Vol. 6o

The Disulphide Bonds of Insulin

By A. P. RYLE, F. SANGER,* L. F. SMITH* AND RUTH KITAIDepartment of Biochemistry, University of Cambridge

(Received 26 November 1954)

In order to deduce the unique structure ofox insulinit is necessary to know its molecular weight. Inprevious papers a value of 12 000 was assumedcsince physical measurements suggested that thiswas the weight of the smallest unit that existed insolution, Recently, however, Harfenist & Craig(1952) have used a new chemical method and havefound a value of approximately 6000. It is difficultto see how this result could be wrong and somerecent physical measurements have shown thatdissociation into units of molecular weight lowerthan 12 000 definitely occurs (Fredericq, 1953;Kupke & Linderstrom-Lang, 1954). It may thussafely be concluded that the molecular weight ofinsulin is 5734, on the basis of condensation of theamino acids present with normal elimination ofwater. This molecule is composed of two poly-peptide chains joined together by the disulphidebridges of three cystine residues. Treatment withperformic acid splits the insulin to two fractions Aand B, which are the oxidized forms of the glycyland the phenylalanyl chain respectively (Sanger,1949a). The sequence of amino acids in these twopolypeptide chains has been determined by partialhydrolysis methods (Sanger, 1949b; Sanger &Tuppy, 1951a, b; Sanger & Thompson, 1953a, b;Sanger, Thompson & Kitai, 1955) and is shown inTable 1. Fraction A contains four and fraction Btwo cysteic acid residues, originating from the threecystine residues of insulin. The purpose of thepresent study was to find which half-cystineresidues are joined together in intact insulin.

For this it was necessary to identify peptidescontaining cystine residues and to determine theirstructure. The procedure used may be summarizedas follows:

(1) Partial hydrolysis of insulin under conditionswhere the disulphide bonds remained intact.

(2) Fractionation of cystine peptides from oneanother. It was not, however, necessary at thisstage to separate them from other peptides notcontaining cystine, since these were separated fromthe cysteic acid peptides during stage 4.

(3) Oxidation of cystine peptides to cysteic acidpeptides.

(4) Fractionation of cysteic acid peptides.* Members of the Scientific Staff of the Medical Research

Council.

(5) Identification of the cysteic acid peptidesfrom the amino acids produced on hydrolysis. Thesepeptides had already been obtained from partialhydrolysates of the fractions A and B, so that theirstructures were known and they could be completelyidentified from their hydrolysis products.From the cysteic acid peptides produced the

structure of the original cystine peptides could bededuced and hence the distribution of the disul-phide bonds in insulin.

During stages (1) and (2) it was essential to avoidany re-arrangement of the disulphide bonds, andthis was the chief difficulty in the work. Initially,hydrolysis was carried out in concentrated hydro-chloric acid and the fact that it was impossible tointerpret the results in terms of a unique structurefor insulin suggested that a random re-arrangementof the disulphide bonds had taken place. This wasconfirmed by studies in model systems and thereaction has been further studied (Ryle & Sanger,1955) and shown to occur in acid and neutralsolution by different mechanisms. The reaction inneutral solution was catalysed by thiol compoundsand could be prevented to some extent by thiolinhibitors such as N-ethylmaleimide (NEMI). Itwas thus possible to use proteolytic enzymes forpartial hydrolysis and the position of one disulphidebridge was found in this way. Experiments usingchymotrypsin and a crude pancreatic extract aredescribed. However, no enzyme could be foundthat would break between the two half-cystineresidues in positions A 6 and A 7 (Table 1) and acidhydrolysis had to be used to locate the remainingtwo disulphide bridges.

Various conditions of acid hydrolysis were triedbut invariably led to re-arrangement until it wasfound that, unlike the neutral reaction, the acid onewas inhibited by thiol compounds. It also appearedto be slower in H2SO4 than in HCI of the same con-centration (Ryle & Sanger, 1955). A rather longtime of hydrolysis was necessary to avoid the pro-duction of large amounts of peptides containing theA 6.A 7 sequence (Table 1), as they do not providethe information required to establish the position ofthe bridges.-t was also necessary to avoid re-arrangement

during the fractionation of the cystine peptides.Initially paper chromatography was used, but it was

541

A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI I955difficult to obtain sufficient material, and frequentlyre-arrangement appeared to have occurred. Thebest results were obtained with paper ionophoresisand in the most recent experiments this method wasused almost entirely.The peptides with reference numbers starting A

orB are those obtained in previous papers as follows:A1, A2, Sanger & Thompson (1953a); Ap, Ac,Sanger& Thompson (1953b); B 1, B2, B3, B4, B5,Sanger & Tuppy (1951a); Bp, Bc, Bt, Sanger &Tuppy (1951b).A preliminary communication ofthe present work

was made by Sanger, Smith & Kitai (1954).

MATERIALS

The insulin used throughout this work was 6-times recrystal-lized cattle insulin (batch 9011 G), obtained from BootsPure Drug Co., Nottingham. Crystalline chymotrypsin wasobtained from Worthington Biochemical Sales Co., Free-hold, New Jersey, U.S.A.

METHODS

IonophoresisThe method used was essentially that of Michl (1951) inwhich the ionophoresis is carried out at high potentialgradients in pyridine-acetic acid buffers on filter papers

CarbonelectrodesK

1 Cooling coil

,Toluene

Filterpaper

Buffer

0 1i 2;0 30 40 cm.

Fig. 1. High-voltage ionophoresis apparatus (Michl,1951).

completely immersed in toluene. This last prevents evapora.tion and cools the paper. It was found that sharper bandswere obtained by this method than by other methodsstudied and the short times required for separations mini-mized the danger ofbreakdown or re-arrangement of cystinepeptides during ionophoresis.The apparatus, which is shown in Fig. 1, was made from

two.battery jars, one fitting inside the other. By having thelevel of the buffer solution higher in the cathode than in the

542

0

E-4

z

K- 0

t¢co

co

0

PV,v

;gSO

ooo00

a-

Er'

Ez

deq

Co

0

co-ct

co

.0

ka3s

co-c

0 2^uat;

.= &,. '3 t;l

I z '4.ow

0

; .. o

THE DISULPHIDE BONDS OE INSULINanode vessel, electroendosmotic flow was to some extentcounterbalanced by hydrostatic flow. Carbon rods wereused for the electrodes and were connected to platinumwires underneath the toluene. Suitably shaped glass rodswere used to prevent the paper from touching the sides ofthe vessels, and the whole apparatus was covered with aglass lid.

Since the buffer (pyridine-acetic acid) is somewhatsoluble in the toluene and large volumes were involved, itwas not convenient to keep changing the buffer for differentexperiments. Two units were therefore employed; the onecontained buffer at pH 6-5 (10 vol. pyridine: 0 4 vol. iceticacid: 90 vol. water), the other at pH 3-6 (1 vol. pyridine:10 vol. acetic acid: 89 vol. water). In some of the earlierexperiments 0-2m acetic acid (pH 2.75) was used as theelectrolyte. A paper up to 20 cm. wide could be used in theapparatus. The material to be fractionated was applied as athin line across the dry paper to within about 1 cm. of eachedge. It was allowed to dry and then laid on a glass platewith a glass rod underneath the line of application. Thepaper was wetted with the buffer to within about 1 cm. oftheapplied material and the buffer allowed to flow in towardsthe line of application from both sides, thus concentratingthe material in a thinner line. It was essential to ensure thatthe buffer flowed in evenly and at the same rate from bothsides and to be certain that the paper was completely wettedbefore putting in the apparatus. Where rather large con-centrations of peptides are applied they sometimes wet veryslowly. If a spot is not wetted by the buffer it is wetted bythe toluene and causes an unevenness offlow. After blottingthe paper to remove excess buffer itwas put in the apparatus.

