Vol. 68 fl-NITROPROPIONIC ACID FROM P. ATROVENETUM 653clear pale-yellow in colour, gradually darkened to clearorange-yellow after 12 days' incubation.The quantitative examination of the mould cultures was

carried out in a similar way to that described in section A,but with the following variations: (a) The residual N in theculture filtrate other than NH3-N (Table 5, column 5) wasnot estimated. (b) Residual glucose in solution was esti-mated polarimetrically after removal of tartaric acid withbasic lead acetate solution (Table 6, column 3). The resultsobtained are given in Tables 6 and 7.

SUMMARY

1. P-Nitropropionic acid, CH2(NO2) *CH2 *CO2H,has been shown to be a major nitrogenous meta-bolite of cultures of Penicillium atrovenetum G.Smith grown on two chemically defined or 'syn-thetic' culture media containing, as sole sources ofnitrogen, the ammonium salts of tartaric, phos-phoric and sulphuric acids in the one medium andsodium nitrate in the other.

2. Over 60% of the ammonia nitrogen that wasmetabolizedbythemouldforpurposesotherthanthesynthesis of its mycelium was recovered as crystal-line f-nitropropionic acid. A maximum yield of2-4 g. of this acid/l. of culture filtrate was isolated.

3. f-Nitropropionic acid is formed in theearliest stages of growth of the mould, reaches amaximum amount before the mould is fully grownand thereafter decreases.

4. The yield of ,-nitropropionic acid obtainedfrom the medium containing nitrate was only about10% of that isolated from the medium in whichammonium salts constituted the sole source ofnitrogen.

5. P-Nitropropionic acid has now been isolatedfrom several different species of plants and micro-organisms, so that it seems probable that the acidplays a significant, but at present undefined, partin their nitrogen metabolism.

We wish to thank the Agricultural Research Council fora grant in aid of this work which enabled one of us (A. S.)to take part in it. Our thanks are also due to ProfessorHenry Rapoport, who, while a guest worker in thisDepartment, kindly arranged for the recorded analysis of,-nitropropionic acid to be carried out by the Micro-chemical Laboratory, University of California, Berkeley,U.S.A.

REFERENCES

Barton, D. H. R., de Mayo, P., Morrison, G. A., Schaeppi,W. H. & Raistrick, H. (1956). Chem. & Ind. p. 552.

Bush, M. T. & Goth, A. (1943). J. Pharmacol. 78, 164.Bush, M. T., Goth, A. & Dickison, H. L. (1945). J. Pharma-

col. 84, 262.Bush, M. T., Touster, 0. & Brockman, J. E. (1951). J. biol.Chem. 188, 685.

Carrie, M. S. (1934). J. Soc. chem. Ind., Lond., 53, 288T.Carter, C. L. (1943). J. Soc. chem. Ind., Lond., 62, 238T.Carter, C. L. & McChesney, W. J. (1949). Nature, Lond.,

164, 575.Gorter, K. (1920). Bull.Jard.bot. Buitenz., series III,2, 187.Gresham, T. L., Jansen, J. E., Shaver, F. W., Frederick,M. R., Fiedorek, F. T., Bankert, R. A., Gregory, J. T. &Beears, W. L. (1952). J. Amer. chem. Soc. 74, 1323.

Hirata, Y., Okuhara, K. & Naito, T. (1954). Nature, Land.,173, 1101.

Merck Index (1952). The Merck Index of Chemicals andDrugs, 6th ed., p. 68. Rahway, N.J.: Merck and Co.

Morris, M. P., Pag4n, C. & Warmke, H. E. (1954). Science,119, 322.

Nakamura, S. & Shimoda, C. (1954). J. agric. chem. Soc.Japan, 28, 909.

Neill, K. G. & Raistrick, H. (1957). Biochem. J. 65, 166.Rebstock, M. C., Crooks, H. M. jun., Controulis, J. &

Bartz, Q. R. (1949). J. Amer. chem. Soc. 71, 2458.Rosett, T., Sankhala, R. H., Stickings, C. E., Taylor,M. E. U. & Thomas, R. (1957). Biochem. J. 67, 390.

Shimoda, C. (1951). J. agric. chem. Soc. Japan, 25, 254.Sidgwick, N. V. (1942). The Organic Chemistry of Nitrogen,

p. 241. Ed. by Taylor, T. W. J. and Baker, W. B.Oxford University Press.

Smith, G. (1956). Trans. Brit. mycol. Soc. 39, 111.

The Separation of Amino Acids and their N-Acetyl Derivativesby Paper Chromatography and Paper lonophoresis

BY J. K. WHITEHEADBriti8h Empire Cancer Campaign, Radiochemical Laboratory, Barnato-Joel Laboratories,

The Middlesex Hospital Medical School, London, W. 1

(Received 20 June 1957)

The success of any method of analysis by isotopic-dilution techniques in which labelled reagents areemployed is dependent upon the complete separa-tion of the derivatives formed both from each otherand also from any excess of the reagent used.During the development of such a method for thedetermination of sub-microgram quantities of

amino acids and peptides, which uses acylatingagents labelled with 3H and 14C and separation ofthe acetyl derivatives by two-dimensional chro-matography, it was found that traces of activeacetate ion appeared in a vital area of the chro-matogram. A satisfactory separation of the N-acetyl amino acids is obtained by chromatography

