TIIE JOURSAL OF BIOLOGKAL CHE~STRY Vol. 247, No. 18, Issue of Ssptember 25, pp. 5928-5934, 1972

Printed in U.S.A.

Epidermal Growth Factor

PHYSICAJ, ASD CHEMICAL PROPERTIES*

(Received for publication, May 3, 1952)

JoH;~ i\I. TAYLOR,~ WILLIAN AI. ~IITCHELL, AIW STANLEY COHES§

Fmn the Depar-tme&s of Biochemistry, Hicmbiolcgy, and Medicine, Valrdeybilt Ullivezity Xchool of JIedici~~e, Nashville, Tennessee 37232

SUMMARY

The major physicochemical properties of epidermal growth factor are reported. The molecular weight of epidermal growth factor is estimated to be approximately 6100 by the methods of sedimentation equilibrium, gel filtration, and minimum molecular weight calculations from amino acid analyses. Epidermal growth factor is a single chain poly- peptide having an asparagine amino-terminal residue and an arginine carboxyl-terminal residue. It is further charac- terized by the absence of 3 specific amino acid residues: lysine, alanine, and phenylalanine. No detectable free sulfhydryl groups, hexosamines, or neutral sugars were observed. Epidermal growth factor has an isoelectric point of pH 4.60. The ultraviolet absorption spectrum is typical for proteins, with an E:%, a t 280 nm of 30.9. The secondary structure, as judged by ultraviolet circular dichroism, is principally nonhelical.

Epidermal growth factor is a polypeptide isolated from the submaxillary glands of adult male mice (I), which stimulates the proliferation alld keratinization of various epidermal tissues in viva and in vitro (2). Among the metabolic events in epidermal tissue affect,ed by EGF’ are: the stimulation of protein and RNA synthesis (3), the formation of ribosomes that are more active in cell-free protein synthesis (4), the conversion of previously existing ribosomal monomers int,o polysomes (5), and the in- duct,ion of ornithine decarboxylase with the concomitant accu- mulation of intracellular polyamines (6). Studies 011 the mecha- nism of action of EGF have recently been reviewed (7, 8).

Although the biologically active form of EGF is a relatively

* This study was supported by United States Public Health Service Grants HD 007% and AM 10833. J. M. T. was a National Tnst,it,llt,es of Health graduate trainee (2.TOl-AM-5441). W. M. M. is the recipient of l Career Development Award from National Institute of Arthritic and Metabolic Diseases.

$ This work was taken in part from a dissertation srtbmitted to the Graduate School, Vanderbilt IJniversity, in partial fulfill- ment of the requirement,s for the degree of Doctor of Philosophy. Present address, Department of Pharmacology, Stanford Univer- sity School of Medicine, Palo Alto, Calif. 94305.

$ To whom reprint requests should be addressed. 1 The abbreviation Ilsed is: epidermal growth factor (FXF)

low molecular weight polypeptide, having a sedimentation velocity coefficient of 1.25 S (l), it can be isolated as a subunit component of a high molecular weight complex (9). In this communication, major physical and chemical properties of the low molecular weight form of EGF are described.

EXPERIMESTAL PROCEDURE

Purijication of EGF

EGF was prepared from homogenates of the submasillar> glands of adult, male, albino mice as previously described (1). To remove trace impurities from EGF, each preparation was subjected to additional gel filtration over columns of Sephades G-75 and Bio-Gel P-IO as described below.

Physical Afethods

Cr’el Filtration-All gel filtration experiments were performed at 5” by the reverse flow method with Sephadex G-75 (Phar- macia) and Bio-Gel P-10 (100 to 200 mesh) (Bio-Rad Labora- tories). The elution buffer consisted of 0.01 M sodium acetate, pH 5.9, containing 0.1 ;M sodium chloride. In a typical esperi- merit, the sample, in a volume equal to 1 to 2(7; of the column bed volume, was added to the column by siphoning. All column eluents were monitored by all ISCO flow monitor at 280 nm.

