{Jnrll'l".,ll'flf){)r/ ./1!1/r/1(/.1 uf r l'll·rilllli)j ,..;,ril'un· 111/d J ui/11111 /nd!lslr.JJ. l'nlll/111' -f . . \'u/11/lf'r :! . . l,u·/1. I!J:l.-,_
Studies on t he O rigin of Sulphur In Wo o l.
II. A Cuprous Mercaptide Method for t he Determination of Cystine or Cyst e ine.
Jl_,
~ D. HI~~~() l'\\ .. B.S1· .. 1\·,,ol lii'Sl';l!l l1 ( IIJi,·c>1. ;t IHI
T . . J. \\'ILhf.:.\ .. )(JIUH::'i. J)_:-;, ... 1·\liilil'll\ Ht·~~·;~n·l, tlfii,.,., .. l•:111pin· .\I ar];pj iII,!-!' lloa rd. ( lndt·r.'IPI"'"I'l.
11.1\T\'t. t-·~taldi . .;[tp([ tl1e ,-alnP of 1l11• :-;ulli,·;\11 IIH·IhrHI, a.; IIHHiific>tliJY tlw author~ (I!J:l.J-l. as a SJlPI·ific a111l lJli;IIJtilalin .. n•;1dio11 for th~
dPtenninatioll of ,.,·,teiiiP or ,.,·sti11e. it J,pcanw JIPI·t·~.;an· to .;tucl, it.; po~~ihle al'l,ji,.;Jtlo11 to t!J;. <tllal~·,.;i.; of ;111im;1l ;tlld. I'P,!-!'!'tahfe lltateJ·i;d~. 'l'llP fir~t .;tPp i11 .;nl'll all ;\llal.~·lit·al Jll·ocpdJII'(' 11·onld oln·iou . .;[_,- i11dudt-· ~ome .pron•,.;_.; . .;twh a.; a1·irl h\dl·ol,.;i.; ~~-IH•rpl,_,. tht•
l'y.;tiJI(• or 1'_\'~t·eilll' of the• r<l\1 lllatt•1iHl lotall.y lihel·;tl<'ll and l[llalltl- l;ti in•]_,. hJ·otl) .. dli i11to ~olutirllt. l11 .;u('h jll'!ll'P.;~e~ t l.1t' iii!Pnsity of al'ill (l·p;tilllellt lllll.;l he :'llftir·iPIII \ll liher;tit• nll i!Jp n · . .;(iiiP 01 1'\'S(<'IflP from it.; protPilt 01 lJl'lliPill-likP t·omplP.'\. but uot .. .;u illll'tl>:>~ a~ io ,[e.;ho_,·lhe , . .~-~Iiiii' lln1.; lilwr;~IPll. It i~ nut thP l'lll'l''"'' of thp 111'!'~1'111 paper lo r·oll;-;i,lPt· lhi~ phas<· of tilt• p1old,·tn, .;illce in,·pstig;~liolt,; itt lhi.; di1·l'l'!io11 ;~n• .;(ill ullllE'l' 11·a\·. th1 llw l'OIII1;1!'\'. i1 ha.; hPt>ll l'OII- si,]PJ·pd ;tdYi_.;;Jhle to n.;.;ume. fo1·· tiJ,. tinH-' hein~·. t.l1al (}H· \J,·tlroh;-;i.;
llf the 1;\\1' IJI;tiPri;~l (';111 he ,.j'fl•l·lt•d lJll;lllti(;tti~·l'l\·. \\'JiHl ,\J;1.; J;t->ell ai111ed ;I( in the pn•.;pn( 11·ork i~ to ti11d a lllethod \1-hPrPhy tht• <·.1.-<tine or l:,pdt•inp :H·IJI<ill_,. pn• . .;ent in ;-;ul'h h_,-d,·ol\.;;lle' may lw dt·lenllined
~ u l'Ce~s f ull,1·.