J. K. WHITEHEADin one direction and ionophoresis in a seconddirection. Under these conditions, the acetateimpurity moves to an area on the paper wellremoved from the other components ofthe mixture.A similar difficulty was experienced with the

spread of active inorganic iodide ion on paperchromatograms in the separation of ""I-labellediodo-amino acids in hydrolysates of thyroid tissues(Gross & Leblond, 1951). With the ionophoretic-chromatographic techniques, the iodide migratesas a discrete spot well ahead of the other activecompounds in the mixture during the ionophoresis.The apparatus described in this paper was

originally developed for the two-dimensionalionophoretic separation of peptide materials attwo different pH values, and it resembles theapparatus used by several authors (e.g. Durrum,1950, 1951; Flynn & de Mayo, 1951; McDonald,Urbin & Williamson, 1951) in which the filter paperis suspended in a moist chamber. The evaporationof water from both surfaces of the paper saturatedwith electrolyte is reduced as far as possible bysupporting protecting papers just above and belowit. These protecting papers are kept saturatedwith water and cooled by banks of cooling tubes.

EXPERIMENTAL

Apparatu8

The apparatus consists essentially of three compartments:two similar electrode chambers connected by a middlecompartment containing the supports for the papers andthe racks of cooling tubes. The main casing of the ap-paratus is made of wax-impregnated wood and the otherparts are mostly constructed of insulation Tufnol board(Tufnol Ltd., Perry Barr, Birmingham) or glass sheet.Perspex sheet would have been ideal for construction, butwas not readily available when the apparatus was originallydeveloped.The essential features of the apparatus are shown in

Fig. 1. Each electrode compartment (20 in. x 4 in. x 2 in.)contains two glass troughs of the type used in chromato-graphy. The lower troughs (1) (20 in. x 1 in. diam. with18in. x i in. slot) arefilled with water to serve the wick end-papers which keep the lower protecting paper saturated.The upper troughs (2) (22 in. x 1 in. diam. with 18 in. x * in.

slot) contain the electrolyte solution and extend at eachend through holes bored through the sides of the compart-ments. Perspex rods (3) around which the platinum-wireelectrodes (30 in. x Io in. diam.) are spiralled are held inposition by rubber stoppers at each end of the troughs.Holes (j in. diam.) are blown in the top of each trough tofacilitate the changing and levelling of the electrolyte.The middle compartment (24 in. x 20 in. x 4 in.) is

fitted with a copper drip-tray (4) to catch any water con-densing on the lower set of cooling tubes (5). This part ofthecooling system consists of 32 glass tubes (A?, in. externaldiam., i in. apart) running transversely across the chamber.A glass plate (6) (20 in. x 16 in. x 1 in.) rests on the tubesand supports three Whatman no. 3MM filter papers whichare kept saturated with water by the wick end-paperswhich dip into the lower troughs in the electrode compart-ments. A I in. mesh made of linen thread (Chadwick'sHeavy Stitching Thread no. 20) impregnated with siliconegrease (Dow Corning High Vacuum Grease) is suspendedj in. above the glass plate and is supported on two Tufnolcross-members and the sides ofthe compartment. The paperon which the ionophoresis is carried out is laid on thisthread mesh overlapping (approx. 2 cm.) and resting on, ateach end, wick papers (40 cm. x 11 cm.) which bend over,pass through slits (18 in. x 1 in.) cut in the Tufnol strips(7) and dip into the electrode troughs.A second mesh (i in. grid) is suspended i in. above the

mesh already described and is supported across a woodenframe extending over all three compartments (25 in. x20 in.). Two Whatman no. 3MM filter-paper sheets(42 cm. x 58 cm.) rest on the thread mesh and are moistenedwith water before each run. Immediately above this upperprotecting paper is a set of 40 cooling tubes (8) (Ain.external diam., i in. apart) supported on the same frame-work as the upper mesh. The delivery tubes for the coolingwater pass through the back of the compartment and arejoined to the tubes on the frame by rubber tubing. Theserubber connexions act as hinges for the upper framework,which can thus be raised to facilitate the insertion of theionophoresis paper before an experiment. The compart-ment is closed at each end by wooden side pieces (9) to thebase of which are screwed the two Tufnol strips (7). The lidof the apparatus consists of a sheet of plate glass (10)(26 in. x 21 in. x i in.).Water maintained at about 10° below room temperature

by periodic additions of ice is circulated from a laggedstorage tank (capacity 8 gallons) by a Stuart Turner pump.The flow is controlled by a Sunvic temperature regulatorthrough a relay switch connected to the motor of the pump.The regulator stem passes through holes bored through the

Fig. 1. Diagram of apparatus in longitudinal vertical section. Numbers are those used in the text.

654 I958

SEPARATION OF AMINO ACIDSfront of the cabinet of the apparatus and the front memberof the upper framework supporting the cooling systembetween the two central tubes. The control is set so that thethermometer laid on the upper protecting papers near tothe regulator stem shows a constant temperature of 50below room temperature.Smoothed rectified current is supplied to the electrodes

from a conventional power pack capable of giving 1200v at60 mA. The circuit includes a variable resistance, volt-meter and milliameter.

