A N A t - y ] r c A L B t o c H E M r s t R y 7 6 . 1 9 2 - : 1 3 ( 1 9 7 6 )

A Gas-L iqu id Chromatograph ic - lso tope D i lu t ionAna lys is o f Cys te ine , H is t id ine , and Tryp tophan

in Acid-Hydrolyzed Protein

PErpR Ferrpn

I)(purtntetr t o l Botur t t ' unt l Plut t t Put l to logt , Mi t l t igan Stute Univt ' rs i t1, ,Eu. ; t Lun.s ing. Michigun 18821

Rece i ved May 10 . I 976 : accep ted Ju l y l . 1976

A gas- l iquid chromatographic- isotope di lut ion method for quant i tat ivede te rm ina t i on o l c ys te i ne ' ( cys t i ne ) , h i s t i d i ne . and t r yp tophan and a nove lmethod of protein hydrolysis are descr ibed. Acid hydrolysis of protein. usingIysozyme as a model , is accompl ished in thc absence of oxygen and in thepresence of 1.2-ethanedi th io l fbr protect ion of t ryptophan. Fol lowing the disul f idereduct ion. cysteine is S-alky lated to protect i t dur ing hydrolysis. The resul tantt ryptophan, a lky lated cysteine. and hist id ine are converted to the methyl estersand then N-acety lated to form vol iLt i le der ivat ives sui table fbr chromatographyon a polar s i l icone column. Correct ion fbr losses is made by an isotope di lut ionassay. The 70-eV mass spectra of the der ivat ized amino acids are presented.Ul t ravio let spectra l data are sho* 'n for 5-carboxyethylcysteine.

There are studies of the determination of amino acids in proteins bygas- l iqu id chromatography (c f . l -3) but the determinat ion of cyste ine,histidine, and tryptophan from hydrolyzed proteins remains diff icult.Cyste ine and t ryptophan are unstable dur ing ac id hydrolys is , whi lehis t id ine is unstable dur ing gas- l iqu id chromatography. There is anextensive body of knowledge concerning amino acid derivatization. thechemical modification of proteins, and ion-exchange chromatographictechniques for amino acid analysis. This knowledge and new methods andtechniques have been ut i l ized to permi t the determinat ion of cyste ine,h is t id ine, and t ryptophan by gas- l iqu id chromatography (g lc) . Tryptophancan be protected dur ing ac id hydrolys is wi th th ioglycol ic (4) ,to luenesul fonic (5) , or mercaptoethanesul fonic ac id (6) , but such reagentsmust be removed by column chromatography pr ior to g lc . In th is s tudy, theuse of the volat i le protect ive reagent d i th ioethane ( 1,2-ethanedi th io l ) isdescr ibed, thus permi t t ing recovery of t ryptophan fo l lowing ac idhydrolys is and i ts determinat ion as N-acety l -O-methyl t ryptophan. Af ter.S-a lky lat ion of the prote in, cyste ine can be assayed in the hydrolysate as.Ay ' -acetyl-O-methyl-S-carboxyethylcysteine and histidine as ly'-acetyl-O-methylh is t id ine. Using these hydrolys is and der ivat izat ion procedures andan isotope dilution method to correct for losses, the method has beenshown to be applicable to lysozyme and chymotrypsin with 75 to 8Wc'

( o p ) r i g h t O 1 9 7 6 b f A c i r d c n r i c l ) r c \ s - t n c .

r \ l i r i u h t s L r l r c p r o c l r r c l r o n i n i r n t l o r n t r c r c r r c d

192

CHROMATOGRAPHY OF AMINO ACIDS 193

recovery of tryptophan and 93 to 98% recoveries of cysteine and histidine,

respectively. The procedure described here for tryptophan. cysteine, and

histidine requires approximately 5 hr prior to hydrolysis and 4 hr after

hyclrolysis, with subsequent glc at the rate of three per hour. Thus' this

procedure together wi th that prev iously des:r ibed ( l ) permi ts a complete

amino acid analysis on a fractional aliquot of the hydrolysate from 300 pr.g of

prote in.

MATERIALS AND METHODS

Methanol and acetyl chloride were purif ied as previously described

( l ) , except the methanol was not s tored over L inde 4A. s ince that causes a

residue to remain rrpon drying. Purif ication of Mall inckrodt AR grade

acet ic anhydr ide was by d is t i l la t ion through a 60-cm Vigreaux column,

and storage was in a ground glass-stoppered bottle at room temperature.

Several hours prior to use approximately 30 mg of anhydrous CaSO., and

30 mg of anhydrous K,CO, were added per mi l l i l i ter of the acet ic anhy-

dr ide. This combinat ion fac i l i ta tes the acylat ion of the indole n i t rogen in

tryptophan and neither disti l lation nor treartment with P,O., substituted for

the anhydrous CaSO.,-K2CO, t reatment . Possib ly , t races of acet ic ac id

react wi th K2CO:r to y ie ld potassium acetate and carbonic ac id. which '

upon dehydrat ion by the CaSO*, vo lat i l izes as CO2.Triethylamine was purif ied from a number of sources by resinifying

yel low impur i t ies wi th KoH pel le ts for 24 hr . fo l lowed by d is t i l la t ion f rom

the KOH pellets and storage in an aluminum foil-wrapped glass-stoppered

bot t le over KOH pel le ts .Acrylonitri le was redisti l led in a nitrogen atmosphere and stored at

5"C in an amber bot t le wi th 0.01% NH{CO:r as stabi l izer .

Ethanedithiol was purchased from Aldrich Chemical Co. and used with-

out further purif ication. Dithioerythritol was from Sigma Chemical Co.

Both of these compounds were analyzed for th io l wi th El lman's reagent (7)

and found to be at least 807c reduced, based on two -SH per mole'

Hen egg whi te lysozyme, 3x crysta l l ized. was obta ined f rom Sigma

Chemical Co. (St . Louis, Mo.) . The desiccated lysozyme was d issolved

in water to a concentration of I mg/ml. The concentration was then

determined using an 8, . ,n ' . , o f 26.5 at 280 nm (8) which indicated the

pur i ty tobe85Vo. The amounts of cyste ine. h is t id ine, and t ryptophan were

then calculated us ing the amino acid composi t ion and molecular weight

data of Canf ie ld (9) . Absorbance measurements were wi th a Cary 15

spectrophotometer calibrated with a standard alkaline potassiLlm chromate

solut ion (10) and found to be wi th in 2% of the standard at 280 nm.

The radioact ive I t {C]cyst ine (30 mCi/mmol) was obta ined f rom

Schwarz/Mann (Orangeburg. N.Y.) whi le the [ l {Clh is t id ine (324 mCi l

mmol) and tryptophan (-57 mCi/mmol) were obtained fiom Amersham/

194 PETER FELKER

Sear le (Ar l ington Heights, I l l . ) . The [1aC]cyst ine was shown to beradiochemically pure by tlc on sil ica gel G plates using propanol:aceticacid:water (4: l: l) as solvent, and its specific activity was confirmed byisotope dilution assay as described below. The specific activity of thetryptophan was determined by measuring the absorbance at 280 nm usinga molar absorptivity of 6070 at 280 nm in methanol and by counting theradioactivity of a small aliquot of the same solution. A specific activity of60.0 mCi/mmol was found in satisfactory agreement with that claimed.An attempt was made to estimate the specific activity of histidine in asimilar manner using a molar absorptivity of 63 l0 in water at pH 0 at211 nm. Ultraviolet-absorbing contaminants present in the [1aC]histidineprecluded such measurements. The specific activity of the histidine wasverif ied with the known histidine concentration in lysozyme.

Combined gc-ms was on an LKB-9000 mass spectrometer interfacedwi th a l -m x 3.0-mm i .d. g lass column packed wi th l% OV-17 on 100/120mesh Supelcoport. The helium flow rate was 25 ml/min, the ionizingenergy was 70 eV, and the molecular separator and the ion source were at240 and290"C, respectiveiy. Mass spectra were recorded with an on-linedata acquisit ion and processing program. The oven temperature wasinit ially 150'C and was programmed to 230"C at 5imin. The flash heatertemperature was about l0"C higher than the oven temperature.

Exact mass measurements were obtained with a Varian MAT CH5/DFinstrument using a direct probe, an ionization voltage of 70 eV, and per-fluorokerosene as a standard.

Gas-liquid chromatography was with a Hewlett-Packard 402 gaschromatograph using an annular effluent splitter. The splitter containsTeflon parts with an upper temperature l imit of 250'C, and this temperaturerestriction mandated a short column of 106 cm to separate S-carboxyethyl-cysteine, histidine, and tryptophan from the other amino acids. Apeculiar feature of the annular splitter was an approximate threefolddecrease in sensitivity after allowing for the split ratio. The decrease insensitivity was attributable to the inside diameter of the flame ionizationdetector jet used with the splitter. The glc column used was 106 cm long,2-mm i.d., with 5% SP240l as the l iquid phase on 100/120 SupelconAW-DMCS. The column had 0.25- in. o.d. , 3-mm i .d. p ieces fused on thedetector end to mate correctly with the splitter and a similar piece fusedon the injection side to prevent the injection needle from striking the glasstube wall. The column was packed to within I cm of the site of needlepenetration and had silanized glass wool on the injection port to preventbackflow of column packing to the injector when removing columns. Theglass wool and the top 2 mm of packing were replaced daily if many injec-t ions were made.