For most purposes a potential of 1500v was applied.Using a 20 cm. wide Whatman no. 3 filter paper, a current of60-70 mA was produced with the buffer at pH 3-6 and of40-45 mA at pH 6-5. Owing to differences in design of theapparatuses the potential gradients produced were 29 and33v/cm. respectively. With no. 4 papers about half thesecurrents are obtained. At the higher currents there isconsiderable heating, and it was necessary to have a coolingcoil in the toluene. To find the position occupied by thepeptides after ionophoresis, marker strips were cut from thepaper and tested with a suitable dipping reagent, as below.

Ninhydrin. A 0.25% solution in acetone was used(Toennies & Kolb, 1951).

CN-nitropruside. To test for the presence of cystinepeptides the CN-nitroprusside test of Toennies & Kolb(1951) was used. This was found to be more sensitive thanother tests investigated, though the colour was rathertransitory, and it was necessary to record the position ofthebands fairly rapidly.

Tyro8ine test. To test for tyrosine peptides the method ofAcher & Crocker (1952) was used.

Chymotryptic hydrolysis (Expt. Ic)Insulin (100 mg.) was suspended in 10 ml. water and

dissolved by the addition of NH, to give a final pH of 8.Chymotrypsin (4 mg.) and 1 mg. of NEMI were added andthe mixture was incubated at 370 for 24 hr. The solution wasthen brought to pH 5'7 by the addition of dilute acetic acid.This produced a precipitate, which will be referred to as the' chymotryptic core'. It is probably essentially the same asthe 'core' studied by Butler, Dodds, Phillips & Stephen(1948) which was obtained from a chymotryptic hydrolysateby precipitation with trichloroacetic acid. The yield of

'core' was about 65 mg. Itwas further purified by dissolvingin dilute NHa, and precipitating at pH 5-7. In certainexperiments the 'core' did not precipitate immediately onbringing the hydrolysate to pH 5B7 and it was necessary toconcentrate to a smaller volume. However, once the pre-cipitate had been obtained, it could not be redissolved nearpH 5-7.The soluble material from the hydrolysate was freeze-

dried before being subjected to fractionation by iono-phoresis.

Hydrolysis with a crude pancreatic extract (Expt. Ix)

The preparation used was a crude 'trypsin' (British DrugHouses, no. 557). It apparently contained several otherenzymes, since several bonds which were outside thespecificity range of trypsin or chymotrypsin were exten-sively hydrolysed.

Insulin (300 mg.) was dissolved in 18 ml. 0-01 N-HCl11 mg. of NEMI was added and the pH was adjusted to 7.8by the addition of 0O1N-NH,. 1-2 mg. of the 'trypsin' pre-paration was then added in solution in a little water, and thepH was re-adjusted to 7-8. After 4 hr. incubation at 370 thepH had fallen considerably and was brought back to 7-8.After a further 17 hr. incubation the pH was 6-8 and thesolution was very cloudy. The digest was then brought topH 6 by the addition of 0-2N acetic acid and centrifuged toremove the precipitated material. On concentrating thesupernatant solution in vacuo a further small precipitateappeared, but this was ignored, and the whole of the con-centrate was applied across a 25 cm. wide strip of Whatmanno. 3 paper and was subjected to ionophoresis at pH 6-5(in buffer containing approx. 10-3m NEMI) at 1500v for12 hr.

Acid hydrolysesA number of different experiments were carried out using

acid with various conditions ofhydrolysis and fractionation.Two such experiments will be described which incorporatethe various techniques used and results obtained. In thefirst (I 1) 10N-HSO at 1000 was used and the peptideswere separated into two groups (I Ic and I 1,) by ion-exchange chromatography and were then fractionated bya number of different ibnophoreses. In the second (12) thehydrolysis was carried out at 370 and the neutral cystinepeptides were separated by two-dimensional ionophoresis.

Experiment I 1. The 'chymotryptic core' (100 mg.) wasdissolved in a mixture of 5 ml. 20N-HSO, 3 ml. glacialacetic acid and 2 ml. water. Thioglycollic acid (3,uA.) wasadded and the mixture heated on a boiling-water bath for45 min.Removal of the HSO4.with Ba caused extensive losses of

the cystine peptides and was much more convenientlycarried out using the basic ion-exchange resin AmberliteIR-4B, 44-100 mesh/in. (manufactured by The Rohm andHaas Co., Philadelphia, U.S.A.) in the acetate form. Acolumn was prepared 8 cm. high in a tube of radius 2 cm.having a tap at the bottom. The resin was equilibratedagainst 20% (v/v) aqueous acetic acid and the hydrolysatepoured on the column. To prevent uneven flow the top halfof the column was stirred with a long glass rod so that mostofthe HAO5 was initially adsorbed at the top ofthe column.The column was then allowed to flow and was developedwith 20% acetic acid. The effluent was collected till a dropon a filter paper no longer gave the ninhydrin reaction.

Vol. 6o 543Q

544 A. P. RYLE, F. SANGER, L75-100 ml. was usually sufficient. In some earlier experi-ments 5% acetic acid was used for elution, but the cystinepeptides were considerably retarded and a greater volume ofeffluent was required. The solution was concentrated to10-20 ml. in vacuo in the rotary evaporator of Craig,Gregory & Hausmann (1950), using a solid CO-methylcellosolve mixture to cool the condensing bulb. The thio-glycollic acid and remaining acetic acid were extracted withether and a small amount of NEMI (2-3 mg.) was added.The solution was brought to pH 5 with aqueous NH3 and

put on a column of Amberlite IR-4B in the acetate form,which had been washed with distilled water until theeffluent attained pH 3-4. The column was developed withwater. Part of the hydrolysate was not retained by thecolumn and was collected and freeze-dried (I 1/3). The pep-tides retained on the column were removed by eluting with20% (v/v) aqueous acetic acid, the solution concentrated,extracted with ether and freeze-dried (I lx).

Experiment 12. Insulin (100 mg.) was dissolved in amixture of 7 ml. 20N-H2S04 and 7 ml. acetic acid containing6,ul. thioglycollic acid and kept at 370 for 17 days. H2SO4was removed on Amberlite IR-4B as described in Expt. I 1and the material freeze-dried in the presence of NEMI.