General techniqueOne-dimensional ionophoresis. The mixtures are applied

in the usual way from a micropipette at points marked3 cm. along a pencil line drawn across the paper (Whatmanno. 2, 58 cm. x 36 cm.). The starting line is usually thecentral axis of the paper, but where all the ions move in thesame direction it is often more convenient for the mixturesto be applied further from the electrode to which the ionsmigrate, thus allowing a longer period of separation.Pencil lines are drawn across the paper parallel to thestarting line and at a distance of j in. on either side. Thepaper is suspended vertically from one end over a drip trayand the area below the starting line is saturated with theappropriate buffer solution by slowly drawing the tip of afilled 50 ml. pipette across the lower parallel pencil line andallowing the electrolyte to flow down the paper and draininto the tray. The excess of buffer remaining on the loweredge of the paper is removed by blotting with dry filterpaper. The paper is inverted and the saturation processrepeated. It is found that with practice this manipulationcan be performed so that the electrolyte fronts meet on thestarting line and very little distortion of the spots occurs.The saturated paper is laid on the thread grid so that

each end overlaps (approx. 2 cm.) and rests on the twoelectrolyte wick papers. The surfaces adhere well togetherand form a satisfactory electrical connexion. The coolingrack is lowered into place, the temperature regulator in.serted, the glass lid restored and the appropriate potentialdifference applied across the electrodes.When ionophoresis follows chromatography the paper is

marked in pencil in the same way as described above. Themixture is applied at a point on the starting line 6 cm.from one edge, and ohromatographed along this axis. Afterdrying, the paper is saturated with electrolyte as describedabove and the ionophoretic separation performed in thesecond dimension. If volatile electrolytes are used, theorder of performing the separations may be reversed.

Two-dimensional ionophoresis. The two starting axes aremarked in pencil on the paper (40 cm. x 58 cm.) togetherwith sets of parallel guide lines as described above. Avolatile buffer (e.g. acetic acid, ammonium acetate,ammonium carbonate, ethylamine acetate, trimethylamine

Fig. 2. Method of marking the paper for the determinationof electrolyte flow. Sucrose is applied at the pointsmarked with small circles.

acetate) of the appropriate pH is used to impregnate thepaper by using the shorter guide lines. Ionophoresis alongthe longer axis is carried out. The paper is removed fromthe apparatus and dried first in a stream of warm air andfinally in the oven. The dried paper is reduced in length to40 cm. The size of strip which is removed from either end ofthe paper in order to leave the separated components of themixture on the remaining square is determined by previousexperiment. The paper is impregnated with a second buffersolution by using the guide lines parallel to the axis of thefirst separation, and placed on the thread grid of theapparatus in the usual way so as to overlap electrolytewick papers which have been increased to the appropriatelength. Ionophoresis is carried out in the second dimension.

Experiments to trace electrolyte movement during iono-phoresis. The movement of sucrose spots placed at differentsites on the paper was followed during ionophoresis as ameans of determining the flow of electrolyte in the paperunder a variety of experimental conditions.Two sheets of Whatman no. 2 paper (58 cm. x 18 cm.)

were marked with longitudinal pencil lines spaced 2 cm.apart. Lines at right angles to these parallels were drawnat 5 cm. intervals on either side of the centre axis as shownin Fig. 2. The papers were saturated with electrolytesolution and placed, 1 cm. apart, on the grid of the appar-atus with the ends resting on the wick papers in the samemanner as described above. A few microlitres of sucrosesolution (5%, w/v) were applied from a capillary pipetteto the mid-points of the cross-lines (marked O in Fig. 2).After innophoresis, the papers were removed from theapparatus and dried in a current of warm air. The sugarspots were developed by spraying the paper with a freshlyprepared mixture of equal volumes of 0-2% (w/v) naphtha-resorcinol in ethanol and 2% (w/v) trichloroacetic acid inwater (Partridge & Westall, 1948). The distance (x cm.)travelled by each spot was measured and plotted againstthe distance (xo cm.) of the origin from the centre line.The potential gradient was determined by measuring the

voltage between points 25 cm. on either side of the centreline with an Avo Testometer. Resistances were determinedwith a Mullard measuring bridge (type E. 7555). A strip ofWhatman no. 2 paper (58 cm. x 36 cm.) was saturated withelectrolyte solution and placed between glass plates. Theends of the paper were allowed to dip into the electrodevessels of the apparatus, which had been removed for thispurpose. The levels of the electrolyte in the vessels weremadeto coincide with lines drawn across the paper at a distanceof 50 cm. apart. The cross-sectional area of the electrolyteon the paper was determined by weighing the papers beforeand after saturation. The weight of electrolyte so obtainedwas divided by the density of the solution and the length ofpaper.

Separation of amino-acidsAmino acids. Commercial samples of amino acids

(British Drug Houses Ltd. or Roche Products Ltd.) wereused without further purification. Stock solutions (1 mg./ml.) were made up in 0-1 N-HCI. Monoiodotyrosine and tri-iodothyronine were prepared by the methods of Pitt-Rivers (1956) and Gross & Pitt-Rivers (1953).

Electrolytes. The solutions used were: 0-025 M-sodiumborate (pH 9.2); M-acetic acid (pH 2-0); ammoniumacetate soln., prepared by adding aq. 0-025N-NH3 soln. to0 025N-acetic acid to pH 9-2. The solution was stored in aglass-stoppered bottle.

Vol. 68 655

J. K. WHITEHEAD

Ninhydrin reagent. Ninhydrin (0 5%, w/v) in butanol-acetic acid (1: 1, v/v) was used.

Solvent system. Butanol-dioxan-aq. 2N-NH3 soln.(4:1:5, v/v) (Gross & Leblond, 1951) was used.