The collection of peaks from glc was accomplished with a pasteurpipet inserted into Teflon tubing connected by means of a Swagelock

C H R O M A T O G R A P H Y O F A M I N O A C I D S

TABI-E I

Sprc t r tc Acr rv r r res o r AvrNo Acros Uspo rN nre Isoropp Dr rur loN MrrHoo

l9,s

l - a b e l c d i r m i n o i r c i d i r c l t l e t l

l r ) p f o t e l nL-rbeled rnlno acrd used l i r r

g l c c a l i b r a t i o n

S p e c i f i c A c l i l i r ! N , l a s s

r c t i \ i t ! ( l ) a d d c d r d d e d ( , 1 ' )

{ t C i i p m o l ) ( c p m , l 0 p I ) " t p g , l 0 A l )

A c t r \ r t ! N 1 a s s

rdded added Rxt io d' ( n m l { ) / l ) r s t s l l i F I ) r l 1 . .

Spccif ic

r c t i \ i t ) r I I )( p C i , a m o l )

5 carboxyeth] ' l

c y s t e i n e

H i s t i d i n e

I r) 'ptophun

t0I1,1

57

I1 .59-s1.r0.0005,1..1(X)

0 . l :q ,0.01.r60 . l 0 l

0 . 6 I I0 . :540 t75

l : . I (x )

I t .15015.090

1.1 ' 19 . I1 . '7 11765..15 I l0

, , A r c i s t e i n e " q u j r ' , , l " n t r .

fer ru le to the ef f luent por t . These Pasteur p ipets were cut to an outs idediameter that f itted snugly into the Teflon tubing. Differences in backflowpressure caused d i f ferent f low rates and detector sensi t iv i t ies, and th isprecluded using glass wool in the upper end of the pipets and required thatthe ins ide d iameter of the p ipets be uni form. Af ter co l lect ion, theradioact ive amino acids were washed into 6-ml sc int i l la t ion v ia ls wi thI ml of d ioxane. F ive mi l l i l i ters of Bray 's solut ion were added and thetubes were counted. Dioxane was found to decrease the counting effi-c iency by 2.5% and to leave only background counts in a second 1-ml d iox-ane wash after eluting 3700 cpm in the first 1-ml of wash.

Evacuat ion of hydrolys is tubes was wi th a Welch Duo-Seal No. 1405two-stage vacuum pump connected wi th 0.5- in i .d . rubber tubing to a dryice-ethanol co ld t rap. At approximately 20 in . f rom the hydrolys is tube,a "T" was inser ted in to the vacuum tubing to a l low connect ion to aBendix- type GTC- 1000 thermocouple vacuum gauge. Between thevacuum gauge insertion and the hydrolysis tube. a heavy-duty Hoffmanstopcock c lamp was used to a l low changing hydrolys is tubes wi thoutevacuation of the entire apparatus. As there was a pressure gradient fromthe hydrolys is tube to the pump, the vacuum transducer had to be near thehydrolys is tube. Wi th th is apparatus, f ieezing a sample, evacuat ing to20 pm, thawing to 70- 100 pm, ref reezing. evacuat ing to less than 20 pm,and seal ing in a f lame requi res about l5 min. For best resul ts . the pumpwas allowed to run for a dav before use.

Ls ot o p e Di I r t t i o rt A s.r'n-r'

Amounts of amino ac ids in the hydrc l lysate were determined by is t r t t rpedi lu t ion us ing the equat ion descr ibed by Ri t tenberg and Foster i l l ) .

Y : € : ! - r ) x .C

r96 P E T E R F E I - K h R

where I is the mass of the unlabeled compound, X is the mass of radio-act ive compound added, Co is the speci f ic act iv i ty of the added radio-

chemical compound, and C is the speci f ic act iv i ty of the mixture iso lated.The in i t ia l and f ina l speci f ic act iv i t ies need not be known. only the rat io

Cr; /C. This rat io can be measured using a g lc wi th atn ef f luent sp l i t ter . The

compound emerging f rom the g lc was spl i t so that a constant f l 'act ion is

col lected. and a constant f l -act ion was burnt in the f lame ionizat ion de-

tector . The counts col lected d iv ided by the peak area are then proport ional

to the speci f ic act iv i ty . When the peak areas and col lected counts were

determined fbr both the in i t ia l and d i lu ted compounds. the rat io Cr. r /C

could be determined. Since the mass of the radiochemical added was

known. the amount present in the sample could be determined. The

accl l racy of th is method depended upon the l inear i ty of the spl i t ter and

upon a stable detector response. The " l inear i ty" of annular sp l i t ters such

as the H-P402 standard spl i t ter has been proven (12) . The major sourceof error res ides in a lowered detector response which could occur when

crude samples are analyzed. leading to erroneously h igh vei lues for the

speci f ic act iv i ty .The isotope d i lLr t ion method was most accurate i f a d i lu t ion of at least

l0- fo ld is obta ined ( l l ) . Consequent ly two radioact ive stocks were pre-

pared foreach amino acid, one of h igh speci f ic act iv i ty . used undi lu ted to

add radioact ive t racer to the prote ins, and another of suf f ic ient ly low

speci f ic act iv i ty to be measured by g lc . The amounts and speci f ic act iv i t iesof the amino acids used are g iven in Table L Since the speci f ic r tc t iv i tyof the amino acid added to the prote in and that of the standard used forg lc cal ibrat ion were d i f ferent . a modi f icat ion of the isotope d i lu t ion equar-t ion wzrs used. wi th the terms having thei r etbove-descr ibed n leanings andthe mul t ip l ier r / used to correct f t r r speci f ic act iv i t ies:

uCtt) - (

c l ) . \ - .

Thus. a is a constant fbr each set of amino ac id stocks and is the rat io ofthe speci f ic act iv i ty of amino ac id added to the prote in d iv ided by the

speci f ic act iv i ty of the amino acid used for g lc cal ibrat ion. Val r - res f i l t 'a areshown i n Tab le l .

Prepurutiort rl l S- [1tC ]Curbo.rtetlr-t ' ft r ' .r/cirtc

A 2 . -5 -m l a l i quo t o f a 30 pC i /m l so lu t i on o f cys t i ne i n 0 .1 N HCI wasdr ied under reduced pressure in a l3-ml conical centr i fuge tube. A solut iono f 2 m l o f 0 .1 N NH+HCO, ] . pH 7 .0 , was added and the tube was son -ica l ly agi tated for l0 min to d issolve the cyst ine. Cyst ine reduct ion wasaccompl ished by addi t ion of -50 p l of a 3.2 ' ,4 (v lv) i iqueous solut ion ofd i th ioerythr i to l . Af ie f I I min of sonic agi tat ion. -s0 p l of a 7.U/r (v /v) so lu-

CHROMATOCRAPHY OF' AMINO ACIDS

tion of acrylonitri le in water was added and agitation continued for I 5 min.

The mixture was shel l f rozen and lyophi l ized to dryness. The a lky lated

cyste ine was then washed wi th 3 x 2 ml of d iethy l ether to remove the

dithioerythritol, and residual NH4HCO3 was removed by heating the tube

to 80 'C in a sand bath. Af ter cool ing, the contents were d issolved in 0.3 ml

of 6 N HCI and transferred to a hydrolysis vial. The tube was washed with

an addi t ional 0.3 ml of 6 N HCI and th is was added to the v ia l . The tube

was evacuated to 15 g,m, as described. and held at 1 10"C for 4 hr tohydrolyze the nitri le to the corresponding carboxylic acid.

Following hydrolysis, the S-carboxyethylcysteine in 6 N HCI was

transferred to a conical centrifuge tube. The hydrolysis vial was washed

twice with 0.7 ml of glass-disti l led water and the washings were transferredto a conical centrifuge tube. The tube was dried in rocuo,2 ml of glass-

disti l led water were added, and the tube again was dried to remove residualHCl . In a pr ior exper iment , t lc on s i l ica gel G (30:13:6, l -butanol :acet icacid:water) showed contaminating radioactive and ninhydrin-positivespots. These contaminating materials were removed by the followingprocedure.

The S-carboxyethylcysteine was dissolved in 0.3 ml of water andappl ied. together wi th two 0.3-ml washes, to a 0.2-ml bed volume columnof Dowex l-acetate at a pH of approximately 5. The column was washedwi th 8.0 ml of g lass-d is t i l led water and the e luate was saved. A l i t t le of the.l-carboxyethylcysteine as well as all of the contaminating ninhydrin-posi t ive and radioact ive compounds appear in th is wash as judged by t lc .The S-carboxyethylcysteine was then eluted from the Dowex columnwith l0 ml of 1.0 N acet ic ac id and dr ied. A check of the column wash af ter

concentrat ion a lso showed the presence of S-carboxyethylcyste ine.Accordingly, the concentrated wash was run over an identical DowexI column. The I N acetic acid elutions were combined, dried, and taken upin 2.0 ml of 0.1 N HCl . Radiochemical pur i ty was checked by t lc as before.Scraping the adsorbent from the plate and counting showed 87Vo of theradioactivity at the Rr of S-carboxyethylcysteine. The remaining countstailed from that R1. The overall yield was 40Vc as judged by counting anal iquot of the radiochemical ly pure S-carboxyethylcyste ine. This low y ie ld

was probably due to the insolubi l i ty of cyst ine in NH'HCO,, at pH 7.0.