l11 a ft-•11· 1';\.'f-' . .; of hig·h l'\· . .;1illl' ('llldt>lli. e.g·. 11'0111 ;IIIII _.;()Jill-' jllll'e Jl1'111Pin~ rivli in n ·.;tin<-', thP ,-olour of the hYdroln;:lle t·atl.'e~ n•n·
li1ile iniE'J'fPrt>nr-e,· allll the ~ulliY;\11 lel'linirp;P a~, iniJll'O\'l'll hy tl{t>
;tuthor.; (JWl.J-) C';tn be ;ipplied ,linTth n11d "·i1h ~<lli.;fal'ior\ re~nlt.'.
\\Tith lo11· 1'\'ctliiP <·otiiPnl- ;tnd a dark-,·olourP<l ln·droln;aiE'. llw Jli'OblE'IIl lwr·r;mes rathn inYo[,·ed. It i.' rJln·iou.'h· ,juite ,ilnpo.;.;ihlt>
lo npph· rlirP<·tl.l' ;I l'olour rPadion to ;t h~·,lrol,·,;;t(p ohlainetl from gras,; o1· SOJII<-' auiJII;Il ti . .;~ue l<lll' in , . .~-~tint-•. a~ an\·nHe 11·ho lw.' h;illlllerl
·• '1'!1 is artir·lc• wn' origina 11~· pn hlisllPri 1n t l1r· J?j,,./, r-mir-ol J ou.n;nl, Y ol. 20.
p. '?Hl !10.'30).
STL'JJII•:S OS THE OI{]GL\ 01' Sl'I:I'Hl·l~ 1::\ \\ OOT,.
such ,;ol uti011 ~ ,,·ill rPadil.\· a p pre cia tP. 'l'o oYercom P thi:s clifficul h· it hac; l1een sugge::;ted by J-lenrici (Hl:)2) that the cystinE' should fir~t be Jll't'Cipitatecl hom :such a solution. Even ;~ftpr m.Lu..:h manipulation it is still difficult ho"·pyer (o ohiai n a soluti.ou sufti.l'iently light lll colour for l'Olorimetric nwthoclt; to lJp appliecl '~'tth :tll;Y uegree of :sati;;factiou. Certain 11·orkers (S11llinw, 192~), a1l(l Sulliva11 mHl Hess, ] !J:iO), haYe :mggt',.;i eel the u:-;e of a dive and animal charco a I ancl other cle('oloring agenh. ~one of the::;e substn nc-es ho"·eyer appears suitable, siucP the cystine in solution i:s eiHwr achorbecl along
"·ith the colouring matter, or it is c;Jtab·ticalb· clPcompo,.;ed ;wcl LleRtroyecl (Bnur ancl \Yunllerly, 19:13). ()thf'r \Yorkers (Lugg, 19:{:}) haYe triecl to in hihit c·olou r (leYelopment cluring hydrol_,·:-;i.s by a deli ng- ,;nc·h reclucing ag-ent,; ;~:-; stannous or titaJlO\Is chlm·ille "·ith equall.1
llll~Ul'CP:-;sful l'E'S1Jlts.
Un the othN hand it has ht'en shmul b1· \'i('kery .and \Yhitt>
(19:12), among~t others, that cy:-;tt'ine, in CJuantities of ~bout 20 mg., can he pl'el·ip[tnte<l quuntii<JtiYely hom solution as the insoluble
c~·steine cuprous mel'capticlf'. Yickery aurl \Vbitf' used cuprous oxide, all(l <letenni nNl the c_1·sti.nr iH the solution by· determining the sulphur coutent of the cuprou~ mercaptide pr<-'cipitate. Hopkiw;
(HJ29) ]JrPC'ipitaie<l glutathione in .a "imilar wa.1· and founcl the c-OllJ- lJOUll c1 to he (\ 0Il, GN" (\,R(~u. l>relimiwuy expe1·i nw nts on th <-' behaYiom· of this c·upro11s menoapti1le "oon lt>d to thf' c·onYiction that it embo(lied <.t reaction ,;uffi.ciently sen,;ibw io enable the separatio11 of qnantities of c·ystine or c;_l·steinf' lll11C'h smaller tlwn tbut JJOStub tecl by Y icker:v rt))(l \'{bit<-'.