Details of procedure. Two-dimensional ionophoreticseparations are carried out with the origin at a point20 cm. from the centre of a line drawn longitudinallyacross the middle of the paper (58 cm. x 40 cm.). Separa-tion is carried out in the first dimension in M-acetic acidsoln. and with the paper placed in the apparatus so that theorigin is nearer to the anode. A potential difference of380v is applied across the electrodes (5.2v/cm.) for 17 hr.(overnight). After drying the paper, parallel pencil linesare drawn 10 cm. on the anode side of the shorter startingline and 30 cm. on the cathode side. The end pieces are

removed from the paper and the remaining square issaturated with the borate solution, or ammonium acetatesolution is placed on the grid of the apparatus so that thestarting line for the second separation is in the centre. Thewick papers from the electrode vessels are extended to theappropriate length. Ionophoresis is carried out overnightwith an applied voltage of 230v (3-2v/cm.). After drying,the paper is sprayed with the ninhydrin reagent. The usualcharacteristic coloured spots show up after heating for3 min. in the oven at 1100.The iodo-amino acids are separated by ascending chro-

matography with the butanol-dioxan-aq. 2N-NH3 soln.solvent system, followed by ionophoresis in 0-025 M-Na2B407 with an applied voltage of 230v (3.2v/cm.) across

the electrodes for 17 hr. The starting line for the iono-phoretic separation is 15 cm. nearer to the cathode fromthe centre of the paper.

Separation of N-acetyl amino acidsN-Acetyl amino acids. Samples of these compounds were

prepared according to the methods of Synge (1939) andGordon, Martin & Synge (1943). Stock solutions (1 mg./ml.) were made up in ethanol-aq. 2N-NH3 soln. (1:1, v/v).

Phenol-ethanol-benzene-water solvent. Liquid phenolB.P. (550 ml.), absolute ethanol (100 ml.) and benzene(100 ml.) were mixed, giving one phase.

Ethylamine acetate buffer. 01 M-Acetic acid soln. was

added to 0.1 M-ethylamine solution in water to pH 9.Bromocresol-green solution. Bromocresol green (British

Drug Houses Ltd.) (0-1 g.) was dissolved in 11. of A.R.acetone to which 5 ml. of 40% (w/ v) formaldehyde solutionhad been added.

Details of procedure. Ascending chromatography was

carried out in the phenol-ethanol-benzene-water solventsystem overnight at 250. No previous equilibration of thepaper with the solvent was found to be necessary. Iono-phoresis was carried out in ethylamine acetate buffer(pH 9) for 4 hr. with a potential difference of 600v (lOv/cm.) applied across the electrodes. The starting line was

placed 15 cm. nearer to the cathode. The paper was dried ina current of warm air, followed by 2 hr. in the oven at 1000to remove as much of the remaining traces of the ethyl-amine acetate as possible. The dried paper was allowed tostand in an atmosphere of formaldehyde in a glass tank for2 hr. The N-acetyl amino acids were shown up as yellowspots against a blue background by the dipping techniqueof Jepson & Smith (1953), with the bromocresol-greenindicator solution. The spots take up the colour of thebackground rapidly, although the use of formalin in the

indicator solution arrests this process long enough tomark the areas in pencil.Attempts to carry out the separations in the reverse

order were unFsuccessful owing to the difficulty of removingfrom the paper the last traces of ethylamine acetate, whichappeared to interfere with the chromatographic separationi.

RESULTS

All movements and measurements occurring in thedirection of the anode are designated as positiveand those occurring in the direction of the cathodeas negative.Kunkel & Tiseius (1951) have shown that, owing

to the intricate channelling of liquid that exists inthe paper, distances (cm.) (1 or d) measured on thesurface do not represent the true distance (1') ofvoltage drop or the true distance (d') travelled by adissolved particle through the paper. The factor 1/1'is determined by using the relationship 1'= Rqk inwhich R is the resistance of the paper, q the cross-

sectional area of the liquid channel and k the conduc-tivity of the electrolyte. For Whatman no. 2 paper,the value for 1/1' was found to be 0-61. Values ofpotential gradients given in this section are as v/cm.measured on the surface of the paper (i.e. v/l).

Movement of electrolyte on the paper

Effect of 8ite on the paper. Fig. 3 shows thecurves obtained when the displacement (x cm.)during constant time (t hr.) is plotted against the