Two-dimen8ional ionophoresi8. Hydrolysate from Expt.12 (50 mg.) was dissolved in 0.15 ml. water and applied ona line near to the anode end of a 18 cm. wide strip of no. 3filter paper for ionophoresis at pH 3-6. A potential of1500v was applied for 5-5 hr. After drying the paper, a stripwas cut from one edge and the position of the cystinepeptides located by the CN-nitroprusside test. The appear-ance of this test strip is shown in Fig. 2. The most slowly

+0 5 10 15 20 25 30 35-Disnce (cm.) travelled from origin towards cathode

Fig. 2. lonophoresis of partial hydrolysate of insulin(Expt. 12): 50 mg./18 cm. wide no. 3 paper; pH 36;29v/cm.; 5-5 hr. In this and subsequent figures repre-senting ionophoretic separations the different types ofshading indicate the colour reaction used to locate thebands as follows: 1, ninhydrin reaction; *, CN-nitroprusside reaction; , tyrosine reaction. The depthof shading represents the approximate strength of thecolour reaction.

migrating material was largely free cystine, so was notfractionated further. The part ofthe paper between 10-6 and28'6 cm-. from-the origin was-then out out-andmaterial elutedfrom it on to a second no. 3 filter paper as shown in Fig. 3. Inthis method one edge of the 'cut' was put in the trough of10% acetic acid, which washed the peptide bands downnear the 'front'. In order to keep this front as even as

possible it was sometimes necessary to pipette drops ofacetic acid on to the paper behind it. The other edge of the'cut' was clamped down on the second paper with a glassplate so that only the extreme edge was in contact. When theacetic acid front had flowed into the second paper to forma band 1-2 cm. wide, the elution was stopped, the banddried off and then subjected to ionophoresis at pH 6-5 for-10 hr. using 1500v. A small amount of NEMI was added to

F. SMITH AND R. KITAI I955the buffer just before wetting the paper, to inhibit inter-change reactions.At the end of the ionophoresis the paper was hung up in

air for about 10 min. to allow the surface toluene to drop offand evaporate. It was then laid on a flat sheet ofglass. A drysheet of no. 1 paper was laid on top of it and rapidly presseddown with another glass plate to make even contact andthus take a 'print'. Both papers were then dried. The no. 1paper was treated with the CN-nitroprusside reagent, andfrom the positions ofthe spots, the cystine peptides could belocated on the no. 3 paper, cut out and eluted.

Fig. 3. Elution of peptide material during two-dimensionalionophoresis.

Structure of cystine peptides

To determine the structure of the cystine peptides theywere oxidized with performic acid to the correspondingpeptides of cysteic acid, which were then fractionated andsubjected to complete hydrolysis. Previously the fractiona-tion had been carried out by paper ionophoresis in aceticacid by which means all the cysteic acid peptides, exceptthose containing a basic amino acid, could be completelyseparated from other peptides (Sanger & Thompson,1953a).More satisfactory results have now been obtained by carry-ing out the ionophoresis in pyridine-acetate buffers atpH 3-6. Here some fractionation of the individual cysteicacid peptides is obtained, which simplifies their identifi-cation.The areas occupied by cystine peptides were cut out ofthe

papers and eluted with 10% acetic acid. The eluates wereput in small test tubes and taken to dryness in a desiccator.A few drops of a solution of performic acid prepared bymixing 1 vol. 33% (w/w) H.02 with 9 vol. formic acid, wereadded. Oxidation was allowed to proceed for 15-30 min.,a few drops of water were added and the solutions taken todryness in a desiccator. Each fraction was then transferredto a polythene strip with a small volume of water and takento dryness again to ensure removal of formic acid.The residues were dissolved in about 1OuAl. water and small

portions (each about 2p.) were transferred with a capillarytube to a sheet of no. 4 paper near to the cathode end andsubjected to ionophoresis at pH 3 6 for 2 hr. at 1500v. Thepapers were dried and developed with ninhydrin.From this preliminary ionophoresis some information

could be obtained about the cysteic acid peptides presentand it could be decided which fractions should be investi-gated further and at what concentration to carry out theionophoresis. Thus, for instance, peptides containing two

THE DISULPHIDE BONDS OF INSULINcysteic acid residues could readily be identified (see Figs. 14,21) and since such peptides could not provide the requiredinformation, they were usually not investigated further. Onthe other hand, fractions giving two peptides each containingone residue of cysteic acid were of particular interest.

For further investigation of the cysteic acid peptides, thewhole fractions were put on paper as a line and subjected toionophoresis as above. A strip was cut from the edge andtreated with ninhydrin to locate the position ofthe peptides,which were then cut out, eluted and hydrolysed. Theresulting amino acids were identified by paper chromato-graphy using phenol-0.3% aqueous NH3.

RESULTS

Chymotryptic hydroly8ate (Expt. Ic)Fig. 4 shows the distribution of peptide bands afterionophoresis of the soluble fraction (30 mg.) fromthe chymotryptic hydrolysate.

|N~~~ ~ ~~~1- II

10 5 0 5 10 15 20+ Distance (cm.) travelled from origin

Fig. 4. Ionophoresis of peptides from soluble fraction ofchymotryptic hydrolysate (Expt. Ic): 30 mg. on 18 cm.wide no. 3 paper; pH 6-5; 33v/cm.; 2 hr.

1 2

p..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

_f .,3+ 5 10 15 20 25 -

Distance (cm.) travelledfrom origin towards cathode

Fig. 5. Ionophoresis of fractions lco, Ic,B and Ic8 (Fig. 4):no. 3 paper; pH 3-6; 29v/cm.; 7 hr.

Band a, which contained no cystine, was re-

fractionated on a two-dimensional paper chromato-gram using phenol-0-3 % NH2/butanol-acetic acid.One main spot was present which proved to beTyr.Thr.Pro.Lys.Ala (Table 2). Band C was

similarly shown to be Thr. Pro. Lys . Ala and band XPro.Lys.Ala. This last peptide had not been ob-tained from the chymotryptic treatment offraction B (Sanger & Tuppy, 1951 b). Its presence issurprising since it would not be expected from theknown specificity of chymotrypsin.

35

Bands ae, ,B and 8 were further purified by iono-phoresis at pH 3-6 (Fig. 5). Both 81 and 82 gavephenylalanine and tyrosine on hydrolysis. Band 82probably contains the free amino acids, since theymove at the same rate on ionophoresis at pH 3-6.Band 81 is probably a dipeptide Phe.Tyr, whichwould be expected to travel slower than the freeamino acids. This dipeptide was not detected in thechymotryptic hydrolysate of fraction B, but mightbe expected to occur from the known specificity ofchymotrypsin.

b

.a b

aa b

+ 10 5 0 -Distance (cm.} travelild from origin

Fig. 6. Ionophoresis of oxidation products of bands al,,B, y and 83 (Figs. 4, 5): no. 4 paper; pH 3-6; 29v/cm.;2 hr. (see Table 2).

The four cystine peptides (cxl, ,B, y, and 83) wereoxidized and the resulting cysteic acid peptidesfractionated by ionophoresis at pH 3 6 (Fig. 6). Theresults are summarized in Table 2. All four gave thesame-peptide (b, Fig. 6) which was neutral at pH 3*6and was characterized by the presence of arginineand a large amount of glycine. Its structure canonlybeLeu .Val . CySO3H. Gly . Glu .Arg. Gly . Phe. -

Phe, identical with peptide Bc 4.The three acidic peptides ocla, fia and ya gave the

same amino acids on hydrolysis but moved atdifferent rates at pH 3-6. Band ya was by far thestrongest and is probably GluNH2 . Leu. Glu . Asp -

NH2. Tyr. CySO3H . AspNH2 (Ac 3) which would beexpected to be a major component. Band ala gaveno ninhydrin reaction, suggesting that itwas apyrro-lidonoyl derivative identical with ya except that theN-terminal glutamine residue had cyclized. Thiswould account for xl moving faster towards theanode than the other acidic cystine peptides as ithas one less amino group. Band ,Ba probably differsfrom ya only in having one amide group less andtherefore being slightly more acidic.On oxidation the only neutral cystine peptide

(83) gave a neutral peptide (83b) identical withoclb, fb and yb, and an acidic peptide 83a havingthe structure CySO2H .AspNH2 (Ac 1).