'.l'he addition of cuprous oxide in the ,;olill form .Uj)]Jearecl some- '"hat eumbrrsome, ancl un;;uitecl for micro-"·ork. Since cnJn·ous ehloricle i:s onl_,. Yery slig-htly soluhlr iu water, initial attempt" were macle 11·ii.h a solution of cqrrow; chloride c1iswhed in potassium acetate ancl ~11·ehc acitl mi:dm·es. AltlJOugll ]li'OJni.sing results "·erf' sometimes nhtaill!ecl "·ith quantitieo; as low ns 1 mg. of c.1·stine reduce<1 to eyc;teine hy lll<-'allS of a few mg. of ziue; dust, the 1·esultc; we1·e some-
"·hat irregular. At this stage it ll'as l'et>olved to tlhul_~· the pl·ecipita- tion of e;yRtine by making use of thr "·ell known wluhilit,- of eUJJrous c-hloride in pota;;~ium chloride soluti.OJJR. A ve1·y intere,.,ting fact "·as
~oou clisconred. \\T orki o.g on 2 mg. (luan ti ties of c_1·st in e per ali.q uot Yolume, the preliminan· reclndinn to c~'steine >Yas omitted ln· merE' accicleut; ho'>YeYer, on aclrling the solution of c·u1n·ou,.; chlm~icle in potassium chlm·icle, th<-' cyst ein P c·uprou" me rca pti elf' " as o bservecl to form as 1·eadib- as if a~l the cystine had been ]Jres<-'nt as cysteine.
In all subsequent expenments the preliminary rt'clnction of cystin<·
to cystf'inf' >n1s the1·efore omittecl. The cu prou~ c hlori tles solution
\ras preparetl frmn 11:erck's Reagent C'Uj)l'OUR chloride. About 0·5 o·.
of the salt "·as shaken up with 1 per cent. HCl to remOYf' moRt of tl~tc·
cupric chloride present, the i usolu ble en prom; chloride allo11·ecl tv Rettlt>, and the supernatant liquirl poured off. This perfecth· whitt' r~sidue wu.!l _the11 dissuh-ecl irt a minimum quantit.1· of 25 ]J~r cent.
h.Cl contammg- 0 · 2 Jlel' cent. HC'l. 'fhe reag-e11t cdtoulcl prefer a hlY be prepared freshl~- befor<-' use, although, if left in a well-stoppt>1 e~l bottlE' it ma~~ he kt'pt for n fp"~ clays.
S. Jl. ROSSOu"· A)>D T. J. WIJ.KE~-JOltJJJ<;N.
On ~tudyi ug the preci lJitation of cy~tine ,,·it l1 thi,; reagent, it 1nt~ fouml that the <·y~tei.ne could iJP quanhtattYely pi·eclJHtatecl at room temperature at h.nlrogen ion cmJcentration,; hehYeen pH 3 · 0 all(l 5 ·5, although the rate of precipitation a1Jpeared to he gTeatest at pH 4·i). It \\as therefore rlecidecl to "\York at a hydrogen lOll ~.;on
centration of appnnimately pH 4·5. Aliquot portio~n.; of 2 lll~··
eystiue di~,;ohetl in Yer.\· <lilute HCl ,,·pre measm·ed mto oO mL centn- fuge-tulws, and a fp,,· chop,; of g-lacial aceti~ acid a<~rle<l to each.
