`
`.'.:0
`40
`.'>O
`30
`Alcohol content in %
`
`Eton Ex. 1061
`10 of 17
`
`

`

`Studies on the solubility of cystine.
`
`45 l
`
`V.
`
`INFLUENCE OF TEMPERATURE UPO~ THE SOLUBILITY OF
`
`CYSTINE, AND THE TIME IN CONNECT ION WITH
`
`T H E SEPARATIO~ OE CYSTINE.
`
`As it is stated in Table IX, in the medium of 0.05 mol of
`NaCl and pH=4.8 cystine was made to crystallize at divergent
`temperatures and then I estimated amount of the residue cystine
`in the filtrate. The results were that the higher tbe temperature
`was the higher was the solubility, although not very remarkably.
`I n the n ext place, I tried to see what ·would be the con(cid:173)
`n ection between the duration of time and the amount of t he
`yield of cystine at 0°0. and pH=4.S. The results are shown
`in the following Table No. X . The time required for complete
`separation, as it will be seen from the table, oscillated according
`to the kinds and concentration of salts dissolved in the medium.
`In the medium containing ammonium chloride or sodium chloride
`(0.05 mol), the separation was complete in one hour at 0°0.
`''' hen the table salt contents were higher, say 2.55 mo], the
`crystallization took place more slowly while the amount of the
`residue cystine in the filtrate was more than in the former
`medium.
`
`TABLE IX.
`
`Influence of temperatnre upon the solubility of cystine.
`
`0.5 g/dl
`0.2 mol
`cystine solu-
`NaOH
`No. of
`tion (in a
`test tube 0.2mol HCl) solution
`cc.
`cc.
`
`NaCl
`concen-
`tration
`mo!
`
`pH
`
`Tempera- Residue
`ture
`cyst ine
`(DC.)
`0.'
`/ 0
`
`1
`2
`3
`4
`5
`
`5.0
`
`"
`,,
`
`"
`''
`
`5.0
`,,
`,,
`,,
`
`"
`
`0.05
`
`4.8
`
`"
`"
`,,
`,,
`
`"
`"
`"
`"
`
`0"0
`
`5"
`10,,
`15 ,,
`20 ,,
`
`0.050
`0.05~
`0.054
`0.059
`0.066
`
`Eton Ex. 1061
`11 of 17
`
`

`

`4-52
`
`L. Okabe:
`
`TABLE X.
`Influence of duration of time upon cystine separation.
`
`Residue cystine contents C %)
`tin various concentn1tion of various salts)
`
`NaCl concentration
`
`NH. Cl concentration
`
`0.05 mol
`
`l
`
`~-5 mol
`
`0.05mol
`
`1 hour
`1 ~ hours
`!!-t
`"
`::; days
`7
`
`"
`
`0.05
`0.05
`0.0-H
`0.048
`0,049
`
`0.07,5
`0.071
`0.069
`0.068
`0.068
`
`0.048
`0.049
`0.047
`0.047
`0.048
`
`I
`
`2.5 mol
`
`0.05
`0.048
`0.049
`0.047
`0.04i
`
`No. of
`test tube
`
`Duration of
`time allotred
`far
`sepftration
`(0°0. and
`pH=4.8>
`
`1
`
`,)
`
`?
`
`,,
`-1
`!j
`
`VT.
`
`STUDIES O~ THE SEPARATION OF CYS1'1NE FR0~1 THE
`HYDROCHLORIC ACID HYDROLYSA.TE OF HAIR.
`
`So L.ir I haye experimented on the separation of' cystine in
`In this section,
`a pure cystine hydrochloric 3cid solution.
`I will deal with the various conditions which influence the
`separation of cysti ne from the hydrochloric acid hydrolysate
`of hair.
`The horse hair was hydrolysed by 20 % h ydrochloric acid.
`Decolorization was effected by the use of animal charcoal. Then
`it was distilleu in yacuum, and the hydrochloric acid was then
`removed as completely as possible. Then the obtained material
`i\·as dissolved in distilled water and an ammonium solution
`was adde<l.
`The amount of the ammonium chloride in the medium at
`the time of th e cystine separation was 1.5 mol, while the I-I(cid:173)
`concentration was varied, and the separation was allmved to take
`place at 0°0. After complete separat.ion took place, the residue
`cystine contents of the medium in the tubes were estimated.
`The solubility-curve was analogous to the ones shown ab:)Ve,
`the only difference being that the solubility of the cystine in
`each tube was f:.ir m ore than that in the experiments made using
`
`Eton Ex. 1061
`12 of 17
`
`