For molecular weight estimations, the columns were calibrated according to the procedures described by Whitaker (10). The protein standards used for calibration were from Mann Research Laboratories, Kit 8109A, with the exception of ribonuclease A (Sigma) and pancreatic trypsin inhibit’or (Worthington). Col- umn void volumes (V,) were determined by using blue dertmn (Pharmacia). Individual PampIe elution volumes (V,) were measured by weight determinations of the eluate. The ratio of V,: VO was experimerltally determined for each protein standard and plotted against the logarithms of the known molecular weights. The resultant calibration curve was then used to estimate unknown molecular weights from experimentally tletel,-

mined elution volumes. Isoelectric Focusing-.Isoelectric points were determilled 1))

the technique of isoelectric focusing at 5” with an LKB model 8121 column accordirlg to the manufacturer’s directions (Tech-

nical Hulletin I-8100.E01), and as described by IIaglund (11). Ampholyte solutions (LKB) of 1.2(;;, were used over the pH range of 4 to 6. Protein solutions containing from 3 to 5 mg were focused for 48 hours with a final limiting voltage of 600

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volts. Fractions of approsimately 2 ml were collected and the I)roteitl content was monitored with an ISCO flow monitor at 280 111n. Measurements of 1’11 on each fraction, at lo”, utilized a IMiometer T’I‘Tl b pH meter with a PHA scale expander at- tachment standardized against p1-I 4 and $1 7 buffers.

CeZ I~lectroplzoresis--niscolltinuous polyacrylamide gel elec- trophoresis was performed according to the method of Davis (12), utilizing 7.5y0 resolving gels at pH 9.5. Gels were stained t’or protein with 0.5%;, aniline black in 7 ‘/; acetic acid.

Extinction Coe$cient-.Measurements of protein concentra- tions were performed with the Spinco model E analytical ultra- c*rntrifuge equipped with a Rayleigh interference optical system in a synthetic boundary cell at 20” according to the conditions described by Chervenka (13). EGF was dissolved in 0.1 M

sodium acetate, pII 5.60. A value of 4.1 fringes per mg per ml was used as the average refractive increment for a typical protein solution (14). Optical densities were measured at 280 nm with a I%eckman I>I- spectrophotometer in 0.1 M sodium acetate, p1-I fi.60, and in glass-distilled water, with a l-cm path length cuvette. So differences in protein absorbance were observed between the two solvents.

Sedimentation Equilibrium Vltracentrifugation-Low speed sedimentation equilibrium was performed with a Spinco model 1.: aualytical ultracentrifuge equipped with a Rayleigh inter- I’erence optical system. Solutions of EGF were exhaustively dialyzed against 0.1 31 sodium acetate, pH 5.60, and a 3-mm liquid colrum of sample was layered over an inert base of per- fluorotribut~larl~ille (FC 43, Minnesota Mining and Manu- fncturing Co.). During the run, the temperature was main- tained at 15” and measurements were made at 31,310 rpm with double sector cells equipped with sapphire windows. Fringe l)ositions were recorded on Spectroscopic II G photographic l)lates (Kodak) sud analyzed with a Nikon model 6 micro- comparator at 50 times magnification. Runs were made for 24 l~ours, equilibrium being assured by a lack of change in the i’riilge concentration gradient on sequential photographs.

The apl)arent weight average molecular weight (M,) over t Irr whole liquid column was evaluat,ed by t,he relatiolrship:

15 pg per ml. The circular dichroism data were recorded hi the instrument directly in terms of degrees ellipticity, 6. The data were then converted to mean specific residue ellipticity, [0]‘, having units of degrees cm2 per decimole according to the relationship:

e (MZZW) ___- Ie” = (10) lc (2)

where JfRW is the mean residue weight of the sample, c the concentration in grams per cm3, and 1 the path length of the sample solution in centimeters. A mean residue weight of 115 was used in the calculations. Secondary structural composition was evaluated from the polylysine model system of Greenfield and Fasman (18).

Chemical and Enzymatic Methods

Amino Acid Analysis-Protein samples of 100 pg were hy- drolyzed in 6 N HCl at 110” under less than 30 p vacuum for different periods of time. The hydrolysates were analyzed ac- cording to the method of Spackman et al. (19) on a Beckman model 120 C analyzer equipped with a range card scale expander and an Infotronics integrator. Tryptophan was determined according to the spectrophotometric method of Goodwin and hhlfm (20).

IIexosamine Determination-Hexoeamilres were analyzed ac- cording to the method of Ford2 on a I%eckman model 120 analyzer equipped with a range card scale expander. Protein samples of 0.1 prnole were hydrolyzed in 4 N HCl at 110” under 15 p vacuum for 6 hours. The hydrolysates were dried under vacuum over sodium hydroxide pellets, then dissolved in 1.0 ml of pH 2.2 citrate buffer, 0.2 RI. The sample solutions were then added to a column, 0.9 X 15 cm, of Beckman type PA 27 spherical resin at 55”. The column was eluted with $1 4.25 citrate buffer, 0.2 M, at a flow rate of 68 ml per hour. Hesosamiues were measured by the ninhydrin reaction, and related to known standards.