'rhese solutions 11·ere then brought to pH 4 · o by aclc1mg cm·efully
10 JH'l" <"Plli. KOH ll:illJg bron_wc.resol grePn (B.J).H.) as an exte:·nal
in<licatm. To the hutferecl ,;olutiou cooled to room tPmperatnre ;J-10 drops of the <.;urpous chloride solutio11 wf're aclclecl "~ith <.;onstant Rtirring. A fine precipitate was ohsenerl to form almost Jlll~tJediatel:J>
an<l coao·ula ted o-ra<hlallv to a flocculent mass. After 40 nunutes tlus precipit~te ~~·as "'centrift;ged, and the ,.;upernatant liquid poured oft.
The precipitate \\"a;;; washP<l \l·ith alc·ohol, again centrifuged, and the alcoholic li.qui<l poured oft. After <lrai ning, the precipitate was rlissoh·ed in 5 ml. J-2 per ceni. HCl, a]l(l tmusfene<l to a 2iJ ml.
mearnuing cylinder of g-oo<l accurac~·, using- abon( L,) ml. of wash water. 2·5 ml. of a 0 per cent. <lcehe acicl ;;;olution 11·ere then adderl, ancl after mixing 1 ml. of 10 per eent. KCXS was a<lclecl in order to precipitate the cuprous eo]Jper. To remo1·e the Yarying amounts of
~;upric copper ah1·ays present at tl1is ,.;tage, n~e \Yas m.a<le of the 1·eactiou of Bi:liiZO (1926) by precipitating the eu1Jric <.;opper as the green s ul1 >ho~.;y:t n ide-copper-pyricli n P compl e:x. At this stage pyricl ine was then·fore nd<lerl 1111til J>H 4 · 5 wa.~ reachetl.
r
ncler these <·oncli- tions no meat>mable aclsorhtiou of cy.~tine hy the precipitate could he lletectecl. A ftpJ· mixing thorong-hl~- a nrl making 11 p to 2,) ml., the preci pita tes were filtered off. 'J1o ol"ercome the buffering action of thP acetic acirl in solution, 1 ml. of 10 per cent. KOH was added to a suitable alirp10t (usually 5 ml.) of the clem· filtrate, and the cystine determined by the moCI.ifiecl SulliYan method as clesr~riherl by the authors in :t prel"ious a rti de (Rossonw anrl J onlen, ] 9;i4).U,.;iug- this method, the effect of l"arying amounts or potassium chloritle on the precipitation of the eysteine l"UJTI"Ol\S mncaptirle was studied, sin<.;e with actual hvclrochloric aci(] liYclrohsatec; the amount of pota::;sium chloride in tl1e aliquot solutio;1 ml~st var:\· with the amount of c~·Rtine present in the material. 'l'hi~ <·OJlsideration is most important, since, as the <·urYe,.; u-·vroclu<·e,l below rearlily show, the euprous mereaptirle IYill partl~· or totally <1is~ohe in an excess of
potas~ium chloricl~, unle,;~ a <·OJTeSJ>On<lingl.Y b1·ge excesfi of l"llJ>l·ouo ebloncle reng-ent JS adrlecl. I-IoweYel·, con~i<lering the fact ihat tht- presence of potaA;;ium acetaie in rebtiYely large quaJttities has been fm1n<~ to rlepress the solubility of the mena}Jti <1e in ]Jotassium chlonde,_ the_ curves show that it is perfectl.1· practic-able to determine ihe cystme 111 ~nu·h solutions as a h_\·chochloric ariel ln·tlroh·sate of sheep's h~oocl. I1_1 clea~ing with gra~:-;e~ howeYer the position ~s some- what thfferent, smce 111 a hydrochlon c ar-id ll"nhol,-sa te a suitahl e aliquot containing a determinable amount of cvs.tine ~-ould ineYitabh contain sueh u large quantit~- of hyclmchlori~ acid that the amou~t of cuprom; ch lorirle reagent nece:;;sarY becomes uniYielrlv an<l impra('-
tica hle. · ·
455
100
1--' ' .a-- - - -
'80
' ' ' ' ' '9
'60
-.
l ml. rcagC'nt
40
1 00
t - - -o--80 - :
' ' \' \
' '
60 - :
'' 2 mi. reagent ' I
9 ~
40
1 00 80
~-'-- - - - --- - - - -
--a--- -
' '
60
4 ml. reagent40
100 80
_cr- - - - - - - -
~-- -
, , ,
60
'40
8ml. reagent (50 ml. solution)
1 t ~ 4 5 6 ~ 1 9 10 1,1
gms. K.Cl. in 40 c.c. solution.