Bioch. 1955, 60

Vol. 6o KA5

A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAITable 2. Peptide8from chymotryptic hydrolyBate of insulin (Expt. Ic)

Amino acids ofoxidized peptides Probable structure

a: CySO3H, Asp,Glu, Tyr, Leu

b: CySO3H, Glu,Gly, Val, Leu, Phe,Arg I.

ot2 Asp, Glu, Leu,Tyr (weak Val)

,B a: CySO,H, Asp, Glu,Tyr, Leu I

b: CySO,H, Glu, Gly,Val, Leu, Phe, Arg J

a: (CySO3H, Asp,Glu, Tyr, Leu

b: CySO3H, Glu,Gly, Val, Leu, Phe,Arg

Tyr, PheTyr, Phe

a: CySO3H, Asp

b: CySO,,H, Glu,Gly, Val, Leu,Phe, Arg

Thr, Ala, Tyr,Pro, Lys

Thr, Ala, Pro, LysAla, Pro, Lys

I

NH3 NH,

Pyr* .Leu. Glu . Asp. Tyr. Cy. AspSS

Leu.Val. Cy. Gly. Glu. Arg. Gly . Phe. Phe

NH2 NH2

Glu. Leu. Glu. Asp. Tyr (Ac5)

See text

NH2 NH, NH2I ~ ~ ~~~~~I

Glu .Leu . Glu .Asp . Tyr . Cy . AspSS

Leu.Val. Cy. Gly.Glu.Arg. Gly.Phe. PhePhe.TyrPhe +Tyr

NH2Cy.AspS

Leu.Val. Cy. Gly. Glu. Arg. Gly.Phe. PheTyr. Thr. Pro. Lys. Ala (Bc7)

1I

Thr.Pro.Lys.AlaPro.Lys.Ala

* Pyrrolidonoyl.

(Bc5)

-O 5 10 15 20 25 30 35 +Distance (cm.) travelled from origin towards anode

Fig. 7. Ionophoresis of crude 'tryptic' hydrolysate ofinsulin (Expt. Ix). Soluble material from hydrolysate of300 mg. insulin on 25 cm. wide no. 3 paper; pH 6B5;33v/cm.; 12 hr.

Hydroly8is with crude pancreatic extract(Expt. Ix)

Fig. 7 shows the positions of the bands obtainedby ionophoresis at pH 6-5 of the crude 'trypsin'hydrolysate. Band vq, which had moved ahead ofmost of the ninhydrin-reactive material, was

thought to be fairly pure and was oxidized directly.When part of the material from bands oc-c andand L was oxidized several cysteic acid peptides

were obtained from most of the bands, indicatingthat they contained more than one cystine peptide.

Band(Fig. 7)

PIaI

461 462 63 p64Y, I Ff IN R

r1 Y2 y3I&- - m B - Y

fl- 42 &3 &4 4t 5C-I 0K

a ,a El s2 O3,a I

+0 5 10 15 20 25 30 35-Distance (cm.) travelled from origin towards cathode

Fig. 8. Purification of bands oc-s from Expt. Ix (Fig. 7) byionophoresis: pH 3*6; 29v/cm.; 6 hr.

The material from band behaved in the same wayas that from band -1, and bands and & were con-

sidered too weak to give any results after furthermanipulations, so these three bands were notfurther dealt with.

546

Band(Figs. 4, 5, 6)

I955

V

8182

83

E

C77

I

THE DISULPHIDE BONDS OF INSULIN

Table 3. Jy8teic acid peptides obtainedfrom crude 'tryptic' hydroly8ate of insuin (Expt. Ix)

Amino acids present incysteic acid peptide

CySO3H, Glu, Gly, Val, LeuCySO8H, AspCySO3H, Gly, Val, LeuCySO3H, AspCySO,H, Ser, Ala, ValCySO3HCySO3H, Ser, GlyCySO3H, Ser, Gly, LeuCySOH, Ser, Ala, ValCySO3H, Ser, Gly

CySO3H, Ser, GlyCySO,H, Ser, Ala, ValCySOH, Ser, Gly, Ala

Assumed structure ofcysteic acid peptide

Leu. Val. CySO3H. Gly. GluCySO3H.AspNH2Leu.Val. CySO8H. GlyCySOSH .AspNH,Same as -bCysteic acid

Leu. CySO3H. Gly. SerSame as 'qb

CySO8H. Gly. SerCySO3H.CySO3H.Ala . Ser. Val.CySO8H

The remainder ofthe material from bands a-e waspurified by ionophoresis at pH 3-6. The separatedcystine peptides, detected by the CN-nitroprussidereagent (Fig. 8) were eluted, oxidized and run againat pH 3'6 for the separation of the cysteic acidpeptides whose positions are shown in Fig. 9.

Band ofcystine peptides(Figs. 7, 8)

a by2

a b

b&20EA MA 11

a b c d

,, , a, , b, c ,--_ 0 5 10 15 20 25 30 35

Distance (cm.) travelled fromorigin towards anode +Fig. 9. Ionophoresis of cysteic acid peptides from Expt. Ix

(Figs. 7, 8): pH 3-6; 29v/cm., approx. 2 hr. FractionsP4, y2 and 81 were run for slightly longer than the otherfractions (see Table 3).

The cysteic acid peptides strong enough to giveresults were eluted and hydrolysed, except in thosecases where one ofa pair ofbands could be identifiedas cysteic acid by its speed on ionophoresis. Theamino acids found in the hydrolysates and theprobable identification of the cysteic acid peptidesare shown in Table 3. Certain peptides (e.g. ,B2 andyl) gave results indicating that they wereprobablycomplex mixtures and that no definite conclusionscould be drawn from them. The results with thesepeptides are not recorded.

Peptides f4b and y2b were identified as CySO8H. -

AspNH2 (identical with Ac 1). y2a can only beLeu .Val . CySO3H . Gly (positions B 17-20), since

these four amino acids do not occur together else-where in the molecule, so that peptide y2 must be

Leu.Val. Cy . GlyS

S

Cy . AspNH2.By analogy it is concluded that fi4 is

Leu .Val . Cy. Gly. GluS

S

Cy.AspNH2,though from its amino acid composition ,B4a couldbe Gly. Ileu.Val. Glu. Glu.CySOaH (positions A 1-6). Bands Sla, 82c and qb all contain the same

characteristic peptide which is present in largeamounts in this hydrolysate. Judging from itsionophoretic mobility it is very acidic and has a

high content of cysteic acid. In order to determineits structure it was subjected to partial hydrolysis.

I rAKrAii VI-A, kmA1 2 3 4 5 6 7

_0 S 10 15 20 25 30 35+Distance (cm.) travelled from origin towards anode

Fig. 10. Ionophoresis of partial hydrolysate of peptideIx'qb: pH 3-6; 29v/cm.; 2 hr.