P R O C E D U R E

Twenty-five microliters of the protein stock solution were pipetted into0.2 ml of d is t i l led water in a 14-mm-diameter hydrolys is ampule. In thecase of lysozyme, th is a l iquot , as determined by uv absorbance, conta ined274 p.g of lysozyme with 18.4 pg of cystine. 2.95 pe of histidine, and23.4 p.g of tryptophan. A 2A/a trichloroacetic acid solution (0.2 ml) wasadded and the tubes were centrifuged for 30 min at 10009. The super-

t97

r98 P E T E R T . E L K E R

natant was removed. 2 ml of ether were added. and the contents of thetubes were mixed wi th a Vortex mixer . Fol lowing centr i fugat ion. thediethyl ether was removed, and residual water and ether were evaporatedat room temperature in a stream of nitrogen. Two mill i l i ters of diethylether were added to the dry protein, and the contents of the tubes weremixed and centrifuged as before. The ether was removed. the protein waswashed again with 2 ml of ether and dried in a stream of nitrogen toevaporate the ether. These washes removed the trichloroacetic acid fromthe p ro te in . Nex t . 0 .3 m l o f 0 .1 N NH*HCO, . pH 7 .0 , and 20 p l o f a3.2Va (v lv) aqueous solut ion of d i th ioerythr i to l were added. Ni t rogen waspassed through the tubes. They were stoppered wi th No. 14 serum capsand shaken on a reciprocating shaker. After 3-5 min, 20 prl of a 7% (vlv)solut ion of acry loni t r i le in water was added to the tubes, which wererecapped and shaken an addi t ional 25 min. The S-alky lat ion was stoppedby adding 2.0 ml of hexane to the suspension. mix ing br ief ly , andcentr i fug ing. The hexane supernatant so lut ion conta in ing the acry loni t r i lewas removed and the hexane wash was repeated. The NHTHCO,, buf ferand protein were taken to dryness at 60'C in a sand bath with a streamof n i t rogen. Di th ioerythr i to l causes in ter l -er ing peaks on g lc and wasremoved f }om the a lky lated prote in by 3 x 2-ml washes wi th d iethy lether .The tubes were p laced in a 80 'C sand bath to remove the remain ingNH1HCO,, . ResidLra l NH{HCO;r was detected e i ther by bubbl ing uponaddi t ion of the 6 N HCI or as needle- l ike crysta ls in the f ina l acy lat ion mixpr ior to g lc .

Next . the labeled amino acids were added to the prote in by f i rs t addinga smal l amount of water to the prote in (0. I ml) and then l0 p l o f each of thestandard labeled amino acids. This mixture was then dr ied at -50"C under astream of Nr . The labeled amino acids. i f d issolved in aqueous methanol ,cannot be p ipet ted d i rect ly in to the 6 N HCI because of vo lat i le impur i t ies(probably formaldehyde) which la ter condense wi th d i th ioethane andinter fere wi th acylat ion.

A 0.5-ml a l iquot of 6 N HCI and 2-5 pr . l o f d i th ioethane (stench) wereadded to the prote in. The outs ide of the tube was lubr icated and inser tedinto the rubber tLrb ing connected to the vacuum pump. The sample in thehydrolys is tube was next f l -ozen in adly ice-ethanol bath and evacuat ionwas begun. When the vacuum gi iuge read 20 pm. the v ia l was a l lowed tothaw until a pressure of 70 to 100 pm was reached. The tube was refrozenand af ter two successive f ieeze- thaw cycles the f iozen hydrolys is tube.at 20 pm pressure. was sealed wi th a f lame. Hydrolys is was accompl ishedin a temperature-regulated heat ing b lock (Lab-Line Inst ruments, MelrosePark . I l l . ) f o r 20 h r a t I l 0 'C .

At the end of the hydrolys is per iod. the tubes were a l lowed to cool .then centr i fuged fbr -5 min to cause the immiscib le d i th ioethane to forma globule in the bot tom of the tube. The fo l lowing procedure was con-ducted in a hood as the stench of even 20 r r l o f d i th ioethane is suf f ic ient

CHRONIATOGRAPHY OF AMTNO ACIDS

T A B L B ]

RtpRoouc ls tL l ry ( )F THE Iso top t D l r .u ' r rou METHoD, ,

t99

Peak areaMater ia l chromato- ( l )

graphed (mg of paper)

Radioactivi ty Ratro( l ) 2 l l

(cpm) (cpm/mg of paper) Mean + SD

Stock solut ionr '

D i l u t ed s tockso lu t i on I '

Di luted stockSo lu t i on I I '

57.66 3 . 4t)4. /

( 4 . /

-59.7

1 .2-53l.-s07l.-s091 . 3 4 11 . 5 7 7t . -s3 l

9tt.4160r 3 6 . 6l -s4 . lr 2 9 . 8t 4 2 . 6

4 | .60449.93ti-s4.883_s4.286_53.41 .1-s l . t29

-s.-57tt6 .4616.-s96.5 .8 I l6.0436.6U r

l . fJ-561.88-s2 . 3 7 71.6091 . t 9 32.43 I

860 + l 3 . l

.1._36 + .Q-59d

17.-s + 0.u

ri7:It67866839816tt90

'1..1-51 . 2 91 . 3 74. 331 . 8 3 '1 . 3 6

1 8 . 9r 8 . 017.11 6 . 9t 6 . 917.0

r i As only 3.6 t r rg was der ivat ized f rom the stock solut ionl ,compared to 100. , { t l t r rg for thedi luted stock solut ion I ' . recorder scales wi th l6- fo ld d i f ferences in sensi t iv i ty were used tctrecord the peaks. The peaks on the less sensi t ive scale rvere then mul t ip l ied by 16.

/ 'Solut ion as obtained l i rm the suppl ier wi th a reported spcci f ic act iv i ty of 30 rnCl i /mmol.' To 100 g.g of unlabeled cyst ine was added 0.297 pCi of the abovc stock solut ion. This

corresponds to 0.4t1 g.g, assuming the manulacturer 's speci f ic act iv i ty to be correct .'1 The mean and standard deviat ion were calculated u ' i thout using the anomalously low

va lue o f J . 83 .' ' l 'h is was a working standarcl known thrn many previous assays to h l tve u spcci f ic

ac t i v i t y o f 0 .60 mC i immo l .+ See lbotnote 11.

to cause compla ints f rom adjacent laborator ies. The top of the v ia l wasbroken and the 6 N HCl. avoid ing the d i th ioethane. was p ipet ted in toa l3-ml conical centr i f i rge tube wi th a ground g lass jo int . Glass-d is t i l ledwater (0.7 ml) was added to the hydrolys is v ia l , and the contents weremixed and again centr i fuged to sedintent the d i th ioethane phase. Thedist i l led water phase was t ransf-erred f l 'om the hydrolys is tube into theconict r l centr i fuge tube. An acld i t ional 0.7 ml of g lass-d is t i l lec l water wasadded to the conical centr i fuge tube, thus d i lu t ing i t about threefo ld. I t wasthen possib le to remove the tube f l -om the hooci and take the conicalcentr i fuge tLrbes to dryness on a BLrchler Evapo-Mix at 60"C. us ing a heatgun or lamp to hasten evaporat ion f ron-r the tops of the cctnnectors.

200 PETIIR F I ILKER

. Fol lowing dry ing. g. l -s ml of g lass-d is t i l led wate l was added to the

tubes. the contents were mixed and the water and a s l lbsequent wash were

transf 'er red to 2-ml conical react ion v ia ls (1) . The 0.-5 ml of d is t i l led water

ancl amino acids in the reaction vials were dried at 70"C with a stream

of n i t rogen in a sand bath. Next . 0 .4 ml of a l i 'eshly (dai ly) prepared solu-

t ion of acety l ch lor ide in methancl l was added. The tubes were capped

with a Teflon-faced culture tube cap, agitated sonically. and placed in a

70"C sand bath for 30 min. The tubes were cooled. then dried at 70'C

with a stream of nitrogen. and 80 pl of acetic anhydride and 20 pl of

t r ie thy lamine were added fbr acy lat ion. The t 'eact ion mixture was capped,

mixed, and p laced in a sand bath at 100"C for 30 min. Af ter cool ing ' the

reaction mixture was reduced to dryness with a stream of nitrogen and,

when almost dry. 30 tri. l of acetic anhydride were added. The reaction

mixture was capped wi th a septum, mixed. and the N-acety l -O-methyl

esters o l ' the amino acids were ready for g lc .