To over('Ollle this difficulty wheu >YOl'kmg wi lh muteri al very low in eystiue, tbe hychochlorie acid
'''"1s
re]Jlaced by sulphuric acid. In testing this modification of the metborl 2 mg. of c:ystine were ad<le1l to fi ml. of 40 pe1· cent. H2SO,, 2·5 ml. g-lacial acetic acid "·ere added and the solution brought to pH 4·5 by neutralising with 10 per cent. KOH. After cooling to room temperature, :)-10 rlrops of the c11prous chloride solution wen• stirrecl in. Pl'ecipitation and flocl'nlahon were rapiil, and JOO per cent. recovPl'Y wa;; ohtainerl rPpeaterlly, eYen after lt•aYing the prel'ipitate to Rtnnrl for 24 hours.S. D. l10SS0u"· .1:'\D '1'. J. '\YJI.I\BJ\-J01U1EN.
This 1n·oeedure of first precipitating the c,ptine as the cysteine cuprous n1ereapticle soon pniYecl most succe:;sful in the treatment of hydroly:-;ntes, 8s1Jeeinll.1· those highly contamiJlate<l with excessive quantitie,; of dark colouring and othe1· matter. On bringing snch hYdrolysate~ to pH 4·5 by mPan;; of acE'h(' ac:irl and J10tasc;ium hydroxide, a largP miwunt of objPctionablP colouriug material wa,.;
j)l'ecipitated an<l r·.entrifuge<l oft. That the mnterialilnJS precipitated eould oednde or eontain no mea::;nraole amount of ey,.;t-ine emt he safely .aceepterl, .~i JH·e nPi t her could cystinP lw found i 11 such pre- cipitates no1· coul<l any lo"s be 1lemonshate<l on arlfling a kno\l·n :~mount of e:vsti11e l·o i liP hy<holysate. On atlllillg' 1 hP cuprou:-;
cb.lori<lP rPagc•itL io tht' !'enlrifuge<l an<l <lef'autP<l solution, a further quantit.v of d<nk-!'oloure<l maierial "·'1" ]Jrecipitate<l along witb thP
<·upi·ou:-; mt'rcaph<lP. On <li:-;sohing the mercapiicle in dilutP hy<ho- chloric ari<l, the areompanying clark material was nlso brought into solution, hut ,,·as n'moYe<l practicalb· eomplPtt'l_,. in tlw precipitahon of the eop1Jer "·iih sui phor·)·m,i<le and [!.Hi cline. The final solutions
"·ere eitllPl' lJPl'fel'ily colomle~~ or Pxhihiterl only the faintest shaw- Yellow v.·hich l1aR .JJO me<l~uralJle influt-nr.;e on the cn;tinP cleten11inn- iion. As an PxamplP of the applicability of the m~thorl, the follmv- Jng- mwlyti.r:al figures might. lw giYPll:-
)lPrino "·ool: 12·:) ppr <·Pnt.
1Jried shePp's hloo<l: 400 mg-. per 100 g.
Y ell<lll. 111 eali P~: 1 O.J. mg. pPr ] 00 g·.
LucPrne hay: RO mg-. JlPl' 100 g.
\Telcl g-r<tss: 20-BO mg. JWl' l()tl g.