on E

o o

~~~~~~t :D

0 0

1~ ~~~4 cI

C CO

-v

-a.-

16 12 8 4Distance of origin fromcentral line towardsthe cathode (cm.)

.7

6.5.4

-32

*1

Distance of origin fromcentral line towards

anode (cm.)

340J

84-'0

6 0 1-5

.7

0

Fig. 3. Variation of displacement of sucrose spots due toelectrolyte flow with distance of the origin from thecentral line. Horizontal scale: distance of origin fromcentral line (xo cm.). Vertical scale: displacement of spots(x cm.). Curve 1 (0), M-acetic acid; curve 2 (O),0.1M-ethylamine acetate; curve 3 (A), 0 025M-borax.Conditions of the experiment are those given in the textfor the routine separations.

656 I958

SEPARATION OF AMINO ACIDSdistance (xo cm.) of the original site from thecentral line of sucrose applied to the paper and runelectrophoretically as described in the Experi-mental section. The net displacement of all sucrosespots applied at sites other than the central axisis towards the middle line. This movement ofelectrolyte from the electrode vessels occurs owingto the evaporation ofwater from the surfaces of thepaper. The straight-line plots in Fig. 3 show thatthis movement is proportional to the distance xo.Sucrose applied to the central axis of the paper isdisplaced in all cases towards the cathode. Thismovement is due to endosmosis.The equation for the movement of the electro-

lyte is x=-AxO+B, (1)

where A and B are constants.Effect of applied heating load!. The slope of the

x/l0 plots (value ofA in equation 1) is a measure ofthe movement of the electrolyte caused by theheating effect of the current passing through thesolution. This flow of electrolyte is reduced byincreased cooling as shown by the decrease in slopeof the curves in Fig. 4 with decrease in temperatureof cooling water. As it was not possible to arrestthis movement completely by cooling methods,for routine separations moderate cooling at 50below room temperature was adopted.

Fig. 5 shows the plots of x/xo at different periodsof time (t hr.) for sucrose movement in 0025M-borax under standard conditions of applied voltageand cooling. Although sucrose forms a complex ion

Distance of origin fromcentral line towards the

cathode (cm.)

Fig. 4. Variation of displacement of sucrose spots due toelectrolyte flow with distance of the origin from thecentral line under different cooling conditions. Hori-zontal scale: distance of origin from the centre line(xo cm.). Vertical scale: displacement of spots (x cm.).Curve 1 (0), cooling water at 10 below room temper-ature; curve 2 (A), cooling water at 50 below roomtemperature; curve 3 (0), cooling water at 100 belowroom temperature. All experiments were conducted inO lM-ethylamine acetate with an applied potential of600v for 3 hr.

42

in borax solution and will show its own constantmobility towards the cathode in these experiments,this movement appears only in the second term ofequation 1. The slope of the lines in Fig. 5 is stilla measure of the flow of electrolyte due to theapplied heating load (w, mw/cm.2).

If xt (cm.) is the distance of the sucrose from thecentral line of the paper at time t then

(2)xt= xo + x,

and substituting in equation 1

x=(A/1+A) xt+(B/l+A).The results shown in Fig. 6 and Table 1 show

that the flow of electrolyte due to the appliedheating load is a linear function of time and thatthe rate of this movement is directly proportionalto the amount of heat energy (w) dissipated on thepaper. The value of the rate of movement/unit ofapplied heating load [A/(1 +A) tw=a] is shown inTable 1 to be a constant irrespective of the electro-lyte saturating the paper and with the apparatusused here has a mean value of 0025 cm./hr./mw/cm.2.The flow of electrolyte due to the applied heating

load is thus equal to awxtt cm.

ET

+4)4

-15sFig. 5. Variation of displacement of sucrose spots due to

electrolyte flow with distance of origin from the centralline, during ionophoresis in 0-025M-borax under standardconditions of applied voltage and cooling for differentperiods of time (0, 3 hr.;-, 4 hr.; A, 6 hr.; A, 9 hr.;o, 14 hr.; *, 17 hr.). Horizontal scale: distance oforigin from the central line (xo cm.). Vertical scale:displacement of spots (x cm.).

Bioch. 1958, 68

Vol. 68 657

(3)

J. K. WHITEHEAD

Effect ofendo8mo8i8. The second term in equation 3relates to the movement of electrolyte due toendosmosis. Table 2 gives the values for differentelectrolytes. The movement is towards the cathodein all the cases studied and is given by Cvt, whereC=B/(1+A) vt and v (v/cm.) is the appliedpotential gradient.

1 51-4

1-3 1

1 -2

1.1

11-0

,- 0-8

0-7

-; 0-6

0-5

0-40-3

0-2

0-1

/ I I I I I I I I0

I I I I I I I I0 2 4 6 8 10 12 14 16 18 20

Time (hr.)Fig. 6. Values of A/(I +A) obtained from the slopes of the

curves in Fig. 5 plotted against time (t).

lonophoretic 8eparation of amino acid8Calculation. Consider the movement ofa charged

ion with mobility U (cm./hr./v/cm.) placed on thecentre line of the paper under an applied potentialof v (v/cm.) for time t (hr.).Equation 3 for the electrolyte displacement may

be written x=a=, t +Cvt.The speed of electrolyte flow

dx= +awxt+ awt d-x+Cv. (4)dt dtThe net mobility of the charged ion on the paper= Uv ± dx/dt

dxt =Uv+ awxt+ awt dxt+ Cv, (5)dt dt