Modi f icat ions of the procedure for denaturat ion. S-a lky lat ion ' hy-

dro lys is . and g lc must be made wi th caut ion. For example, ' 'C- labeled

cyst ine cannot be added pr ior to d isu l f ide reduct ion and a lky lat ion owing

to the solubi l i ty of S-cyanoethylcyste ine in ether . Whi le the solLrb i l i ty was

only l -5 ng/ml . th is was suf f ic ient to y ie ld an apparent ly greater d i lu t ion

of the isotope. At tempts to remove d i th ioerythr i to l wi th an ether wash

af ter hydrolys is contaminated the ac id wi th peroxides. so that . upon

taking the hydrolysate to dryness. the t ryptophan was destroyed. Thus

i t was necessary to synthesize S- [1 'C]carboxyethylcyste ine and to add

i t j us t p r i o r t o hyd l ' o l ys i s .We also at tempted to modi fy the acylat ion condi t ions to avoid a brown

color formed dur ing acylat ion. This was dgne by heat ing the acylat ion

mixture for longer times at lower temperatl lres, but it was found that

when t ryptophan was fu l ly d iacety lated, the react ion mixture was yel low

in standards and darker brown for prote in hydrolysates. In f -act , when

prote in hydrolysates d id not become brown dur ing acylat ion for 30 min

at 100'C, i t ind icated that e i ther the K' ,CO, or CaSO., was not anhydrous.

Acry loni t r i le wi l l ar lso a lky late h is t id ine (13) wi th pro longed heat ing at

h igh pH. and thus the dry ing of the a lky lat ion mixture and a lky lated

protein in NH*HCO,, buffer must be done car.rt iously' We compared

lyophi l ized a lky lat ion mixtures wi th a dupl icate dr ied at 60"C wi th a st ream

of nitrogen. No differences were noted. The hexane wash following

S-alky lat ion was not essent ia l but i t y ie lded c leaner chromatograms.

RESULTS

Ac'curur'y' u nd Re protlu r: i hil i t l ' oJ' t l t e glt '- l s ot ttp a Dilut io n M e t hod

The precis ion of the method is shown by the data used to determine

the speci f ic act iv i ty of the 1 'C- labeled cyst ine (Table 2) . The man-

CHROMATOGRAPHY OF AMINO ACIDS

r o

Frc. l . Gas- l iquid chromatography of approximately 0 '7. 0 9. and l 3 t r rg ' respect lvely '

o f S - [ ' 'C ] ca rboxye thy l cys te i ne . h i s t i d i ne . and t r yp tophan . (A ) 1 .07 -m- l ong r 2 -mm- i ' d g l ass

col t rmn wi th -57 SP240l on 100/120 AW-DMCS was user l in combinat ion wi th an annular

spl i t ter . The f low rates for n i t rogen. hydrogen. and ai r were 60' 12' and -500 ml;min '

respect ively. chromatography was done isothermal ly at 200"c. wi th the f lash heater and

detector both at 210'c. The col lect ion of the peaks was done in . ' t ime windows" wi th the

ai t l ofa stopwatch. The bars under the peaks indicate the peak areas col lected. ' fhe

retent ion

t imes in minutes are grven abovc the peaks. (B) Gas- l iquid chromatogram of an i r l iquot of

the der iv i t izet l lysozyme hyclro lysate. Approximately 36 ptg tota l of amino aclds were

in - i ee t e t l . .A I I o t he r c t r nd i t i r t n : we l c l l \ i n I A ) .

ufacturer's reported specific activity of the stock solution was 30 p"Cil

prmoi. After reiluction. S-alkylation, and 6 N HCI hydrolysis, as would

be tlone for an unknown. the resultant S-carboxyethylcysteine was

chromatographed, using the effluent splitter. the peak was collected and

counted. and the amount of S-carboxyethylcyste ine (as peak area) was

201

202 PETER FELKER

determined. As seen in Table 2, the speci f ic act iv i ty , in these arb i t raryunits, was 860 -t- 23.3 counts/min per mill igram of charl recorder paper.Next , 0 .297 prCi of th is s tock. corresponding to 0.48 g,g, assuming themanufacturer's reported specific activity to be correct, was diluted with100 pg of unlabeled cystine. Derivatization and chromatography of thediluted stock yielded a speciflc activity, in arbitrary units, of 4.36 -r 0.059cpm/mg of chart recorder paper (Table 2). The isotope dilution equationcan be used to solve forX. the amount of labeled amino acid added.

: 0.-s09 rrg.(860/4.36) - r

and this corresponds to the value of 0.48 pg given by the manufacturer.Next, another sample of [1aC]cystine, previously calibrated and known

to have a specific activity of 0.60 pCil p.mol, was derivatized, chromato-graphed as above, and found to have an arbitrary specific activity of 17.5cpm/mg of chart recorder paper (Table 2). The specific activity of thestock [1aC]cyst ine solut ion can be calculated as

(860/17.5) x 0.60 :29.5 p"Ci lp .mol ,

which again accords with the manufacturer's reported specific activityof 30 pCilpmol. It should be noted that replicates for the values of Table 2zrgree within 5% of the mean.

C ltronttttogt'uphy, o.f Amino A<'id Stundords

The glc recorder tracing for a standard mixture of S-carboxyethyl-cysteine, histidine, and tryptophan and for the corresponding acids froma hydrolysate of lysozyme are presented in F ig. L In a standard mixture,S-carboxyethylcysteine, histidine, and tryptophan are separated and yieldwell-formed peaks with a slight trail ing edge. Since the chromatographywas isothermal , S-carboxyethylcyste ine y ie lded a sharp peak c lose to thesolvent front, while the tryptophan peak was broader. If the monoacylatedderivative of tryptophan was present, it would occur between histidineand t r yp tophan bu t c l ose r t o h i s t i d i ne . I f t he reac t i on m ix tu re con ta in ingmonoacylated tryptophan was held at room temperature. that peak woulddimin ish in area, whi le the d iacy lated peak would increase.

The glc tracing of a lysozyme hydrolysate showed that most of theamino acids, other than the three being studied by th is procedure, occurredin the solvent f ront . These of course, can be assayed by the procedurepreviously descr ibed ( l ) . The except ions are tyros ine, which occurs justpr ior to h is t id ine, and a poor ly shaped lys ine peak. Between the lys inepeak and tryptophan was a major peak which is not in the synthetic aminoacid mixture of l7 amino acids. The very smal l peak immediate ly pre-ceding tryptophan was perhaps a tryptophan acid hydrolysis degradation

X : Y :

(ColC) - |

100

CHROMATOGRAPHY OF AMINO ACIDS

TABLE 3

De.. t rnvtNet. roN aNn Cl lcur-ATroNS op CysrnrNr. . Hrs l rorNr. aNn TnvpropH.qNtN Lvsozyvp- sy Gas-Lreuro CHnorvl rocnapnv lsorope DrrurroN

203

C a l i b r a t i o n r l a n d a r d s

A m l n o r c i d "

(mg ol

p a p e r ) ( c p m )

( c p n l l

n rg o l paper ) . {vc rage

( m g o f

p r p e r , ( c p m l

( c P m l

rng o f paper l Average

. l -carboxyethyl-

c v s t e i n e

H i s t i d i n e

Trl ptophan

1 7 . 8 9 3 :

1 t . . 1 i l 8 7

1 9 . 6 l l l :

. 1 0 . 9 l l 5 l

1 7 . 0 1 + i.1,1.1 106l,11. i I 096

t 5 . 8 r 0 r 0

6 0 . I l l t 6

6.1.0 t t5l

5 9 . 6 l : 0 9

5 5 . . 1 : 1 1 6

.10 .0 5 I l50.1t 69 I. { 1 . 6 5 9 74 1 . 1 7 t 8

1.1 t ' l .r{8.0 10.t.17 . s t76 l1.1 15.11

5 1 . 1 I t . 1 t6 6 . 9 l T l l t56 .7 1 .1095r,t.9 l l t i l

I 1 . 9

l l . 6

1.1..1

t 5 . 1

Ir0I6il11.'r l5l

" l l ! s u b s t i t u t i n l l t h c i i v e r a g c v a l u e s o l C o r n d ( i n t h c i \ ( ) t o p c d i l u t i r ) n e q u a t i o n a n d u s i n g t h e v a l u e s l i r r a . a n d - \ ' l - r o ml ' a b l c I o n e c r n c a l c u l a t c :

Y i e l d ( . 7 )

L \ \ l c r n c

l { i : t i d i n e '

I r \ n l o p h r n

( 4 q l ) l l t l r I h r a q t : i

( l - l 0 )

( l l - b ) ( l . l . i )I { ) ( } r r I l t 9

( - r 5 t ) l

( l : 0 ) ( 1 6 . i )I l r l l r | { '

I l J . - j

l x I 91..1

I t g

product , a l though i ts speci f ic act iv i ty was not as h igh as the maintryptophan peak. The bars underneath the peaks indicate where col lect ionof the g lc ef f luent took p lace and, as ageneral ru le. co l lect ion was begunI min before the glc peak emerged and continued I min after the peak.

The data of Table 3 show the determinations of peak areas and radio-act iv i ty for the g lc chromatogram of F ig. l . The calculat ions in Table 3show that cyste ine. h is t id ine, and t ryptophan. respect ive ly . were re-covered wi th 93, 9t l , and 7-5% of theory. Theoret ica l va lues were obta inedfrom the absorbance of unhydrolyzed prote in at 280 nm and the calculat ionof the amount of amino ac id that should be present in the hydrolysate.These percentages are not relative but are an absolute measurement of whatshould have been in the prote in pr ior to hydrolys is . Hist id ine and cyste ineare wi th in the overal l exper imenta l error whi le t ryptophan is substant ia l lylower.