T n the Sll!'!'e:-;~ful PXe<·utioll of thP nwthod, ~ome experience i,.;
undouhterll:v necessm-,v. If too lnrge an f'XCPSR of tl1P cuprous chloride rPagent lw a<l<lerl io ihe sulplturic aci<l hy<holysatPs, ;;;ome cuprous chloride mny he preci pita ted along with t h P m t'l·ca lJtide. Excessive quantities of i his cnprou,; chlol'icle nwkf' the r.liswlution of the mereap- tide in the clilute hydrochloric ncid mort' r.litlicult to !'Ontrol, and ma,'l' further I'f'Snli i u Home !'_ystine being I't'ta in P<l h~- the r.uprous <·hlori rle.
In Ruch cases the rliA'irulty may lw rt'mP<liP<l h~- aclrling- a fpw mg.
of zinc clust to the h.'·<hochloric acir.l ,;olntion alH1 thus coJJYel'ling the eupr·ous e hlori.rle into metallic coppt'r. In such r·ases ho\1-eYer the filtrate, after a<lding Fmlphocynni<it' .an<l pyridine, must he left to stand OYernight to allo\\· an.'· cyst Pine formed to be I'Poxi<lised tv cystinf'. An.v zinc sol uti on i,; practically <·ompletely removed by pyridine at pH 4·!>; thP residual haces thnt ma:-· remain in solution have 110 pffect on the determin:ttiOJI h~· 1lw SulliYan meihorl, .as also shown by Pntnt_,, (1000).
J!ETHOD.
'l'he method, em]Jlo:-·i ng :mlphuric aci<l, mny be summariRe<l as folJo\\·s:-
A su1tnhlP q~tantit_~- of rhiPcl mntt'rinl 7d11s S timt's it~ \\·eig-ht of 40 per ct'nt. TT)iO, hyrlrolysPll for 24 hours at 12.50 (oil-hath tem- l1Prahue).
J{v<hol,v;;;ate filtPrecl hot nncl mnrle np io Yolnnw.
407
STUDT"J•;S ON TI-m OIUGIN OF Sl"LPHlJR IN \\"OOL.
A suitable aliquot measurecl into a 50 ml. cenhi fuge-tube.
2·5 ml. g1aci.al acetic a<"icl artcled to this and brought to pH 4·5 with 10 per cent. KOH.
If precipitate is shown here, centrifuge and wash with acetic acid KOH. mixture of pH 4·5 (fimd ,-olume to he approximately 40 ml.).
Stir in a fe"' drops of cuprous chloride reagent.
LeaYe until flocculatim1 setR in (a few minutes to 1 hour).
Centrifug-e and "·ash with alcohol.
DiRsohe in 5 ml. ] per cent. HCl.
Transfer, with washings, to a 25 ml. measurmg cylinder.
Acld 2 · 5 m l. 5 per cent. acetic a ci.d, and nux.
Add 1 ml. 10 per cent. KCKS and mix.
Add pyridine to pH 4 · :) a nc1 make up to Yol ume.
:Filter.
Determine cy~tiue in suitable aliquot ( U · 4 mg-. cystine), by methorl of SulliY.an as mo(lifierl hy the authors, addir1g 1 ml. of ] 0 per cent. KOH to <·mtnterad h\tffering- effect of acetic aeid.
DrsccsswN oF )IECI'LI.KlS:\L
The exact nahtre of the cuprous mercaptide precipitate has not been determined. Indications (Preisler and Preisler, 1932) have been obtained that the mercaptide wbieh is either-
NH2 ~H2 NH2
I I I
CH- CH2- S- Cu, CH - CH2 - S - Cu or CH- CH2- S- Cu- Cl.