Xeutral Sugar Determination-Protein samples (0.1 pinole) were analyzed for neutral sugars according to the gas chroma- tographic methods described by Lehnhardt alld Winzler (21).

2 R7’ 1 AC& Amino-terminal Residue Analysis---The Edman deg;l,adation

M, = (1 - Bp)w2 b2 - a2 Co

(1) procedure was performed essentially as described by l)eLange et ul. (22). The allilinothiazolino~~es were hydrolyzed directly

iI1 which R is the gas constant, T the absolute temperature, v t )I(-’ partial specific volume of the protein, p the solution density, w the angular \-elocity in radians per s, b and a the radial posit,ions ill rentimeterh of the base and meniscus, respectively, of the liquid colunllr, AC,b the relet,ive concentl,ation gradient in fringes llrtween b and a at equilibrium conditions, and Co the initial collcentratioli iu fringes. The value for Co n-as established ill a srl)arat,e run using a synthetic boundary cell. The partial si)rcific volume of EGF was calculated from the amino acid c’ompo.qition according to the method of McMeekin and Marshall (13).

Sribseque~it determinations of A/ W at multiple points in the liquid column were evaluated by methods previously described (16, 1 T). Lillear regression lines relating In concentration and the square of the radial distalIce were calculated by the method of least squares.

Circular L)ichroisnz-;\Ieasurernellts of the circular dichroism from 250 to 195 nm were performed on a Car>- model 60 spec- t roi&rimeter equipped with a model 6001 CI) accessory. Es- I)erirnents were carried out at 20” with a l-cm path length cell

to form free amino acids, which were analyzed on the amino acid analyzer. Hydrolysis was carried out at 150” for 20 hours under vacuum wit,11 6 N HCl with 0.1 ‘1; P-rnercaptoetllarlol. In addition, the phenylthiohydantoin derivative of the amino- terminal residue xl-as analyzed by gas chromatography on SP-400 resin and by thin layer chromatography on silica gel fluorescent- indicating plates (23).

Leucine aminopeptidase (Sigma) treatment of EGF and S- aminoethylated EGF was carried out according to the procedure of Light (24). A substrate to enzyme ratio of 25:l by weight was used, and the reaction mixture was incubated at 40” for 5 hours. The reaction products were esamined with the amino acid analyzer.

Carboxyl-terminal Residue dnalysis-l>iisol)rc,1,\-1 fluorophos- phate-treated bovine pancreatic cal,bosSl)el)t,idase A and diisopropyl fluorol~l~o~pliate-treated porcine llancreatic car- bosyl)eptidnse B were obtained from both Worthington alld Sigma. Reactions of EGF with carboxypeptidases A and I< were performed according to the method of l\mbler (25). In the rractiorl with carbosypeptidase A, a substrate to enzyme

with EGF dissolved in glass-distilled water at a concentration of 2 J. Ford, Vanderbilt University, unpublished method.

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5930

1 I I I , 1 , , , , , , , , , ,

3 2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8

LOG MOLECULAR WEIGHT

-

FIG. 1. Estimation of the molecular weight of epidermal growth factor by gel filtration on Sephadex G-75. Samples containing from 4 to 8 mg of protein in 4 ml of elution buffer (0.01 M sodium acetate, pH 5.9, and 0.1 M sodium chloride) were applied to a Sephadex G-75 column (2.5 X 90 cm) equilibrated with the same buffer. The flow rate was 3.8 ml per cm2 per hour and 4.ml frac- tions were collected. The protein standards used in calibrating the column are indicated on the graph. The arrow, indicates the experimentally determined ratio of elution volume (V,) to void volume (V,) for EGF which was used in estimating its molecular weight.

ratio of 40: 1 by weight was used, and the reaction mixture was incubated at 37” for 4 hours. In reactions with carboxypeptidase B, substrate to enzyme ratios ranging from 40: 1 to 400: 1 by weight were used, and the reaction mixtures were incubated at room temperature for up to 12 hours, with samples for analysis being removed at various time intervals. Reaction products were examined with the amino acid analyzer. @-Phenylpro- pionic acid (Eastman), an inhibitor of carboxypeptidase A, was added to the working stock solution of carboxypeptidase B for some of the experiments, as recommended by Potts (26).

Cathepsin C Digestion---EGF was treated with cathepsin C (bovine spleen dipeptidyl aminopeptidase I, from Schwarz), in the presence of fl-mercaptoethanol according to the procedure described by McDonald et al. (27). The reaction mixture, con- taining 0.5 pmole of EGF with a substrate to enzyme ratio of 40: 1 by weight,, was incubated at 37” for 6 hours. During the course of the reaction precipitation was observed. The reaction mixture was passed through an hmicon (YYI-2) filter to remove high molecular weight components, treated with leucine amino- peptidase (4O:l w/w) for 1 hour at 37”, and examined on the amino acid analyzer with lithium buffers. The latter allows the resolution of amidated carboxyl side chain amino acids (28).