dxt awxt Uv Cv (6)dt 1-awt l-awt 1-awt

Solving equation (6)(l-awt) x = Uvt+Cvt+K, (7)

when t= 0, x= 0, then K = 0, and

Ui l-awt)xt a (8)vt

orUvt + Cvt

Xt (1l-awt)If the charged ion starts at a point xo from the

central line, then in equation 7 when t =0, xt=oand K = xo, and so equation 7 beconmes

xt(l-awt) = Uvt+ Cvt+x0.From equation 2, x,= xo+ x, and

U= (I-awt) xawxo C (10)vt v

9

or(Uvt + Cvt) awtx0(1-awt) (1-awt)

Table 1. Effect of applied heating load to the electrolyte flow in the paper

Symbols are those used in the text. The values for w are calculated from the values of 12R/area (area ofpaper=50 x 36 cm.2). _ A A

Electrolyte0-05M-Sodium carbonateN-Acetic acid0-Im-Ethylamine acetate0-025M-Borax

t(hr.)17176

I(mA)5-604-90

20-117 3-8517 5-8517 8-5017 12-0

104 R(ohms)3-719-703-653-833-833-833-83

w(mw/cm.2)

0-651-488-190-330-751-543-07

A-0-242-0-358-0-430-0-124-0-220-0-369-0-596

A(1+A) t- 0-0188-0-642-0-126-0-0083- 0-0166- 0-0335- 0-0868

A(1 +A) tw-0-029-0-022-0-016-0-025-0-022-0-024-0-025

Table 2. Mobility of electrolyte due to endosmos8iSymbols are those used in the text.

Electrolyte0-l -Ethylamine acetateM-Acetic acid0-05m-Sodium carbonate

t(hr.)6

1717

B(cm.)-2-30-2-00-1-75

A-0-430-0-358-0-242

v(v/cm.)10-05-02-1

B(1 +A) vt- 0-066-0-041-0-065

11958658

SEPARATION OF AMINO ACIDS

cS-~ 0 01 CO 0 c. 01-01C6_o4 1Co 01 c .ce

0 I Ir- .- -4 r

as e 0 lb 10 - CoS 0 0C) I; +.5

d^ CoXo 01 o 0sc-- Co0411CoIIII

J._

C Co C- CO

m -

O e0

^ o c;> o ~~~>40 --4

_ 0

C-

04

Qo Q

*;3 B

o ~o

a

C)

E .1*r= OPm

11

_ e CO 10

-. r- -4 r- P-

* . . . . . .

0- - MCOC

co

CO o*1 *0.1*

10o

10 0L-

Co Co Co Co Co Co Co Co

0 00

(

c9

1t0 10c

Bc; c;-Ei

_z~ qc

_0 bX

, sCo14

I_ to _qdC) 0 to

CO ~COI -~

CC-- C- C-C_

-ld t-

P- *4 *- .- *- P-

If x and xo have the same sign, i.e. the chargedion starts at a distance x0 cm. from the centre lineon the same side as the electrode towards which itwill migrate, then equations 10 and 11 hold for allvalues of t. If x and xo are of contrary sign (e.g. theseparation of amino acids in acid buffer and theseparation of N-acetyl amino acids in ethylamineacetate), then equations 10 and 11 hold only formovement from the origin to the centre line.Movements beyond this point are governed byequations 8 and 9.

Table 3 gives the displacements of a number ofamino acids in 0*025M-borax run electrophoretic-ally under various conditions found experimentallyand the corresponding values when calculated fromequations 9 and 11.

Consider the separation of two ions with mobili-ties Ua and Ub placed on the paper at x0 cm. fromthe central line and with displacements of xa andXb cm. respectively in time t hr. From equation 11,if x., b. and xo are of the same sign or if of contrarysign and x. <Xb<xo,

(U -Ub) vt

1- awt(12)

cX _~ Thus the separation is independent of the site oforigin provided that movement does not proceedbeyond the centre line. This is borne out withinexperimental error in the values for the displace-

o ments of some amino acids in 0 025M-boraxstarting at the centre and at points 5, 10 and 15 cm.

on the cathode side of the centre line given inTable 4.

.o Fig. 7 indicates the pattem obtained on a two-dimensional ionophoretogram made under the

conditions given in the Experimental section(M-acetic acid and 0.025M-borax). The abbrevia-

4 tions for the names of the amino acids shown inthe figures are those used by Brand & Edsall(1947).

Fig. 8 gives the chromatographic separation ofe. 00 the iodo-amino acids from a number of other amino

acids in butanol-dioxan-aq. NH3 soln. solventsystem and ionophoretically in 0-025m-borax in theother direction. The iodo-amino acid spots in Fig. 8are filled in black. Inorganic iodide ion moves off

st the paper during ionophoresis under the conditions

described.

Separation of N-acetyl amino acid8

Fig. 9 indicates the pattern obtained in separat-ing a mixture of N-acetyl amino acids chromato-graphically in phenol-benzene-ethanol-waterfollowed by ionophoresis in ethylamine acetate

>> solution as described in the Experimental section.Acetate ion runs well removed from the othercomponents of the mixture.

42-2

Vol. 68 659

S

0

10010

'.

... 0

0

00

0

0 '.

..1 .

S

C0,0

H

660 J. K. WHITEHEAD I958

Table 4. Diaplacements of 8ome amino acid 8pot8 on paper ionophoretogram88tartinJ at difference origin8 on the paper

Symbols are those used in the text. Experimental conditions: electrolyte, 0-025M-borax; pH =9-2; w= 1-54 mw/cm.2;v=3-2v/cm.; t=17 hr.

Displacement (cm.)

Amino acidAlanineGlutamic acidGlycineHydroxyprolineLeucineLysineMethionineThreonineTyrosine

xO=0 cm.-3.3+ 16-2-1-4-0-8-3-6-17-2+ 1-0+ 5-20-0

=- 5 cm.-0-7

+18-8+0-8+1-8-1-6-17-0+3-1+7-4+2-1

xO= -10 cm.+0-9+ 19-9+2-6+3-0+0-5-13-0+4-5+8-6+3-3

xO= -15 cm.+2-2

+21-6+3-4+4-4+1-4-11-4+5-4+ 10-3+5-1

Try-Glya aAlas-

Methionine Vatl 7

suiphone Met Leu

0 * * Thr Ty *pheGlu Cy2S2 MethionineCY2 2

sulphoxide Hypro

Asp

11I1I1I 1I I I Ii I II

E_

-o

10 -0

-o

0-0

co

4)

C

0

E

0

._2in

10 5 0Displacement towards the anode (cm.)

Fig. 7. Two-dimensional ionophoretogram of 18 aminoacids made under the conditions given in the Experi-mental section. The abbreviations for the names of theamino acids are those used by Brand & Edsall (1947).