COOCu
I I
COOH
I
COOH
is apparently associatecl with either cupTous or en pnc chlonde, or both. \Vhatever the composition of the precipitate, it is generally accepted that all the c~·stine is present in the rerluced form as the copper salt of the merca]ltan cysteiuf'. It therefore appears that the cystine rer~cts with the cuprous chloride according' io the follo"·ing reaction:-
NH2 NH2
(l)
I I
CH- CH2- S -S- CH2- CH
+
I I
COOH COOH
{
NH2
I
---+- 2.
cf -
CH2-S - -Cu~
COOH j
+
2 CuCl2 .i:i. D. ROSSOU". AND T. J. \YILT~EN-.TORDE!'.
In the case of the preformed cysteine, the reartion would naturally be:--
(NH2
1
(2) 2
~ ck -
CH2 - SH+
I I j
LCOOH
r
~H2I I I
-+-2~ C~-CH2- S- Cu i +2HCl.
LCOOH
J
These reactions He em to proceed t1uan titaiiYely in feebly acid solutions (pH :l· 0-5 · 5) in the p1·eseuce of au excess of cuprous chloride. In <liHwlving the precipitates in <lilute hy<lrochloric acid, the initial ~tate of the c;p<tine should lw tht- redueed form. _Expen- mPntal <lata r:an lH' a<lduce<l howeYei· to ~ho11· that its conYersion into the oxidised form is Pxtremel.v- rapi<l in ~t\ch aci<l ~olutions. In tl1e normal cotuRe of analysi~ all the c.ysteine is quantitatiYPly conYeried into cysiin<> lr1· thP time the coppe1· i~ precipitated witb sulphocyanide and pyri<line, !:'Yell wl1eu open1tions are ,;pt-ecle<l up to tlw maximum limit \Yith the object of detcding an.1· m10xirlised c:n;tein<:'. It appeal~
that the tranRformation to cystine alreacl:Y occm·s rluring-the p1·ocess of
<lissolution in hydrochloric acid, since, eYen after adding small quantities of 11inc powder uo c.pteine could he cleteeted. Thif' beh:wiour of the mercaptide suggest.~ that it is precipitated as R
complex contaiui ng cupric c hl ori <le. In the presence of hydrochloric acid, anrl in the absence o£ excess CUJn·ous chloride, the reaction (1) is therefore reversed, the reaction being forced to completeness by the precipitation of the cuprous copver as sulphocy;anicle. The observation of Anrlre\1 s (J 933) on the cat.a.l)·tic oxidation of cysteine h.'· copper must probably he explaine1l in a similar wa:v. ·
In the case whe1·e a relatiYely large excess of ziuc dust i,; adclecl to the solution of the men-apti<le ill dilute hydrochloric acid, it waR fouml possible. by rapid >~·ork, to obtain practically all the <oystine in the reduced form. Since in the SulliYnn metho<l the heatment "·iih r:vanide 1·esults in the formation of onlY one molecule of cvsteine
£~·om one mol<>cule of <·;~·sti n e, and si nee, f~1· <lll prn cti cal pm:po.seR one part by \\·eight of cystine is theorehcall:1· t-quiYalellt to one part h.v weight of cysteine, the SulliYan methorl in realii;~· determines onl;~·
half of the cyRtine wlum preRent as r·y~<tiJlP. \Vhen present as c.ysteine therefor<>, a colom· intensit.Y <louhle that of tht- ~inn<lanl should he obtained. In actual experiment colmn intensitie::; rouJHl <lhout 192
"·ere obtainecl with the standard ai 100. On allo\\·ing the solution hom \\hich the eopper had been j)recipitntNl to stanrl fm· a fnrth<>r four hours, the inten~it.,· of the SulliYan r·olonr dropped to 150, while a_fter 20 hmns it had clr·oppecl further to 100, showing that h:v that tune all the rysteine ha<l heen conYerterl into cYstil!e. Since some extra r:ti ons "·it h chloroform according to Bi a11zo ,;till 1·eyealecl minute quantihes of copper in the soh1tion, the conYersion sPems to haYe he<>ll Pffeded catalytically .