Material from band -1b obtained from a digest of135 mg. of insulin similar to that described abovewas incubated at 370 for 40 hr. in 11-7N-HCI, andafter removal of the HCI was subjected to iono-phoresis at pH 3-6 for 2 hr. The positions of thebands detected by ninhydrin are shown in Fig. 10.A sample of cysteic acid moved at the same speedas band 6. The identities of the components of the

various bands are given in Table 4. These show that-1b has the structure CySO3H. CySO3H. Ala. Ser. -

Val . CySO3H (from positions A 6-11). It is probable3&2

Band(Figs. 7-9)

f,4af,4by2ay2bSla81b82a82b82c82d-)aqb7c

V~A M

Vol. 60 51,47

I r4K4 MA VAFSAm

A. P. RYLE, F. SANGER, L. F. SMITH AN]) R. KITAI

that it does not contain -the serine residue frompositionA 12, since such apeptide would be expectedto give large amounts of free serine on acid hydro-lysis, whereas only small amounts were obtainedfrom peptide qb.

Table 4. Peptides obtained from partial hydrolysateof peptide Ix-)b

Band (Fig. 10)1234567

StructureAlanineSerineSer. Val. CySO3H (A lC3)CySO3H.Ala (A ly3)CySO.H .CySO.H.Ala (A 281)Cysteic acidCySO.H.CySO,H

The other main cysteic acid peptide from thecystine peptide ) is qa([CySO3H, Ser, Gly]). Thesethree residues only occur together in positionsB7-9 so that it must be CySOsH.Gly.Ser. How7)b and 71a are linked together is not clear from theseresults since 'qb contains three cysteic acid residues.The two main oxidation products of peptide 82

are CySO3H . CySO3H . Ala. Ser . Val .CySO5H (82c)and 82b which must be Leu .CySO3H . Gly. Ser.Peptides 82a, 82d, and Xc were present in only smallamounts and their composition is not certain.Their ionophoretic mobilities make it unlikely that82a and 82b are CySO3H . Gly. Ser (71a).

Acid hydrolysisIn choosing conditions for acid hydrolysis a

number of factors had to be considered. In Table 5are listed the various peptides of cysteic acidencountered after oxidation ofa partial hydrolysate

Table 5. (Cy8teic acid peptide8from oxidized partialhydrolysate of 'chymotryptic core' of ins8ulin

Distancemoved towards

anode onReference ionophoresis

no. in at pH 3-6previous (Fig. 14)

Peptide papers (cm.)His.Leu.CySO3H Bly4 -1.0His.Leu.CySO3H . Gly B4fi1 -1.0Gly. Ileu. Val. Glu . Glu .CySO3H A 2a7 9 0Leu . CySO3H .Gly BlocS 10 0Ser.Val.CySO8H A 1C3 10X5CySO3H.Ala A ly3 12-5Leu.CySO3H B loc6 13.5Val.CySO3H A 1v2 13-5Glu.Glu.CySO3H - 13-5CySO3H.Gly Blal 14*5Glu.CySO,H A ll 15.5Glu.CySO3H .CySO3H .Ala A 2yl 19*5CySO3H.CySO3H.Ala A2yl 22-5Glu.CyS03H.CySO3H - 24-0Cysteic acid - 28*0CySO8H . CySOIH - 30-0

ofthe 'chymotryptic core'. From these it is possibleto calculate that ninety-five different cystinepeptides would be expected before oxidation. Ofthese, twelve contain a half-cystine residue (CyS) onone side of the -S.S- bond, and provide no in-formation about the distribution ofS .S- bonds.Seventy-two containthe sequence Cy. Cy intact andare also of no use, whereas eleven contain a peptidesequence on both sides of the -S .S- bond and areof thetype required in the present study. In order toavoid the very complicated mixture that would beproduced if there was much of the Cy .Cy sequenceleft intact it was necessary to hydrolyse for as longas possible, but ifthe hydrolysis were carried out fortoo long the peptides containing a CyS residue wouldpredominate and also there would be more danger ofthe interchange reaction occurring.Using model systems it was shown that less inter-

change occurred in H2S04 than in HCI of corre-sponding concentration (Ryle & Sanger, 1955).Although at high concentrations H2SO4 is lesseffective as a hydrolytic agent, it appeared that itwould nevertheless be an advantage to use it.Insulin is insoluble in aqueous H2S04 80 that it wasnecessary to add acetic acid to dissolve it.In an initial experiment 3-7N-H2S04 in 25% (v/v)

acetic acid was used. After 2 days at 370 a precipi-tate started to form and was separated after 5 days.This 'acid core' represented about 30-40% ofthe insulin and on oxidation gave the peptidesGly . Ileu .Val . Glu . Glu. CySO3H .CySOH .Ala andPhe .Val . Asp. Glu. His. Leu. CySO3H . Gly, besideslarger peptides whichwere not identified. It seems tobe a mixture of large peptides which are less solublethan insulin. It could also be precipitated withwater from a hydrolysate that had been obtained bythe action of 5N-H2S04 in 50% (v/v) acetic acid for1 day at 370, so its formation was not caused by amass action effect due to its insolubility.

In later experiments 1ON-H2S04 in 50% (v/v)acetic acid at 370 was used for hydrolysis. The rate ofhydrolysis of insulin in this medium is given inTable 6.

Table 6. Rate of hydrolysis of insulin iniON sulphuric acid

Amino N (Van Slyke nitrous acid method, 11 min.reaction) as % of value after hydrolysis for 48 hr. at 1050(see Peters & Van Slyke, 1932).

Reagent1ON-H2SO4 in50% (v/v) acetic acid

lON-H2SO4 in30% (v/v) acetic acid

Temp.(0) Time37 4 days

10 days14 days

100 23 min.35 min.60 min.11 hr.

hydrolysis263749406680105

548 I955

THE DISULPHIDE BONDS OF INSULINE la|

A B 'C I'Ds E'vFOG H~ I+30 , -25 20 15 10 5 0oDistance (cm.) travelled from origin towards anode

Fig. 11. lonophoresis of fraction llx: 90 mg. on 20 cm.wide no. 3 paper; pH 6-5; 33v/cm.; 1-75 hr.

To obtain sufficiently simple peptides at 370 itwas necessary to continue the hydrolysis for at least14 days, so in some experiments to save time atemperature of 1000 was used. The correspondingrates of breakdown are also shown in Table 6. Therewas slightly more disulphide interchange at thishigher temperature, but it did not significantlyaffect the results (see Ryle & Sanger, 1955).

--~ ~ --IIj_____+30 25 20 15 10 5 0-Distance (cm.) travelled from origin towards anode

Fig. 12. Fractionation of band lIlaJ (Fig. 11) by ionophore-sis: no. 3 paper; pH 6-5; 33vfcm.; 12 hr.

Band of cystinepeptides(Figs. 11, 13)A -I--BIJZ-IC1 I

Band ofcystine peptides.(Figs. 11, 12)

B |[

1 1 3

G *

;: 112 23

I I _*p 1,

+0 5 10 ls 20 25 30Distance (cm.) travelled from origin towards cathode

Fig. 13. Further ionophoretic fractionation of peptidesfrom Ila (Figs. 11, 12): pH 3-6; 29v/cm.; 6 hr.

C3.DiJ

D3JIElr

F1IF2F3rG I

G272H

KIK1-iK2

K3LIL2iL3

M1lEZ

a b

Ir -- - ---- . .0.1

; It..4 u~~~~~1. H.