The repl icates of Table 3 appear in the order in which they werechromatographed. Chromatography of the standard and of lysozyme wereal ternated beginning wi th the standard. This was necessary as the speci f ic

204 PE' fER FELKER

. fABLE 4

Rr-Lerr vp AsuN oeNc e on Pnour N enr krN s r n r u e 70-eV M.css Fnrcur.n.r .qr roN ParrunNsoF rHE Dr l vE ruv t - Es re n o r A tETyLAT I -D .S -C .qReo rye rHyLCys rE INF

Ion ttt i t ' (c/c\

M -M-CH, rOM-CO:CH.JM-CO:CH,r-CH,rCO + HM-2 (CO?CH3) -CH. rCoM-CH,CO:CH,M- (CH] ) ]CO:CH. ]M-S (CH! ) :CO:CH. ]M-CHrS(CH?):CC)!CH.|M-CHCHTS(CH: ) !COrCH, lCH]S(CH,) ,CO?CH,I( cH r ) : s ( cH : )L

cH !s ( cHr ) !cH,rcoNH!cozcH]cH,rcociHsc).rsrU nassigned

U nassigned

26.r ( I .7)1 1 1 l l 5 l

104 (66.0)t62 (7.9\l0 l ( -5 .9 )1 9 0 ( l l . 2 )176 ( l . -s )1 4 4 ( 1 0 . - 5 )l -10 (7 . -3 )1 7 ( 1 0 . 8 )l l 3 ( t 3 . 2 )8r i (31 .6)74 (8 .1 )-s9 ( 16 .9)-s9 ( 16 .9)13 ( 100.0)

172 ( . {8 . l )l . {0 ( t0 .8 )l l l ( 1 3 . 0 )

act iv i ty has a s lc lw upward dr i f t . par t icu lar ly not iceable for prote inhydrolysates. This upward dr i f t was not due to bet ter co l lect ion of radio-act iv i ty but to lowered detector response fb l lowing repeated large prote inhydrolysate in ject ions.

Since the t ryptophan value for lysozyme was low. i t was decidedto check f ree t ryptophan recovery fo l lowing ac id hydrolys is . Thus14.8 p.g of t ryptophan (as judged by the uv molar absorpt iv i ty) wasadded to a synthet ic mixture of l0 p.g of each of l7 amino acids. A valueof 14.3 g.g was found by the method repor ted here. When 14.8 pg oftryptophan was added directly to a glc reaction vial and chromatographedaf ter der ivat izat ion, but wi thout hydrolys is . a value of 14.4 g.g was found.Thus the low t ryptophan recovery f rom lysozyme hydrc l lys; r tes was notdue to losses dur ing der ivat iznt ion but . rather- . to a preferent ia l destruct ionof the pept id ica l ly l inked t ryptophan of lysozyme which was not accom-panied by a compensatory destruct ion of the labeled f iee t ryptophanadded.

Muss Spectromctrv

Gas- l iqu id chromatography of h is t id ine. S-carboxyethylcyst ine. andtryptophan as the l / -acety l -0-methyl esters had not prev iously been

CHROMA |OGRAPHY OF AMINO ACIDS 20.5

reported. and thus we wished to conf i rm the st ructures by mass spec-

t rometry. Of par t icu lar in terest were resolv ing the quest ion of whether the

imidazole n i t rogen of h is t id ine was acylated and establ ish ing the nature

of the two peaks resulting from incomplete derivatization of tryptophan.

As wi l l be shown. h is t id ine ex is ted as the d iacy l der ivat ive wi th an acyl

group on the imidazole n i t rogen as wel l as the a-amino gro l lp . Tryptophan

may exis t as both a monoacyl and d iacy l der ivat ive, having an acety l

group on the indole n i t rogen as wel l as the a-amino group. However, us ing

the der ivat izat ion process descr ibed in th is paper, t ryptophan exis ted

sole ly as the d iacy l der ivat ive. The st ructure of our putat ive synthet ic

S-carboxyethylcyste ine was a lso conf i rmed.Mass fragmentation patterns of the N-acetyl-O-methyl esters of

h is t id ine, t ryptophan, and S-carboxyethylcyste ine were s imi lar to the

ly ' - t r i f luoroacety l -O-buty l esters (14) and the N-heptaf luorobutyry l -

o- isoamyl esters ( l ) . Molecular ions were found for a l l der ivat ives

stucliecl here. Cleavage occurred between carbons 1 and 2 of the amino

acid ester to lose the f ragment CO2CH:r (mle : -59) but the assignment

was equivocal and could be CH:]CONH2. Evidence f rom previous work

wi th other der ivat ives (1.14) ind icated that ester c leavage was favored

over the CH,]CONH, loss. Cleavage a lso occurred at the amide l inkage'

resul t ing in loss of the acety l group. UsLral ly th is loss occurs wi th hydrogen

extract ion. so that the net loss was 42 rather than 43. The loss of the acety lgroup of ten occurs together wi th loss of the ester funct ion to g ive a net loss

o f 1 0 1 .The acety lated d imethyl ester of S-carboxyethylcyste ine y ie lded the

ions shown in Table 4. Loss of the ester funct ion was common, to y ie ld

an intense ion at nt le : 204. lons resul t ing f rom loss of one ester and one

acety l funct ion to y ie ld nt le : 162 were seen. as were ions resul t ing f rom

loss of both ester funct ions as wel l as the acety l funct ion (mle : 102) .

Several smal l ions were present , which inc luded the th ioether l inkage

mle - 74. 88. and 133. An intense ion occurred at mle - l l2 torwhich an

exact ass ignment has not been made. Exact mass measurement of the

mle : l72 ion showed i ts mass to be 172.0193, corresponding to C7HrO3S,

wi th a calculated mass of 172.0193. Since n i t rogen was not present in the

nt le : 172 ion, c leavage must have occurred between the n i t rogen and the

d-carbon. I f the n i t rogen departed in the f iagment CH' ,CONHr(-59) . th is

would have y ie lded an rn le :204, which d id occur and which had a

re lat ive in tensi ty of 66%, . The assignment of rhe mle:204 ion was

equivocal , as th is could have resul ted f rom loss of the ester funct ion.

I f . in conjunct ion wi th loss of CH:rCONH, ion, a methoxy ion was a lso

lost ( -31) , th is would y ie ld an mle : 173, and there is ev idence for the

methoxy loss as evidenced by the ion at ntle : 232. Loss of one hydrogen

fr -om the f ragment resul t ing f rom a CHiCONH, and the CH,, loss would

have y ie ldecl an nt le : 172 wi th an empir ica l formula of CrHrO,S. That

206

ReL,q . . l l v t A guNoeNcE on

Pet . . r rnNs oF THE

PETER FELKER

'fABl-E .5

PnovrNENr lons rN lsp 70-eV Mass Fnacve NTATToNDrecervL .qrn l Mnmvr - Esrnn op Hrsr ro rNe

nt le ((%)

M+M-CH.rCO + HM-CH.jO!CM-CH,rO?C-CH. ]CO + HCH.JCONHCHCO!CHi]M-CH,JO,C-2(CH3CO) + 2HIm" + CH'CO-HIm + CHrCHI m + C HI m + HCH,]CO

253 ( 18 .0)2 r r ( 1 6 . 0 )t94 (26.0)l-52 (61.7)130 (26 .0)l l0 ( -s0 .0)i l0 (_50.0)9-5 il 0.8)8 l ( 6 2 . 2 )69 0-5 .0)13 ( 100.0)

" Im . im idazo le . r n l e : 68 .

f ragment could have exis ted as a l inear or r ing st ructure s imi lar to thatpostu lated by Gelp i et t t l . (14) for a cyste ine-der ived mass spectra l ion.

The predominant ions of the 70-eV mass spectrum of the l/-acetyl-O-methyl ester of h is t id ine are shown in Table _5. The molecular ionas wel l as the molecular ion minus the acety l and ester funct ions wereprominent . The ion (mle :2 l l ) resul t ing f rom loss of an acety l groupfrom the molecular ion was present in h is t id ine but was not present ine i ther the t ryptophan der ivat ives or in the s-carboxyethyl der ivat ive.Acyl imidazoles were very labi le (15) . and acylated h is t id ine was the mostdiff icult of amino acids to gas chromatograph. indicating that acetyl lossfrom the molecular ion was from the imidazole ring rather than the a-aminogroup. Of in terest was the ion mle : l l0 . This could have ar isen f romloss of both acety l funct ions ( -42) and loss of the ester funct ion ( -59)to yield mle : I 10. or it could have been assigned to the acylated imidazoler ing. As the acylated imidazole dng was labi le , we bel ieve the mle : l l0ion resulted from loss of both acetyl function and the ester function.

Whi le Moodie ( l6) demonstrated an acylated h is t id ine by d i rect probe,and Gelp i et u l . (14) presented the gc-ms of the monoacylated h is t id ine,there has been no prior gc-ms confirmation of a diacylated histidinestructure. The gc-ms data presented in Table -5 show that the observedgc peak was diacylated.