`

`Studies on t~ie solubility of cystine.
`
`453
`
`a pure cystine solution .
`This fact induced me to consider that in tho hair h ydro(cid:173)
`lysate, there are certain sub.stance which remarkably interfered
`with tho separation of cystinc.
`I n our next experiment, I employed ammonium and caustic
`sodn for the neutralization of the hair hydrolysate. The am(cid:173)
`monium chloride and the NaCl contents were both 1.5 m oJ.
`The separation was nllowcd to take place nt pH =-L'3 mid (1°C.
`After complete separation the Ii.quid was filtered a~1d the r esidue
`cystinc contents of the filtrate wore c::-timatcrl. T n the ammonium
`<.:hl oride solution, the residue cy:,tinc conteuts were 1.2G ,~;;, but
`in the table salt solution they "·ere l.lH JC. It WftS that 'NaCl
`did more to interrupt the separation of cyst.inc thau ammouium
`chloride.
`By the use of alcohol in the proportion of :Jo .9$, the rc.sid uc
`cystiue contents were 1. 05 Ji, agai11st the control 1. ~6 36.
`As for the connection between the duration of time ai1cl the
`complete separation of cy~tinc it ·was fou1 1d t.hat in the prc.;,c11co
`of a 1.5 mol, NaCl the re.3idue cystine contents of the m edium
`was 1.61 Jt after one hour and 1.35 J;S after 2-! hours. On 1he
`contrar y in a 1.5 mol NHiCl solutio11, the separation of cystine
`~rns independent of the d uration of time, because even after an
`elapse of 24 h oun, the furth er dccrc:tsc of the rcc,idue cystiuo
`contents was only 0.0~ J,C.
`
`VII.
`
`0:'-< ~IY DJPROVED :'IH~THOD OF C'YSTI~E- PREPARA1'10'.'s .
`
`'1'/i c cln>J poi11t8 of my i.mprorcrl mctliorl.
`A.
`(1) A 20 % sollltioP, of hyrlrochloric aci,l ·was cmJ.ilO!Jrcl
`for the hydrolyznlinn of hnir. A higher concentrntio1t thn.1 1 20.~(i
`would be unnecessary, because as in tbP- routi1te method of the
`use of filming h ydrochloric acid for hydrolyzat.ion , t.lw JICl
`concentra tion of the mctlium would soon become about ~rn Jl$.
`Eveu tho boiling slirmld be ma<le ~rith the fla',\k providel1 with
`tho reflux condenser.
`
`Eton Ex. 1061
`13 of 17
`
`

`

`454
`
`L. Okabe :
`
`'l.'hc l1ydrolysatc was distilled i.n a vacuum in order to
`(2)
`eliminnte as much HCl as possible and a syrup-like fluid was
`obtained. By doing so the alkalies employed for tho neutrali(cid:173)
`zation ·were reduced to a Yery small amount in order to eliminatn
`Urn i,iterrupting action of the salts produced, which would
`n ecessarily arise from the abundance of the medium.
`(3) The 'use of aninionium water for the lowe1·ing of ilw
`H-ion co11cc11tration of th e hyclrolysatc of hair. This wtts be(cid:173)
`cause by the routine use of either caustic soda or Nn2COJ there
`would naturally be produced table salt by tho process of neutra(cid:173)
`lization, which if in a considerably high co11ce11trat:011 would
`i nterrupt tho separation of cystine.
`In this cas(•, therefore, it
`woul<l be 1,ccessary to n<ld a copious amount of water to an-
`11ihilate the iufluencc of NaCl ·which would on the other hanu
`c:1.use the loss of a large amount of cystiJJc, ,d1ich would remain
`i11 tho medium as residue cystinc.
`If, however, ammonium
`water should be employed for the neutralization, there would
`be product•ll ammonium chloride which would :Jct Jess against
`the iocpn.ration of cystinc auu the consequently where is a tolerably
`larger amount of yield. Agaiu, if on the other hand in the
`mAnipulatiou of the n eutraliz,1tion by the use of caustic soda
`solution, the medium should be made a1kaline by error, there is
`more or less danger of destroying cystine. Moreover an isomeric
`substa nce woulJ. be produceL1, which is hardly crystalizable.
`Ammonium would not incur such an um\'elcome effect. Lastly,
`ctntmOn 'm.1n chloricfo iuoHlcl a.-u.gment the solitbil,i,ty of tyros,i.n
`a nd ,i11.rn1·c pure cystimi . This wn,s the reason for employing
`am111011ium water for the u cutralization.
`(--!) By the aclclition of (f,lcohol in the proportion of 30 %
`I not 011ly coulLl accelerate a cystine separation but lower the
`cystino solnbility, which brings a higher yield of cystine.
`']'he pl[ of the mediiirn -was ma,de 4.8 by the ,use o-f
`(5)
`the indic.ato1· ln·omcresol-green (0.04 % B.C.G. Ml-i1,tion). Up
`to pH 6 .0 there was nothing remarkably different in the sepa(cid:173)
`ration of cystine, and yet it would be nearing the optimal
`
`Eton Ex. 1061
`14 of 17
`
`