DISCUSSION

Electrolyte flow in the paper and itseffect on ionophoretic 8eparation8

Under ideal conditions, with no evaporation ofwater from the electrolyte saturating the paper,there is a linear relationship between the distancemoved by a charged ion or particle during iono-phoresis, the time of current flow and the potentialgradient placed across the paper. In the moist-chamber type of apparatus these conditions rarelyoccur, although McDonald (1952) was successfulwith his water-jacketed apparatus filled with moisthelium to conduct the heat away from the paper.Mead (1955), using a similar sugar-grid method tothat described above for the determination ofelectrolyte flow during paper electrophoresis,showed that the rate of evaporation depends on thedifference between the temperature of the paper

1-0

0-9

0-8

07 Z

0O6 TTri-iodothyronine$ rx

j05 oThyroxine 04 o0-4

Phe* Leu]0-3 DMonoiodotyrosine Met0 Tr4 4 0-2

Di-lod,* T~Serf Tyr Yal -I|tyrosine Thr rQ pr9Gly>o I

15 10 5 0Displacement towards the anode (cm.)

Fig. 8. Separation of iodo-amino acids from 12 otheramino acids by chromatography (vertical axis) inbutanol-dioxan-aq. NH, soln. and ionophoresis (hori-zontal axis) in 0-025M-borax under the conditionsdescribed in the text. The iodo-amino acids are shown asfilled-in spots.

and that of the film of moisture on the cover of theapparatus. The evaporation from the free surfaceof the paper and the consequent electrolyte flowcould be diminished by cooling the glass plate onwhich the paper rested, in the apparatus describedby Mead (1955). The results shown in Fig. 4indicate that evaporation is reduced similarly inthe apparatus described here by increased cooling,although total elimination of this effect is notpractical. Durrum (1951) showed that the electro-lyte flow due to evaporation occurs from each endof the paper and that the rate decreases as thebuffer approaches the centre line. The results givenin the previous section indicate that this variationis linear with respect to the distance of the pointof arrival of the electrolyte from the centre line,the time of current flow and the applied heating

load to the paper. This relationship is also inde- tion of a given mixture of compounds. McDonaldpendent of the electrolyte used. (1952) has shown that this effect is more markedThe flow of electrolyte from anode to cathode when the filter paper is encased between glass

due to electro-endosmosis must also be taken into plates or immersed in a solvent than when it isaccount when measuring mobilities of ions or freely suspended in a moist chamber. He sug-particles or when considering whether ionophoresis gested that the ions are able to travel through thecan be employed as a practical method of separa- water sheath surrounding the paper when it is

________________ freely suspended, whereas they have to move

through the intricate channels of buffer in theA,cetates body of the paper when it is encased between

-" <plates or immersed in solvent.The components of a mixture of ions or particles

20 Cy.S03H having only small differences in mobility may have_ Asp to travel comparatively long distances before the

18 _ Glu Gly separation of spots on the developed ionophoreto-E ~ ~ Ala gram is sufficient for practical use. This separation is. 16 - so Pro obviously limited by the length of paper, and anyo~ _ _ThrCY2S2 Leu additional movement by electrolyte flow sets a

14 - Ser Met Phe further limitation. To offset this effect the mixture- 12 ~ Tyr Lys should be placed on the paper as far as possible12-Tyr n _His from the electrode to which the ions will migrate.

10 Arg Fig. 10 gives an illustration of the separation ofoi -aspartic acid and threonine. The straight lines8 8- show the movement of these compounds withoutE the evaporation effect. The shapes of the curves

V.!2 6 - reflect the decreasing accelerating effect of evapora-

tion to movement as far as the centre of the paper_4 and the increasing retarding effect to movement

2 _ beyond this point._ These disadvantages of the moist-chamber

I I I I I I apparatus due to electrolyte flow can be reduced to0 01 02 03 0A4 05 0-6 0-7 0-8 some extent by careful choice of buffer. McDonald

RF In benzene-phenol-ethanol-water (1952) showed that the mobility of a compoundFig. 9. Separation of N-acetyl amino acids by chromato- increases with decreasing ionic concentration. Thegraphy (horizontal axis) in benzene-phenol-ethanol- value of the applied heating load is lower for a givenwater and ionophoresis in 0-05 M-ethylamine acetate voltage with buffers of low ionic concentration.(pH 9) under the conditions described in the text. The patters obtained in the two-dimensional

ionophoretic separations are similar to those ob-Xcwcxd tained by Durrum (1951) and Mead (1955) and the

25 , s order of displacement of the iodo-amino acids run

,/ , in 0 025m-borax (pH 9.2) is the same as that ob-o E 20tained by Lissitzky (1954) with veronal buffer0~ ~ ~ .- ¶27(pH 8.6).

v 2 4 6 8 10 12 14 16Time of current flow (hr.)

Fig. 10. Variation of displacement of aspartic acid andthreonine (x cm.) with time (t hr.). Conditions of experi-ment: solvent, M-acetic acid; potential gradient, lOv/cm.; x0 + 20 cm. Broken lines show the movement of thespots in the absence of evaporation.