The 70-eV mass spectra of the mono- and d iacety lated methyl esterof tryptophan are given in Table 6. The spectrum of the cliacetylatedder ivat ive was s imi lar to that of the monoacety lated der ivat ive. Themolecular ions had s imi lar in tensi t ies. as d id the ions resul t ing f rom lossof the ester funct ion, mle : 201 and 243. As has been repor ted i l .14) .

C]H ROMAI 'C )GRAPHY OF . {M INO ACIDS

'fABt_E 6

RnL , r t r v r - AsuNo , \Nc 'E op Pno l r rN r . x ' r ' I oNs r l rH r_ 70 eV Mass Fnnc l rENTAT I ( )NPa r r r -Rxs o r MoNo- .LNo D lnc r - l t r . . \ rEo MEr r t t L l r s tEas o r Tn r -p l opHa l :

201

krn

Monoacylt ryptophan(n t l c (% l )

Diacylt ryptophan(nt le (Vc\ \

M 'M-CH,OICM-CH. ,O?C-CH. ,CO + HATCOCH,-HA r + C H , N HA r + C H N HA r + C H

A r - H C N

160 (-5.2)l0 l ( r9 . -5 )t -59 ( -s . t )172 (0 .0 )l -59 ( 5. -1)| 5 8 ( 1 . 8 )1 4 3 ( t . 5 )130 ( 100.0)1 0 3 ( 5 . r )

302 (6.0)1.13 ( r{r.8)l 0 l ( r 9 . 5 )t72 (10.7)r -s9 (4.0)1,58 (2.0)143 ( r . 4 )t . r0 (100.0)103 (3.9)

t ryptophan undergoes indole r ing expansion us ing the methylene oncarbon 3 to fbrm the quinol in ium ion. The qLr inol in ium ion. mle : 130.was base peak in both the mono- and d iacety lated t ryptophan mass spectra.f n Table 6. the quinol in ium ion is designated as Ar wi th mle : 130.

The d iacety lated t ryptophan loses the acety l group on the indoleni t rogen but acety l loss f rom the molecular ion was not observed. Lossof acety l together wi th loss of ester were prominent in d iacety latedtryptophan , 19.5% relative intensity versus 5.3% relative intensity formonoacety lated t ryptophan. The loss of an acety l group f rom the indoleni t rc lgen was accompanied by H extract ion f rom the acety l to y ie ld a lossof 42, rather than 43. Such H extract ions have been demonstrated wi tht r imethyls i ly l der ivzr t ives of indoles us ing deuterated t r imethyls i ly la t ingagents (Axel Ehmann, personal communicat ion) .

A character is t ic ion for d iacety lated t ryptophan is nt le :172. whichis the acety lated quinol in ium ion. Exact mass measurement of Lhe nt le: 172 ion determined the mass to be 172.0764. The calculated mass forCr lHl0NO is 112.0762. which is the empir ica l formula for the acety latedquinol in ium ion. This ion was miss ing f rom the monoacety lated massspectrum. The subst i tu ted quinol in ium ion was not base peak as was thecase wi th the per f luoroacyl amides ( l . l4) . The acety l amide should bemore labi le to ms condi t ions than the corresponding per f luorc lacy latedamide .

The uv spectra S-cyanoethylcysteine and S-carboxyethylcysteine have notpreviously been repor ted and are presented in F ig. 2 wi th cyst ine forcompar ison. The uv spectrum of s-carboxyethylcyste ine is unusual inhaving a sharp peak at 256 nm and a shoulder at 2-5 I nm. The S-cyano-ethy lcyste ine uv spectr -um had no corresponding peak. This 256-nmabsorpt ion in S.-carboxyethylcyste ine may have ar isen t rom a cyc l icst ructure in which the amino group cyc l ized wi th the carboxyl ic ac id.

20tt PETER FET-KER

L O

o .9

o . 8

" o 6

o

4

o . 4

o . 2

o . l

2 20 24o t* t*"""".i$rti"tt,

F r c . 2 . U l t r av i o l e t abso rp t i on spec t ra o f S -ca rboxye th . v l cys te i ne . .S - cyanoe thy l cvs te i nc .

and cyst ine. The S'-carboxyethylcysteine and. ! -cyanoethylc.vstc ine were dissolved in g lass-

dist i l led water at a concentrat ion of I mgi ml . Cyst ine was used at a conccntrat ion of 0 '5 mgi ml

in water. The cyst ine concentrat ion u 'as:rchieved b-v adjust ing the pH to l i . -5 wi th NH{OH.- f he

uv spec t ra we re measu red and reco rdcd w i t h a C lu l ' l 5 \ pee t l onh t ) l ( rme te l

Evidence for this band occurred with glutathione with characteristic peaks

at 268.5 and 261.0 nm in 12 N HCI (17) ' and these peaks have been

ascr ibed to cyc l ic s t ructures. ln g lutath ione these absorpt ion peaks

occurred only in ac id ic solut ions, whi le in S-carboxyethylcyste ine the

2-56-nm peak occurs at pH 6 in d is t i l led water .

D ISCUSSION

A method has been developed for determining cysteine' histidine, and

tryptophan in prote ins us ing lysozyme as exper imenta l mater ia l . This .

together with previously published procedures for determination of the

remain ing 17 amino acids ( l -3) , made possib le a g lc assay for the 20

prote in-associated amino acids. A number of procedures for determinat ion

of cyste ine, h is t id ine, and t ryptophan was tested. For example. determin-

ation of cysteine as cysteic acid after performic oxidation of the protein

CHROMATOGRAPHY OF AMINO ACIDS 209

showed that as l itt le as 20 p.g of cysteic acid could have been derivatized

using 50 pl of bis(trimethylsilyl) tr if luoroacetamide with 1% trimethyl-

chlorosilane at 150'C for 5 min. This derivative was not stable in a mixture

of the other amino acids. As performic acid oxidizes tryptophan to N-

formylkynurenine, tryptophan determination is precluded on performic

acid-oxiclized samples. Thus, a modification of Cavins and Friedman's( l8) procedure for S-alkylation was adopted in which cystein was deter-

mined as .S-carboxyethylcysteine. This procedure is compatible with

histidine and tryptophan analyses.Numerous methods for glc of histidine were attempted, including

preparation of the trimethylsilyl derivative with chromatography on

SP 2100, OV-17, or SP2401 and preparat ion and chromatography of the

ethoxyformic anhydride derivative of the histidine methyl ester ( 16). The

tr imethyls i ly l der ivat ive was unsat is factory s ince i t y ie lded two fused

peaks on g lc whose stabi l i ty decreased wi th increasing retent ion t ime.

The ethoxyformic anhydride derivative of the methyl ester init ially worked

wel l . but th is der ivat ive was more sensi t ive to contaminat ion in the

injection port than the N-acetyl derivative.Since aliphatic diamines can be chromatographed without derivati-

zat ion on very basic columns (19) . g lc of h is t id inol on lVc 'Carbowax20 M plus 0.25% KOH on 80/100 Gas Chrom A was at tempted. Hist id inol

appeared, as a smal ler peak than expected at 200"C on a 2-5-cm column

with the above packing. Underivatized tryptamine gave a larger peak

at about 160"C. Several workers have reported the efficacy of acetic

anhydride as a chaser in the syringe for obtaining histidine as the per-

fluoroacyl anhydrides (2,3). so we attempted chromatography of the l/-

acety l methyl ester of h is t id ine. This was super ior to other methods,par l icu lar ly on a polar co lumn such as OV-17 or SP 2401. An SP 2401

column was adopted s ince e lut ion was at 20"C lower than OV-17.The major diff iculty with the method as here described was the 20 to

2-57e loss of pept id ica l ly l inked t ryptophan dur ing ac id hydrolys is . This

loss does not seem attributable to incomplete hydrolysis or to uncompensated

losses during derivatization but to the greater acid instabil ity of peptidically

l inked tryptophan than free tryptophan, with the result that the isotope

dilution method did not totally correct for losses of protein-derived

tryptophan. The labi l i ty of t ryptophan to ac id is not to ta l ly understood.

There are indications that ions such as Cu2+ and Fe:r* are responsible

for the degrirdation in acid (20) but it has also been reported that aldehydes

formed dur ing hydrolys is react wi th the t ryptophan (21) . In a color imetr ic

assay for tryptophan described by Spies and Chambers (22) and based

upon the Ehr l ich react ion (23) ,p-d imethylaminobenzaldehyde was reacted

wi th t ryptophan under very st rong acid ic condi t ions in the presence of

an oxidant. The colored product was complex and the mechanism

appeared to be condensation of the aldehyde with carbon 2 of the indole

2 r 0 P E T E R F E L K E R

ring to fbrm an acid-stable product (24). This would explain the effectof metal salts and aldehydes on tryptophan destruction but leaves unex-pla ined the greater instabi l i ty of pept id ica l ly l inked t ryptophan.