`

`Studies on the solubility of cystine.
`
`455
`
`crystallization point of tyrosin, n.ncl it would be apprehended
`that tyro.sin might be crystallizaecl together with cystine, and
`therefore to avoid this inconvenience, I made the reaction of the
`mctlium pH 4.S n.t the time of the separation of cystinc.
`
`B. The proccd11re.
`\Vashed and dried hair of t.he horse or hnman being can
`also be employed. But if wool should be employed, especial care
`should be taken to remove the fat completely. lnb 100 gm. of
`hair are added 300-500 cc. of a 20 Jo HCl solution. The mixture
`is boiled in a round basis flask provided with the reflux con(cid:173)
`denser over a flame covered with asbestos mesh work for 7-10
`hours. 10 - 20 gm. of animal charcoal is then aJdecl and the
`contents are every now and then stirred and still further heated
`for about one half hour to completely decolorize it. Then they
`are cooled down and filtered through the folded filter paper. The
`filtrate is then distilled in vacuum in order to remove as much
`HCl as possible.
`,vhC'n the liquid is almost like syr11p, it is
`then dissolved in about 100 cc. of n·atcr and then about 50 cc.
`of a 90 % alcohol arc n.dded. To the mixture is then added a
`10 % ammonium. water solution to lower the H-ion concentration.
`Then the hitherto brown colored fluid of the hair hydrolysate
`becomes bluish. vVhcn this sudden change of colour is noticed,
`the adding of tlie mnmonium water c;1,n be suspcudecl and roughly
`speaking, tho re:,,ction of the liquid is then almost near the
`Jesircd value of pH = 4.S. The accurate pH is, however, always
`made by the colorimetric test by aL1ding a drop of a 0.002 .%
`solution of the indicator " B. C. G. " in a small amount of that
`liquid (1.0 cc.).
`The rcguhl,tion of the reaction is accomplished by the use
`of either ammonium water or acetic acid to make the pH -value
`of the medium just 4.8. Then the hydrolysate is coole,1 in the
`ice and table s:-1.lt mixture at 0°0. for ~ hour3, during ,vhich
`time the contents are stirred every now and then to crystallize
`the cystine in the solution.
`(If the crystallization shoulJ be
`
`Eton Ex. 1061
`15 of 17
`
`

`

`456
`
`L. Okabe:
`
`flllowed to go on for 2-! hours, the yield will be somewhat aug(cid:173)
`m ented). Then the filtration is carried out _by a sucking ap(cid:173)
`pnratu~. The material obtained is the curded cystine. The
`curded cysti11c t.hus obtained is then dissolved in.as little amount
`of a 10 /0({ mnmollium water as possible and then the solution
`If the liquid
`is m;;1.dc 2 times as much with distilled water.
`does not clccolorize completely 1 about 2 gin. of animal charcoal
`is a<ltlcd and stirred to effect dccolorization. It is then :filtered
`fllld by adding enough of 36 .%· acetic acid to lower the H-ion
`concentration, the solution thus obtained does not contain pig(cid:173)
`~uents as found in the initial hydrolysate of hair, and conse(cid:173)
`quently 1hc desired pH Yalne can be obtained by testing with
`a piece of paper soaked with 0.04 J-G " B. C. G. solution".
`The regulation of the pH value is made after the same
`m an11er as in the preparation of the curded cystine. Then
`cooling and crystallization is allo'wed to take p lace. Cystine thus
`obtained is a pure preparation. It is then filtratell with the
`sucking apparntus :1ud "·::tshecl with distilled water and dried.
`The total amount of cystine yield was 6.4 grams. The cystinc
`contained in horse hair as it was estimated after Okud a's method
`was 11.07 %. This result obtained by my improved method
`proveJ that 60 JG of the cyst.inc contained in the horse hair had
`been recoYcrcd. This was the maximum yield obtained from
`h orse hydrolysate. By further distillat.ion of the filtrates, more
`cystinc can be obtflille,1.
`
`VIII.
`
`co~CLUSIO)l".
`
`I 11 meLlia in which is dissohed salts of certain fixed
`1.
`concentration the solubility of c-ystine Yaries according to the
`H-ion concentration of the mcJia. And the solubility m iuimum
`of cyst.inc conforms with the isoclcctric points as supported by
`Mi chael i s a11cl others.
`2. The solubility of cyst,iHc varies according to the con(cid:173)
`ccutratioi:s and kinds of snlts dissolved iu the media. NaCl,
`(NH~\SOi nn<l Nr2SO. nugme11t the solubility of cystine, while
`
`Eton Ex. 1061
`16 of 17
`
`

`

`Studies on the solubility of cystine.
`
`457
`
`NH4Cl and (NH.)C2H302 h aYe no affect.
`3. By the addition of ethyl-alcohol the cystinc-solu bility
`is decreased.
`4. Under 20°C, the degree of temperature has little effect
`upon the cystine-solubility.
`5. The solubility of cystinc in the hair-hyJrolysr1,te is quit,3
`different from that of a pure cystine-solution.
`
`REFERENCES .
`
`. Michaelis, L. (1911): Bioch. Z. 30, 1-43.
`Okuda, Y. (1925): J. of Bioch. 5, 2li.
`Sano, K. (19~6):
`:Cioch. Z., 16B, 1-l.
`
`Eton Ex. 1061
`17 of 17
`
`