Ia b -----a1 .|1 §1

- U.

. 1

-0 5 10 .15 20 25 30 35+Distance (cm.) travelled from origin towards anode

Fig. 14. Ionophoresis of cysteic acid peptides from Ilo(Figs. 11-13): no. 4 paper; pH 3-6; 29v/cm.; 2 hr. (seeTable 7). All bands coloured with ninhydrin.

Experiment I1. Using Amberlite IR-4B atpH 3-4 the hydrolysate was separated into twofractions 1le and II,. The former, which was

adsorbed on the column, contained acidic and someneutral cystine peptides, whereas fraction I1contained the remaining neutral and basic peptides.Most of the neutral non-cystine peptides were infraction -II, as would be expected (Consden,Gordon & Martin, 1948).

H,-E

Vol. 6o 549

m

A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI

Table 7. Peptidee8fromfractiom Ilac

Amino acids of oxidized peptides Probable structure of cystine peptidesGlu . Glu .Cy

CySO3H, Glu |G SCySO3H S

CyGlu . Glu .Cy

CySO3H, Ser, Val G lSCySO3H, Glu S

Ser.Val.Cyr ~~~Glu .Cy

CySO3H, Glu I SCySO3H S

Cy

CySO3H, Asp fCySO,H { S

Cy.AspGly. Illeu. Val. Glu. Glu.Cy

CySO,H, Glu, lieu, Val, Gly SCySO3H, Ser, Val S

I Ser.Val.Cy( Glu.Cy

CySO3H, Val SCySO3H, Glu S

I Val.Cy( Glu.Cy

CySO,H, Ser, Val SCySO,H, Glu S

I Ser.Val.CySame as peptide C3.Same as peptide D2.

CySO,H, Ser, ValCySO,H, ValCySO,H, GluCySO,H

CySO,H, Ser, ValCySO,H, ValCySO8H, GlyCySO3H, Giu

CySO3H, Ser, ValCySO3H, Glu, ValCySO,H, Glu, Ala

CySO,H, AlaCySO3H, Gly

CySO,H, Ser, ValCySO3H, GiyCYS03H

I955

CY CyS S

s S

Glu.Cy.Cy + Glu.Cy.CyS S

S S

Ser.Val.Cy Val.CySame as peptide D2.

Cy.Gly Cy.Gly~~ ~~8S

I S SGlu.Cy.Cy + Glu.Cy.Cy

S S

Ser.Val.Cy Val.CySame as serine peptide F3.

Cy.Gly

Cy.AlaCy.GlyS

~ S

Ser.Val.Cy

550

Band(Fig. 14)

AaAb

BlaBlb

ClaClb

C2aC2b

C3aC3b

D IaDlb

D2aD2b

D3El

FlaFlbFlcFld

F2

F3aF3bF3oF3d

GIG2aG2bG2c

HaHb

JaJbJo

THE DISULPHIDE BONDS OF INSULIN

Table 7 (cont.)

Amino acids of oxidized peptidesCySOsHCYS03H I

CySO3H, GlyCYS03H

CySO,H, AlaCySO8H

CySO8H, Ser, ValCySO3H, ValCySO3HCySO3H

CySO,H, Ser, ValCySO,H, Val, LeuCySO3H, Gly

CySO3H, Ser, ValCySO,H, AlaCySO3H, LeuCySO3H, Ala

CySO3H, Ser, ValCySO3H, Leu (weak Val)CySO3H

Probable structure of cystine peptides

Cy.GlyS

S

CyCyS

S

Cy.AlaCyS

S

Cy.CyS

Ser.Val.Cy

I

MainlyLeu.Cy

S

S

Cy.AIa

Leu.CyS

S

SCy.oy

S

Ser. Val!. Cy

CYS

S

+ Cy-CyS

Val.Cy

(I2H)

Leu.CyS

S

+ Cy.Cy

S

Val. Cy

Fig. 11 shows the ionophoretic separation offraction 1x at pH 6-5. The main neutral bandIlacI was eluted and iefractionated at the same pHfor a longer time (12 hr.) when it separated into a

number of different fractions (Fig. 12). The acidiccystine peptides (B-G) were not adequatelyseparated and were refractionated by ionophoresisat pH 3-6, as also were most of the neutral peptides(Fig. 13). Fig. 14 shows the ionophoretic separa-

tions at pH 3-6 of the oxidation products of thevarious cystine peptides. Not all the ionophoreseswere in fact run for exactly 2 hr. but the diagramhas been drawn as if they were, so that all samplesof a particular peptide are found on the same

vertical line. After ionophoresis the variouspeptides were identified by their amino acid com-

position and the results are given in Table 7.The various peptides encountered in this work are

listed in Table 5 together with the distance theymove on ionophoresis atpH 3-6 under the conditionsof Fig. 14. A number ofthese peptides had not beenencountered in previous work with the oxidizedchains, and were only revealed by the more efficientionophoretic method used here. Thus Glu. Glu. -

CySO3H (IlocAa) was identified as a peptide con-

taining a high proportion of glutamic acid withcysteic acid and moving considerably slower on

ionophoresis than Glu. CyS03H. Glu. CySO3H. -

CySO,H (IlxFlc, IIcxF3d) was a peptide moving atthe characteristic rate of peptides having tworesidues of cysteic acid, and CySO3H . CySO,5H(IlxL1d, I1xMlc, etc.) was found as a band movingfaster than free cysteic acid but giving only cysteicacid on hydrolysis.From their ionophoretic rates and composition it

was possible to identify the cysteic acid peptides,and the structures of the cystine peptides given inTable 7 were deduced from them. Where a peptidewith two cysteic acid residues was produced on

oxidation it was not possible to deduce the structureof the original cystine peptide from the experi-mental results and the formulae given were derivedwhen the structure of insulin was known.During the purification of the cystine peptides at

pH 3-6 (Fig. 13), weak CN-nitroprusside-reactingbands were frequently observed, moving towardsthe anode or slowly towards the cathode. Theiracidity suggested that they were cysteic acid pep-

tides. As they also contained an intact -S.Sbond, they were probably derived from the pep-

551Vol. 6o

Band(Fig. 14)KlaKlb

K2aK2b

K3aK3b

LlaLlbLleLld

L2aL2bL2c

L3aL3bL3¢L3d

MlaMlbMlo

A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI

tides containing the Cy .Cy sequence by partialoxidation during the purification procedure. IIxKlis probably such a peptide. The others were too faintto investigate.

Fig. 15 shows the ionophoresis of fraction I1,B.

l~ ~ ~ ~~I .w -L

+ 0 5 10 15i 20 25 -Distance (cm.) travelled from origin towards cathode

Fig. 15. Ionophoresis of fraction Il,: no. 3 paper; pH 6-5;33v/cm.; 6 hr.

The two basic peptides B and C were not present insufficient concentration to refractionate, so wereoxidized directly and subjected to ionophoresis atpH 3*6 (Fig. 16). The results are given in Table 8.It is probable that neither of these were pure butconsisted of the peptides shown in Table 8.

L-~ ----'D E+ 0 5 10 15 20 25 30_Distance (cm.) travelled from origin towards cathode.

Fig. 17. lonophoresis of fraction I, A (Fig. 15): no. 3paper; pH 3'6; 29v/cm.; 7 hr.