Subsequent to complet ion of most of these studies, we at tempted tolower the redox potential of the hydrolysis solution to a point wherealdehydes would not be fbrmed to attack carbon 2 of the indole ring,and the oxidation of carbon 2 of the indole ring might have been prevented.The redox potent ia l o f a lky l th io ls is -0.3-5 V at pH 7. but the potent ia lis +0.03 V at pH 0. Thus we added I mg of t in metal (E0 is -0.136

at pH 0) to the hydrolys is v ia la long wi th 2-5 p lof d i th ioethane. Immediate lyaf ter p lac ing the hydrolys is tube in the heat ing b lock. the t in metal wasconverted to stannous chloride and hydrogen gas. The stannous chloridewas removed from the hydrolysis vial by taking advantage of the factthat d i th ioethane is i r chelator of t ransi t ion metals (25) at pH 2, whi lei t does not chelate in 6 N HCl . Thus, af ter hydrolys is . the hydrolysatewils removed from the dithioethane with a micropipet and taken to drynessseveral t imes to remove the bulk of the ac id. A smal l vo lume of d is t i l ledwater and 2-5 g,l of dithioethane were added to the hydrolysate whichcaused the SnCl, to precipate. After centrifugation, the supernatant wastaken to dryness, and the der ivat ives were prepared as usual . This pro-cedure increased tryptopharn recoveries to approximately 86% andel iminated the smal l peak on g lc of the lysozyme hydrolysate immediate lypreceding t ryptophan. This observat ion a lso lends credence to the factcarbonyl oxidation products may be responsible for tryptophan degra-dation.

Recently Nakai and Ohta (26) have repofted that B-oxindolylalanineis the primary degradation product of tryptophan and that tryptophandegradation results fiom reaction of tryptophan with disulfides. Disulfidesmay wel l cause t ryptophan degradat ion, but th is cannot be the sole causeof t ryptophan degradat ion, as carbohydrates a lso cause t ryptophandegradat ion. Moreover. in our s tudies a l l th io ls were a lky lated pr i t l r tohydrolys is . yet degr t rdat ion st i l l occurred. The proposed st ructure for thetryptophan degradation product (26) suggested that the amino shift fromthe a-carbon on the tryptophan side chain to the B-carbon in the tryptophandegradat ion products occuned at the t ime of hydrolys is of the pept idebond, and th is might expla in the d i f ference in lab i l i ty between f iee andpept id ica l ly l inked t ryptophan.

We cannot r igorously exc lude the possib i l i ty that the low t ryptophanrecovery wers the resul t o f incomplete hydrolys is . but (a) we found nosigni f icant d i f ferences in t ryptophan recovery when the hydrolys is t imewas var ied f rom l9 to 24 hr . (b) vary ing the d i th ioethane concentrar iontiom l0 to -50 pl per 0.-5 ml of 6 Nr HCI had no effect on tryptophanrecovery. and (c) increasing the number of f l 'eeze- thaw cycles for oxygenremoval f i 'om one to five increased yield of tryptophan only about -57r.

CHROMATOGRAPHY OI - AMINO ACIDS 2 I I

An obvious solution to this diff iculty would be the use of peptidically l inked[ '+Cl- t ryptophan rather than f ree [14C]- t ryptophan in the isotope d i lu t ionmethod.

A problem that merits further study is the varying diff iculty of reducingdisul f ides to th io ls . We found that d i th ioerythr i to l g ives bet ter reduct ionof TCA-denatured lysozyme than does mercaptoethanol but we have notstudied other prote ins.

The potent ia l prec is ion of the g lc- isotope d i lu t ion method is great .The precis ion is probably l imi ted by constancy of the f lame ionizat iondetector response. Wi th improved detector s tabi l i ty , s tandard deviat ionsof the order of l7c of the mean should be achieved. We have obta inedimproved stabi l i ty by cont inuous operat ion of the detector .

In a prote in hydrolysate, other amino acids in ter fered wi th the h is t id inederivative such that the histidine peak area was not always proportionalto in ject ion volume or other amino acid peak areas. Nonetheless, theisotope d i lu t ion method corrects for these losses. This ind icates that thespl i t ter must be on the detector s ide of the column and not on the in jectors ide .

A modification of the method was fbund helpful when dealing with alow-cysteine protein, for example, soy protein which contains only l57cof the cyste ine found in lysozyme. Hydrolysates of soy prote in conta inedcontaminat ing substances wi th retent ion t imes of 2.2 and 2.6-5 min at200'C, which in ter fered wi th the methyl ester of S-carboxyethylcyste inehaving a rention time of 2.-5-5 min. Preparation of the rr-propyl esters,by the method ident ica l to those descr ibed for the methyl esters. y ie ldedpeaks at 4.22, 5.40.6.90, and 19.0 min at 210"C for der ivat ized S-car-boxyethylcyste ine, tyros ine, h is t id ine, and t ryptophan, respect ive ly . Theretention times of the unknown contaminants were 2.20 and 2.62 min andwere thus easi ly e l iminated. I t was a lso fbund that subst i tu t ion ofchloroform:methanol (70:30. v /v) for the ether wash fo l lowingS-alky lat iongave improved recover ies of t ryptophan f iom soy prote in.

Despi te the above-descr ibed d i f f icu l t ies. the potent ia l accuracy, re l ia-b i l i ty , convenience. and rapid i ty warrant cont inued ef for t and develop-ment . As of now, a recovery cot ' rect ion must be made for t ryptophan,but excel lent recover ies of cvst ine and h is t id ine are possib le.

S U M M A R Y

A novel hydrolys is and gas- l iqu id chromatographic- isotope d i lu t ionamino acid analys is procedure is descr ibed which permi ts the determin-at ion of cyste ine (cyst ine) , h is t id ine, and t ryptophan in ac id hydrolysatesof prote in. Acid hydrolys is is in 6 N HCI for 20 hr at I l0"C in an iner tatmosphere, using dithioethane as a protecting agent for tryptophan. Priorto hydrolys is . d isu l f ides are reduced wi th d i th ioerythr i to l . and sul fhydry ls

212 PETER F EI-KER

are a lky lated wi th acry loni t r i le . Labeled t ryptophan, h is t id ine. and S-carboxyethylcyste ine are added. and the amino acids are methylated,.A/-acylated. and chromatographed on an SP-2401 column at 200"C.Recover ies of cyste ine and h is t id ine f rom lysozyme hydrolysates were.respectively, 93 and 98%,. and tryptophan recovery was about 807r.Methods for improving t ryptophan recovery are proposed.

The 70-eV mass spectral fragmentation patterns fcrr the derivatizedamino acids nnd the uv absorpt ion spectrum fbr 5-carboxyethylcyste ineare presented.

ACKNOWLEDGMENTS

This work u 'as supported by Nat ional Science Foundat ion Grant GB-40t i2 l -X to Professor

Robert S. Bandurski . The author thanks Dr. C. C. Su,eeley f i r r use of the mtrss spectrom-

eter faci l i ty (PHS RR-00480) and Mr. Jack Hirr ten and Mr. Bernd Sol tmann. respect ively.

lbr per lbrnr ing the low and high resolut ion mass spectrometry. The author is a lso indebted

to Professor Robert S. tsandurski and Dr. Arel Ehmann 1or thei l valLtable counsel and

adv i ce . z rnd t o Ms . B renda Gouche r f b r he r he lp i n t hc nu rnL t sc t i p t p r cp i r r i r t i r r n .

i \ot t 'u t l t l t t l in l tnxr l : l t has been fbund that thc stahi l i t l of the t ler ivut ivcs is improved b-v

keep ing t hc acc t i c anhyd r i dc i n a g l ass s toppe red ho t t l c i n a des i cca lo r ove t -P rO , and da i l y

r cmov ing wha t i s r cqu i r ed . F l ush ing anc l c vacua t i ng t he reac t i on v i a l s u i t h t l t ' y n i t r ogen . as

p rev ious l l ' desc r i be t l ( l ) . f u r t hc r i n rp roves t he s tab i l i t l o f t he de t ' i va t i ves .

R E F E R E N C E S

l . Fe f ke r , P . . and Bandu rsk i . R . S . ( 1975 ) Ano l . B i o t l t t n t . 67 . )15 .2 . MacKenz ie . S . L . . and Tenaschuk . D . ( 1971 ) . l . ( h r t t n t u tog r ' . 97 , 19 .3 . M o s s , C . W . . I - a m b e r t . M . A . . a n d D i a z . F . J . ( 1 9 7 1 ) J . C l t n t t r t u t o q r ' . 6 0 . 1 1 . 1 .. 1 . Ma tsuba ra . H . . and Sasak i . R . M . ( 1969 ) B i t t t l t t t r t . B i t t p l t t s . Rc . s . Con t t nu r t . 35 , 197 -5 .- s . t - i u . T . Y . . and Chang , Y . H . ( 1971 ) . 1 . I l i t t l . Chen t . 216 ,284 ) .6 . Penkc , B . . Fe rcncz i . R . . and Kovacs . K . ( 1974 ) Ana l . B i r t t l t un . 60 , . 1 ,5 .7. El lman. G. L. (19-59) Arc l t . Bi r t t l tern. Biopl t r .s . 82,7( \ .13 . Sobe r . H . A . ( 1970 )u r Handbook o f B iochem is t r y - . Se lec ted Da ta t b r Mo lecu la r - B io l ogy .

9 .1 0 .l l .1 2 .

l l .

p . C t i 3 . Chem ica l Rubbe l Co . . C leve land . Oh io .Canf ie ld. R. E. ( 1963) . l . Bio l . Cl tcm. 238,2698.Haup t . C . W. ( l 9 -51 )J . R rs . N r r l . Bu r . S tu r t d . iA , 4 l ' + .R i t t enbe rg . D . . and Fos te r . G . t - . ( 19 .10 ) J . B i o l . C l t cn t . 133 ,737 .Condon . R . D . . and E t t r e , L . S . ( l 96 t i ) r / r I ns t r umen t i r t i on i n Cas Ch roma tog raphy .