. 'rhe rapi<l conYersion of the r·~·steine in the mercaptidt- into cy,;hue was furt!1e1· <lemonstraterl h;~· precipitating the cy~teine from a stanrlarrl cyRteme hyrlrochlori<le so]ntion "·ith cuvrous chloride, and compari11g thP colour intPnsih- of th<> th11s heate<l c.'·steine "·ith that
459
ST"CDIES OJ'\ THE OHIGI~ OF SULPHUR IN \YOOT •.
of the cysteine hychochlori<le stanclanl <lirectly treated by the SulliYan method. The evsteine from the mercauticle \Yas founcl to be onlv half that of the "f'ysteine treated directly~ ·
The fact howewr that in the hvdrochloric acid solution of the mercaptide the cystine can be quantit;tively reduced by means of zinc dust, aml the mo<lified Sullivan method applied to this cysteine solu- tion, is considered to be of o>ome 1wactical importance, since double the normal colour intensity is obtained. Along these lines it has been found ppssible to determine the cystine contents of grasses ancl other material extremely lmY iu cystine. These an<l other results will be reporterl in a following paper.
Su:wniARY.
1. A. method for the micro-determination of cystine in hiologieal or plant material has been described. 'rhe dried material is hydro- lysecl with 40 per cent. sulphuric aei<l aml the cystine precipitated by cuprous chloride as the cysteine cuprous mercaptide. 'fhe cystine content of thiR is <letermined by the morlifierl SulliYan method.
2. 'L'he interfering colouring matter is automatically eliminate<l.
3. The method is quick and comparatively easy.
RliJFF;RENCl~S.
ANDREWS, J. C. (1933). The oxidation of cystine m aeicl solution. .l.H.r'., Vol. 100; Sti. Prot., Vol. '27, p. 4.
BAUR, E., AND \YUNDERLY, K. Cl833). Dber die Hydrolyse Yon Aminosiiuren durch den vViisserig0n Auszug a us Knochenkohle. Biochern. Ztsch r., Vol. 262, p. 300.
BIAZZO, H. (1926). A.nn. Ohi.11L. A.JIJJl. 16, 2 (1926). From ElHhjem and Lindo"·, .J.B.O., Vol. 81, p. 437.
HENRICI, YI. (1932). Cystine and sulphur content of bushes and grasses in a Karroid area (Fauresmith). l8tl1 Brp. Dir. Vet. Ser. An. lr1d.,
r
ofS.A., p. 579.
HOPKINS, F. G. (1929). On glutathio.ne: A HeinYestigation. J.B.O., Vol. 84, p. 269.
LUGG, J. \V. H. (1933). Some sources of error in the estimation of cysteine and cystine in complex materials when acid hydrolysis is employed.
Biochern. J., Vol. 27, p. 1022.
PHF.ISLRR, P. W., AXD PHEISLEH, D. B. (1932). The mechanism of the production of thiol acids R-S-H and sulfonic acids H-SO,-H from dithio acids (R-S-S-R). J.B.C., Vol. 9.'5, p. JSl.
PRUNTY, F. T. G. (1933). 'rhe determination of cystine in biological material.
Biochem. J,. Yol. Z7, p. :l87.
ROSSOUW, S. D., AKD WILKEN-JORDEN, T. J. (1934). Studies on the Origin of the sulphur in wool. I. A study of the Sullin1n technique for cystine. Ond.er.stepoort J. Vet. Sc. ancl An. Incl., Vol. 2, No. 1, p. 361.
SULLIVAN, M. X. (1929). Studies on the biochemistry of sulphur. V. Tl1e cystine contrnt of l!haseolin. Snppl. No. 80 to U.S. P11bl. He!klth Rep
(1929). .
SULLIVAN, YL X., AXD HESS, W. C. (1930). Studies on the biochemistr~'
of sulphur. VII. The cystine content of purified proteins. Stippl. No. 86 to U.S. Publ. Health Rep. (]930).
VICKERY, H. B., AND WHITE, A. (1932). The use of cuprous mercaptide in the determination of cystine. J.B.O., Vol. 99, p. 701.