Band(Fig. 15)IB Im a

C Tt~~WC4 4

a b:c d*- I A .-I .-a

-10 5 0 5 10 15Distance (cm.) travelled from origin

+

Fig. 16. Ionophoresis of oxidation products of fractionsI1,B and I1,C (Fig. 15): no. 4 paper; pH 3'6; 29v/cm.;1 hr. (see Table 8).

, 12 30 5 10 15 20 25

Distance-(cm.) travelled frpm originFig. 18. One-dimensional chromatogram of fraction IliE

(Fig. 17): no. 3 paper; n-butanol-acetic acid-water(4:1:5, by vol.). Run for 36 hr. at 250 and solvent allowedto drip off the bottom of the paper.

Table 8. Peptide8fromfraction IfI

Amino acids of oxidized peptidesNo CySO3HCySO3H, Gly, His, LeuNo CySO3HCySO8H, AlaCySOaHNo CySO3HCySO,3H, His, Leu,(weak Gly)No CySO3HCySO3H

CySO3H

CySO,H, Glu, Ser, Gly(all very weak)

CySO3H

CySO3H, AlaCySO3H

CySO3H, Ser, ValCySO3H

CySO,H, LeuCySO3H

I

f

I-

Structure of cystine peptideHis. Leu. Cy His.Leu.Cy.Gly

S + S

S S

Cy. Ala Cy.Ala

His.Leu.CyS

S

Cy

His.Leu.Cy. Gly+ S

S

Cy

CyS

S

Cy

Cy.. ~~S

S.Ala.Cy

CyS

S

Ser.Val.CyLeu. Cy

S

S

Cy* Peptide E 2a moved considerably faster on ionophoresis at pH 3.7 (Fig. 19) than CySO3H.Ala (e.g. in peptide IllK 3a).

It seems probable that it is Ala. CySO3H formed by inversion during hydrolysis. This reaction has in fact been shown tooccur under similar conditions (Tuppy & Bodoi 1954).

Band(Figs. 16, 19)

BaBbBcBdBeCaCb

CcCd

D

Ela

Elb

E 2a*E 2b

E3aE3b

E4aE4b

552 I955

THE DISULPHIDE BONDS OF INSULINThe main neutral band I1,BA was refractionated

by ionophoresis atpH 3 6 (Fig. 17) to give two bandsD and E. D was free cystine, whereas E was furtherfractionated by one-dimensional paper chromato-graphy in butanol-acetic acid (Fig. 18). Fig. 19shows the ionophoresis of the oxidized peptides andthe results are listed in Table 8.

Band of cystinereptides(Figs. 17, 18)D

El-la b

E2 Na

E3a

E4a

-O 5 10 15 20 25 3--Distance (cm.) travelled from origin towards anode

Fig. 19. Ionophoresis of oxidized peptides from IlflA(Figs. 15, 17, 18): no. 4 paper; pH 3-6; 29 v/cm.; 2 hr.(see Table 8). All bands coloured with ninhydrin.

+

20 gn-0o(U

oo 015

%.- C10 10 .0a r-> 0

-S64

C-5Q

-25 20 15 10+Distance (cm.) travelled from origin towards

cathode during first ionophoresis (pH 3-6; Fig. 2)

Fig. 20. Diagram of two-dimensional ionophoresis ofpartial acid hydrolysate ofinsulin (Expt. 12). First run as

shown in Fig. 2. Second run: pH 6-5; 33v/cm.; 10 hr.;spots located by ON-nitroprusside test.

Experiment 12. Fig. 20 shows the distribution ofthe cystine peptides in the two-dimensional iono-phoresis experiment, and Fig. 21 is a diagram of theionophoreses of their oxidation products at pH 3-6showing the distribution of the cysteic acid peptidesproduced. Some of these were eluted, hydrolysedand their amino acid composition determined. Theresults and the deductions made are summarized inTable 9. The probable structure ofmost of the otherpeptides could be deduced from the position of thecysteic acid peptides on the pH 3*6 ionophoresis.

Spot ofcystine peptides(Fig. 20)AlTBIM

a, ; i

E I

F ia b

G7H

ta Dt__ _

a bj~~~~~~~L-

Q 5 .10 15 20 25 30 35 40Distance (cm.) travellid from origin towards anode +

Fig. 21. Ionophoresis of cysteic acid peptides fromExpt. 12: no. 4 paper; pH 3-6; 29v/cm.; 2-3 hr. (seeTable 9).

DISCUSSION

A good fractionation ofneutral cystine peptides wasobtained by ionophoresis at pH 6-5 for a relativelylong time (Figs. 12, 20). This would appear to be dueto the unusually low pK of the amino groups ofcystine residues in peptides. Greenstein, Klemperer& Wyman (1939) found values of 6-36 and 6-95 forthe pK' values of the amino groups of cystylbis-glycine, whereas the corresponding values forbisglycylcystine were more normal (7-94 and 7-94).Thus peptides such as

Cy .AlaS

S

Cy. Gly

(Fig. 20, spot L)

are somewhat acidic at pH 6-5 owing to theamino groups being partially discharged and

Vol. 6o 553

cG( F

Cj7 cQ)

A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI

(FigiBand

Table 9. Peptide8 from Expt. I2

s. 20, 21) Structure of cysteic acid peptideAa Tyr. Leu. Val. CySO,H (B2az21)Ab Leu . Val . CySO3H (B la8)Ac Ser. Val. CySO3HAd Tyr. CySO3H (A la7)Ae Leu. CySO,H + Val.. CySO3H

Ba Leu.Val. CySO,H (Bloc8)Bb Tyr. CySO,3H (A la7)

Ca Ser . Val. CySO3HCb Val. CySO3H

D Same as band IlaLlE Same as band IlxMlF Same as peptide I laK2 + I laK3G Same as peptide IlocK3

Ha CySO.H.AlaHb Leu.CySO3H

Ia Leu .CySO3H. Glylb CySO,H .Ala

J Peptide I LzJ

K -*

L Same as peptide I xH

Structure of cystine peptideMixture of peptides of type

CySS

X.CyLeu .Val.Cy

1~~~~~~~I Tyr.Cy[ ~~~~~Val.Cy

f Peptide IlfPE3+ s

Cy

Cy.Ala

I Leu.CyCy.Ala

I SS

Ser.Val .Cy

SProbably (} .Cy.Gly

Cy.Gly

* The cysteic acid peptides were not hydrolysed, but the structure was deduced from their ionophoretic rates.

move relatively rapidly on ionophoresis, peptidessuch as Cy.Ala

SS (Fig. 20, spot H)

Leu.Cy

containing only one free amino group of cystine areless acidic, whereas those like

Leu .Val . CyS (Fig. 20, spot B)

Tyr . Cy

are almost neutral. In this way the neutral cystinepeptides could largely be separated from each otherand also from other neutral peptides not containingcystine.Two main cystine-containing peptides (1c83 and

Icy, Table 2) were present in the water-solublefraction of the chymotryptic hydrolysate. Theiroxidation products had been encountered in studies

on the fractions A and B, and their structures,which are shown in Table 2, establish the presence ofa disulphide bridge joining the half-cystine residuesin positions A 20 and B 19. This is confirmed by theresults with the crude 'tryptic' hydrolysate, inwhich

Leu . Val . Cy. GlySSCy .AspNH2

and Leu.Val . Cy . Gly. GluSSCy .AspNH2

(Ixy2)