(K ruge rs . J . r ' d . ) p . 9 l l . Ccn t r c r . E i ndho r . ' en . - f he

Ne thc r l ands .B o s s h a r d . H . R . . . l o r g e n s e n . K . H . . a n d H u n r b e l . R . E . ( 1 9 6 9 ) E t r r . J . I l i o c l r t ' n . 9 ,

f . 1 . Ge lp i . 8 . . Kocn ig . W . A . . G ibe r t . J . . and Oro . J . ( 1969 ) . l . C l t r o t r t u t t t , q r . . " l l r . 7 , 60 .1 .l - 5 . B rucc . T . C . ( 1963 ) i r i Mc thods i n Enzymo log . v (Co lo * i c k . S . P . . and Kap lan . N . 0 . .

eds . ) . Vo l . 6 . p . 606 . Academ ic P less . New Yo rk .16 . Mood ie . I . M . ( 1971 ) J . ( - l t r t t r t t u t o ,q t - . 99 , . 19 - \ .

1 7 . I s h c r w o o d . F . A . ( 1 9 . s 9 ) i r r G l L r t a t h i o n c ( C r o o k . E . M . . e d . ) . p . l 5 . C a m b r i d g c U n i v e r s i t yPress. Cambridge. England.

18 . Cav ins , J . F . . and F r i edman . M . ( 1968 ) J . B i t t l . C l t un t . 213 ,335 '7 .19 . Am ine Ana l ys i s . Bu l l e t i n 737 l l 973 l Supe l co I nc . . Supe l co Pa rk . Be l l e l bn te . Pa .20 . B lock . R . J . . and Bo l l i ng . D . ( 191 -5 ) i r r The Am ino Ac id Con rpos i t i on o f P ro te i ns

and F ' oods . p . 82 . C . C . - l ' homas .

Sp r i ng f i e l d . I l l .

212 PETER FElKtrR

are ar lky lated wi th acry loni t r i le . Labeled t ryptophan, h is t id ine, and S-carboxyethylcyste ine are added. and the amino acids are methylated,l / -acy lated, and chromatographed on an SP-2401 column at 200'C.Recover ies of cyste ine and h is t id ine f rom lysozyme hydrolysates were.respectively, 93 and 98%, and tryptophirn recovery was about 807r.Methods for improving t ryptophan recovery are proposed.

The 70-eV mass spectral f iagmentation patterns for the derivatizedamino acids and the uv absorpt ion spectrum fbr S-carboxyethylcyste ineare presented.

ACKNOWLEDGMENTS

This rvork was supported by Nat ional Science Foundat ion Grant GB-40t i2 l -X to Professor

Robert S. Bandurski . The author thanks Dr. C. C. Sweeley fbr use of the mass spectrom-

erer taci l i ty (PHS RR-00'{ f l0) and Mr. Jack Harten and Mr. Bernd Sol tmann. respect ively.

for per lbrming the low'and high resolut ion mass spectrometry. The author is a lso indebted

to Prof 'essor Robert S. Bandurski and Dr. Axcl Ehmann lbr their valuablc counsel and

ac l v i ce . anc l t o Ms . B renda Go t r che l f o r - he r he lp i n t hc manusc r - i p t p repa ra t i on .

No t t , u t l dL ' d i t r p r t x t f : I t has been f i r unc l t ha t t hc s t ah i l i t l ' o l ' t he de l i va t j ves i s i n t p roved by

keeping the acet ic anhl 'dr ide in a g luss stopper-ed bot t lc in a t lcs iccator over PrC)- , and dai ly

remov ing wha t i s l equ i r ec l . F - l ush in - r1 und evacua t i ng t he reuc t i on v i a l s r r i t h c l r l n i t t t t gen . : r tp rev i ous l y dcsc r i bcd ( l ) . f i r r t he r imp roves t he s tab i l i t l ' o f t he de t ' i vu l i ves .

t .2 .3 .1 ..5.6 .7 .6 .

9 .

I 0 .i l .t 2 .

l l .

R E F E R E N C E S

Fc lke r . P . . and Bandu rsk i . R . S . ( 197 - \ ) Anu l . B i o thu r t . 67 . 115 .MacKenz ie . S . t - . . and Tenasch r - r k . D . ( 1971 ) . 1 . Ch r t t n t u top r ' . 97 .19 .Moss . C . W. . Lamber t . M . A . . and D iaz . F . J . ( 1971 )J . C l t r t t n t u t r t g r - . 60 , 13 .1 .Ma tsuba ra . H . . and Sasak i . R . M . ( 1969 ) B i t t t l t t n t . B i t t p l t t ' s . R t . t . ( - on t t r t u r t . 35 , 197 -5 .L i t t . T . Y . . a n d C h a n g , Y . H . ( 1 9 7 1 ) . 1 . B i t t l . C l t c n t . 2 1 6 , ) 8 4 2 .Penke . t s . . Fe rencz i . R . . anc l Kovacs , K . ( 1971 ) Anu l . B i oc l t e rn . 60 , . 1 . 5 .

El lman. G. L. ( 19,59) Arc l t . Biot l t t 'n t . Biopl t r .s . 82,7O.Sobe r . H . A . ( 1970 ) i r r Handbook o f B iochcm is t r - v . Se lec tec l Da ta t b r Mo l ccu la r B io l ogy .

p . C t i 3 . Chem ic i r l Rubbe r Co . . C leve land . Oh io .Canf ie ld. R. E. ( 1963) J. Bi t t l . Chen. 238,2698.Haup t . G . W. ( 19 -52 ) . 1 . R t ' s . Nu t . I J r r r . S tund .48 , ' 11 ' 1 .R i t l enbe rg . D . . and Fos t c r . G . L . ( 19 ' { 0 ) J . B i o l . C l t u r t . 133 ,737 .Condon . R . D . . and E t t r e . l - . S . ( 196 { i ) r / r I ns l r umen t i r t i on i n Cas Ch roma tog raphy ' .

(K ruge rs . J . ed . ) p . 9 l l . Cen t r c r . E i ndhoven . f he Ne thc r l ands .Bossha rc l , H . R . . Jo r -gensen . K . H . . and Humbe l . R . E . ( 1969 ) F ) r r . J . B i o t l t u r t . 9 ,

1 4 . C . l ; i . E . , K o e n i g . W . A . . G i b e r t . J . . a n d O r o . J . ( 1 9 6 9 ) . l . ( - l t r o n t u t o g t . . ! r ' r . 7 , 6 0 . 1 .

1 5 . B r u c e . ' f . C . ( 1 9 6 3 ) l r M e t h o d s i n E n z y m o l o g l ' ( C o l o w i c k . S . P . . a n d K a p l a n . N . O . .eds . ) , Vo l . 6 . p . 606 . Academ ic P ress . New Yo rk .

16 . Mood ie . I . M . ( 1971 ) J . Ch ro rnu t r t r r ' . 99 . . 19 - s .

1 7 . I s h e r w o o d . F . A . ( 1 9 5 9 ) u r G l u t a t h i o n e ( C r o o k . E . M . . e d . ) . p . I - s . C a m b r i d g e U n i v c r s i t yPress. Cambridge. E,ngland.

f 8 . Cav ins . J . F . . and F r i ec lman . M . ( l 96 t t ) . l . B i o l . C l t u r t . 243 ,33 -s7 .19 . Am ine Ana l - vs i s , Bu l l e t i n 737 (1973 \ Supe l co I nc . . Supe l co Pa rk . t se l l e l i r n t e . Pa .20 . B lock . R . J . . and Bo l l i ng . D . ( 19 .1 - s ) i r r The Am ino Ac id Compos i t i on o f P ro te i ns

and Foods . p . 82 . C . C . Thomas . Sp r i ng l i e l d . t l l .

CHROMATOGRAPHY OF AMINO ACIDS

21 . M i t che l l , H . H . , and Hami l t on . T . S . ( 1929 ) i r The B iochem is t r y o f t he Am ino Ac ids ,ACS Monograph No. 48. p. 103. Chemical Catalog.

22 . Sp ies , J . R . , and Chambers , D . C . ( 1948 ) Anu l . C l t en t . 20 ,30 .

23 . Eh r l i ch . P . ( 1901 ) Med . Wochens ( / r . l 5 l .24. Remers, W. A. . and Brown, R. K. (1972) in The Chemistry of Heterocycl ic

Compounds , I ndo les Pa r t One (Hou l i han . W. J . . ed . ) . Vo l . 25 . p . l 0 -5 , W i l ey I n te r -sc i ence . N . Y .

25 . Ha r r i s . C . M . . and l - i v i ngs tone , S . E . ( 1964 ) u i Che la t i ng Agen ts and Mc ta l Che la tes ,(Dwyer. F. P. . and Mel lor , D. P. . eds.) Academic h 'ess, Nevv York.

26. Nakai , T. , and Ohta. T. (19761 Biochint . Biophls. At tu 420,?.58.

213