SUMMARY

1. An apparatus for two-dimensional paperionophoresis or for one-dimensional paper iono-phoresis to be followed by paper chromatographyin a second dimension is described.

2. A method for determining the flow ofelectrolyte at different points on the paper isdescribed. Equations expressing the relationshipof the flow of electrolyte with experimental condi-tions of potential gradient, duration of currentflow and the applied heating load are given. Theeffect of this movement of buffer on the paper onthe separation of mixtures of ions or chargedparticles is discussed.

Vol. 68 SEPARATION OF A31INO ACIDS 661

662 J. K. WHITEHEAD I9583. A separation of 21 amino acids by two-

dimensional ionophoresis is given.4. A separation of monoiodotyrosine, di-iodo-

tyrosine, tri-iodothyronine and thyroxine from15 other amino acids and an inorganic iodide ion isdescribed.

5. A separation of 18 N-acetyl amino acids byone-dimensional paper ionophoresis followed byone-dimensional chromatography is also given.The apparatus was originally constructed and developed

at the Cereals Research Station, St Albans, Herts, and theauthor is grateful to Dr T. Moran, C.B.E., Director of theResearch Association of British Flour Millers, for his en-couragement and to Mr G. G. Grindley for technicalassistance. The application of the apparatus to the separa-tion of N-acetyl amino acids was part of a research pro-gramme supported by the British Empire Cancer Campaignand the author would like to thank Professor F. Dickens,F.R.S., and Professor J. Roberts for their interest, andMr D. Beale for technical assistance.

REFERENCES

Brand, E. & Edsall, J. T. (1947). Annu. Rev. Biochem. 16,224.

Durrum, E. L. (1950). J. Amer. chem. Soc. 72, 2943.Durrum, E. L. (1951). J. Colloid Sci. 6, 274.Flynn, F. V. & de Mayo, P. (1951). Lancet, 261, 235.Gordon, A. H., Martin, A. J. P. & Synge, R. L. M. (1943).

Biochem. J. 37, 79.Gross, J. & Leblond, C. P. (1951). Endocrinology, 48, 714.Gross, J. & Pitt-Rivers, R. V. (1953). Biochem. J. 53, 645.Jepson, J. B. & Smith, I. (1953). Nature, Lond., 172, 1100.Kunkel, H. G. & Tiselius, A. (1951). J. gen. Physiol. 35, 89.Lissitzky, S. (1954). C.R. Acad. Sci., Pari8, 238, 1167.McDonald, H. J. (1952). J. chem. Educ. 29, 428.McDonald, H. J., Urbin, M. C. & Williamson, M. B. (1951).

J. CoUoid. Sci. 6, 236.Mead, T. H. (1955). Biochem. J. 59, 534.Partridge, S. M. & Westall (1948). Biochem. J. 42, 238.Pitt-Rivers, R. V. (1956). Chem. & Ind. p. 21.Synge, R. L. M. (1939). Biochem. J. 33, 1916.

Determination of Amino Acids by Double Isotope-dilution Technique

BY J. K. WHITEHEADBritish Empire Cancer Campaign, Radiochemical Laboratory, Barnato-Joel Laboratories,

The Middle8ex Ho8pital Medical School, London, W. 1.

(Received 20 June 1957)

Radioactive tracers in the form of labelled reagentsare proving more and more useful in the micro-analysis of compounds occurring in biochemicalmaterials. Keston and his co-workers successfullyemployed p-iodobenzenesulphonyl chloride labelledwith either 131J or 35S as a general reagent for actionwith amino acids. The simple form of the quanti-tative tracer method as originally used by Keston,Udenfriend & Cannon (1946, 1949) with 131I-labelled p-iodobenzenesulphonyl chloride requiresthe complete separation and recovery of thederivatives. This disadvantage was overcome byKeston, Udenfriend & Levy (1947, 1950) byadding a known quantity of the 36S-labelled p-iodobenzenesulphonyl amino acid after the pre-paration of the p-[111I]iodobenzenesulphonylderivatives in the sample to be analysed. In thisdouble-tracer technique, a value for the amount ofamino acid initially present could be calculated froma measurement of the 3S/131I ratio for the purifiedderivative. This estimate was independent of lossesfrom the point in procedure when the knownamount oflabelledamino acid derivative was added.The main disadvantage of applying this method

to routine use is the frequent preparation of thep-iodobenzenesulphonyl chloride necessitated by

the comparatively short half-lives of the labellingisotopes. It was thought that acetic anhydridelabelled with 14C and 3H would prove more satis-factory as a general reagent in double-isotope-tracer methods. Avivi, Simpson, Tait & White-head (1954) employed these reagents for the acyl-ation of the hydroxyl groups of hydrocortisone andaldosterone (electrocortine) in the estimation ofthese compounds in human peripheral blood. Thispaper describes the extension of the method to theassay of submicrogram quantities of amino acids inprotein hydrolysates.

EXPERIMENTAL

Reagents[1-14C]Acetic anhydride. This compound was originally

prepared by heating inert acetic anhydride and anhydroussodium [1-14C]acetate in a sealed tube at 1400 for 4 hr.Under these conditions complete exchange of acetyl groupstake place.

This reagent is now obtainable from the RadiochemicalCentre, Amersham, at a specific activity of 2 mc/m-mole.A stock solution of the labelled reagent diluted with re-distilled inert compound to a specific activity of 40ac/m-mole was dissolved (5 mg./ml.) in redistilled toluene andstored in a glass-stoppered bottle.