(Ix#4)

were detected and with the acid hydrolysate inwhich

Leu .Val . Cy

S (12B)Tyr. Cy

55;4 I955

I

THE DISULPHIDE BONDS OF INSULIN

was found. In the chymotryptic hydrolysate, theremaining cystine residues are present in theinsoluble 'core' and in the crude 'tryptic' hydro-lysate they are present in peptides Ixvq and Ix82 inwhich CyS. Gly. Ser and Leu. CyS. Gly. Ser res-pectively are coupled with CyS . CyS . Ala. Ser . Val. -CyS. Neither enzyme preparation split between thethree CyS residues of the glycyl chain.A number of neutral peptides from the acid

hydrolysates gave CySO3H .Ala on oxidation.These were:

Cy. GlyS

S

Cy . Ala

(IlaoH)

Leu. Cy

s (12H)Cy .Ala

Leu. Cy. Gly

and S (12I).Cy .Ala

CySO3H.Ala was found in the basic fraction I1,Blinked with His.Leu.CySO3H and His. Leu. -

CySO3H . Gly. All these peptides establish that thereis a disulphide bond between positions A 7 and B 7.

This only leaves the half-cystine residues inpositions A 6 and A 11 to be considered and theymust therefore be joined together. This is proved bythe presence of relatively high concentrations of theacidic peptidesSer. Val.Cy Val.Cy

S (IlaD2) and S (IlaDl)Glu.Cy Glu.Cy

and confirmed by the peptidesGlu. Glu. Cy

S (IlaBl)Ser. Val . Cy

Gly . Ileu .Val. Glu . Glu. Cy

and s (IlacC3)

Ser. Val. Cy

A considerable number of peptides containing theCy .Cy sequence were detected. Since theirstructures could all be explained on the basis of theabove results, they added confirmation and thefact that no peptides were detected which could notbe explained showed that no appreciable disulphideinterchange had occurred during the experiments.The results are summarized in Table 10, which

gives the complete structure of insulin. Thisstructure contains a ring, in which the disulphidebond in positions A 6-A 11 is included. Clearly toform such a bond the polypeptide chain must befolded in such a way that the two half-cystineresidues are close together and this fact must be

0

E-

EH

fw

E-4

EH

Z~~~~~

Ps0

-

0

3-o a

- .

41 1

Z .5

Vol. 6o 555

556 A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI I955taken into account when considering possibleconfigurations for the polypeptide chain in thisarea. It is interesting that the ring is the same sizeas the similar disulphide ring found in oxytocin(Tuppy & Michl, 1953; du Vigneaud, Ressler &Trippett, 1953) and vasopressin (Acher & Chauvet,1953; du Vigneaud, Lawler & Popenoe, 1953),which suggests that it may have a possible structuralor biological significance.

SUMMARY

1. Insulin was subjected to partial hydrolysiswith chymotrypsin, with a crude pancreatic extractand with acid under conditions in which the di-sulphide bonds were stable.

2. Cystine-containing peptides in the hydroly-sates were separated and their structure determinedafter oxidation to cysteic acid peptides. Paperionophoresis at high potential gradients in pyridine-acetic acid buffers was found useful for the separa-tions.

3. From the structure of the cystine peptides thedistribution of the disulphide bonds of insulin wasdeduced and is shown in Table 10.

A. P.R. is indebted to the Medical Research Council fora Scholarship for training in research methods.

REFERENCES

Acher, R. & Chauvet, J. (1953). Biochim. biophy8. Acta, 12,487.

Acher, R. & Crocker, C. (1952). Biochim. biophys. Acta, 9,704.

Butler, J. A. V., Dodds, E. C., Phillips, D. M. P. & Stephen,J. M. L. (1948). Biochem. J. 42, 116.

Consden, R., Gordon, A. H. & Martin, A. J. P. (1948).Biochem. J. 42, 443.

Craig, L. C., Gregory, J. D. & Hausmann, W. (1950).Analyt. Chem. 22, 1462.

Du Vigneaud, V., Lawler, H. C. & Popenoe, E. A. (1953).J. Amer. chem. Soc. 75, 4880.

Du Vigneaud, V., Ressler, C. & Trippett, S. (1953). J. biol.Chem. 205, 949.

Fredericq, E. (1953). Nature, Lond., 171, 570,Greenstein, J. P., Klemperer, F. W. & Wyman) J. (1939).

J. biol. Chem. 129, 681.Harfenist, E. J. & Craig, L. C. (1952). J. Amer. chem. Soc.

74, 3087.Kupke, ID. W. & Linderstr0m-Lang, K. (1954). Biochim.

biophys. Acta, 13, 153.Michl, H. (1951). MIh. Chem. 82, 489.Peters, J. P. & Van Slyke, D. D. (1932). Quantitative Clinical

Chemi8try. Vol. 2, p. 385. London: Bailliere, Tindall andCox.

Ryle, A. P. & Sanger, F. (1955). Biochem. J. 60, 535.Sanger, F. (1949a). Biochem. J. 44. 126.Sanger, F. (1949 b). Biochem. J. 45, 563.Sanger, F., Smith, L. F. & Kitai, R. (1954). Biochem. J.

58, vi.Sanger, F. & Thompson, E. 0. P. (1953a). Biochem. J. 53,

353.Sanger, F. & Thompson, E. 0. P. (1953b). Biochem. J. 53,

366.Sanger, F.,Thompson, E. O. P. &Kitai, R. (1955). Biochem,J,

59, 509.Sanger, F. & Tuppy, H. (1951 a). Biochem. J. 49, 463.Sanger, F. & Tuppy, H. (1951 b). Biochem. J. 49, 481.Toennies, G. & Kolb, J. J. (1951). Analyt. Chem. 23, 823.Tuppy, H. & Bodo, G. (1954). Mh. Chem. 85, 807.Tuppy, H. & Michl, H. (1953). Mh. Chem. 84, 1011.

The Structure of Pig and Sheep Insulins

BY H. BROWN,* F. SANGERt AND RUTH KITAIBiochemical Laboratory, University of Cambridge

(Received 26 November 1954)

Insulins from a variety of different animal speciesshow the same biological activity (Scott & Fisher,1940) and immunological behaviour (Wasserman &Mirsky, 1942). They have the same crystalline formand mixtures of different insulins behave as a singlesubstance in the phase-rule solubility test (Lens& Evertzen, 1952). However, in a preliminarychemical study of pig and sheep insulin it wasshown that, whereas the general structure wassimilar, there were certain differences in individual

amino acid residues (Sanger, 1949 b). Harfenist &Craig (1952) have recently analysed the differentinsulins, and found differences in the contents of theamino acid residues given in Table 1. No differenceswere found for the other amino acids.The complete sequence of amino acids in cattle

insulin has 'recently been determined in thislaboratory (Sanger & Tuppy, 1951 a, b; Sanger &Thompson, 1953a, b), and the present paperdescribes similar studies on pig and sheep insulins.Since the larger part of the molecule was the samefor all three species it was justifiable not to deter-mine the amino acid sequence unequivocally ineach case, but only to identify lower peptidesembodying each residue in the molecule. Where

* Damon Runyon Cancer Research FeHow. Presentaddress: Department of Surgery, University of Wisconsin,U.S.A.

t Member of the Scientific Staff of the Medical ResearchCouncil.