S-Rminoethylation-The disulfide bridges of EGF were re- duced with P-mercaptoethanol in 8 M urea and then treated with ethyleneimine according to the method of Cole (29). The extent of S-aminoethglation was determined by amino acid analysis. The color value for a standard solution of S-aminoethylcysteine is approximately 910; of that for lysine (30).

Suljhydryl Group Determinalion---The sulfhydryl reagent, 5,5’-dithiobis(2-nitrobenzoic acid), was used to assay for the presence of free protein sulfhydryl groups. Samples containing 0.2 pmole of protein were analyzed in pH 8.0 sodium phosphate buffer, 0.1 M, containing 8 M urea (Mann, ultrapure) according to the procedure described by Ellman (31).

Protein Determinations-The protein concentrations of various

I I I I I , , ,

3.8 4.0 4.2 4.4

LOG MOLECULAR WEIGHT

A-l

FIG. 2. Estimation of the molecular weight of epiderrnal growth factor by gel filtration on Bio-Gel P-10. Samples containing from 2 to 4 mg of protein in 2 ml of elution buffer (0.01 M sodium acetate, pH 5.9, and 0.1 M sodium chloride) were applied to a Bio- Gel P-10 column (2.5 X 40 cm) equilibrated with the same buffer. The flow rate was 3.5 ml per cm2 per hour and 4.ml fractions were collected. The protein standards used in calibrating the column are indicated in the graph. The arrow indicates the experimen- tally determined ratio of elution volume (V,) to void volume (V,) for EGF which was used in estimating its molecular weight.

solutions were estimated by the procedure of Lowry et al. (32) with bovine serum albumin (Armour) as a standard. Concen- trations were also measured by using the experimentally deter- mined extinction coefficient.

RESULTS

Physical Properties

Gel Filtration-The molecular size of epidermal growth factor, isolated according to the method of Cohen (l), was estimated by gel filtration on a column of Sephadex G-75. The column had been previously calibrated with proteins of known molecular weight according to the procedure of Whitaker (10). Fig. 1 shows the elution position of EGF on the calibration curve, indicating that it has a molecular weight of approximately 3,000. Since this number was considerably lower than the estimate of approximately 15,000 that had been reported earlier (l), it was felt that the possible adsorption of EGF to the Sephadex gel (a cross-linked dextran) could account for the discrepancy. There- fore, gel filtration was repeated with a calibrated column of Bio- Gel P-10 (a polyacrylamide support). The results, shown in Fig. 2, indicate that EGF has a molecular weight of approsi- mately 7,000. Since the same elution buffer and closely similar operating conditions were used in all gel filtration experimentS\,

it is reasonable to conclude that EGF does indeed adsorb to Sephadex gel. Smooth, symmetrical elution peaks were ob- served in each experiment. Since it is unlikely that two proteins of the same size would show the same adsorption phenomenon with Sephadex gels, all subsequent experiments described in this communication were performed with EGF which was re- chromatographed on Seghadex G-75 and Bio-Gel P-10 as de- scribed above, as a final purification procedure.

Sedimentation Equilibrium-.Ilr order to resolve the apparent discrepancies concerning the molecular weight values for EGF, low speed sedimentation equilibrium was performed using the Rayleigh interference optics system. Snalysis of the fringe displacement over the entire liquid column indicated a molecular

weight of 6,400 and revealed no significant heterogeneity. A linear plot of In concentration versus r2 is illustrated in Fig. 3. A small upward deviation of the plot at the base, showing a molecular weight of 8,500, is indicative of minimal heavy mate- rial, probably due to sample aggregation.

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2.6.

2.4

1.8.

In c 1.6 -

l.O-

0.8.

0.6 Ii/ I

o.4 MENISCUS

47 48 49 50 51 52 53

r2

FIG. 3. Low speed sedimentation equilibrium plot of In fringe concentration against the square of the radial distance (T) for epidermal growth factor.

Partial Specific Volume-The partial specific volume (fi) of EGF was calculated according to the method described by McMeekin and Marshall (15) and found to be 0.69 cm3 per g.

Extinction Coe#cient-The value for E::,,, at 280 nm of the epidermal growth factor was determined to be 30.9. The protein concentrations were calculated from the interference fringe counts in the ultracentrifuge synthetic boundary cell.

Isoelectric Point--The isoelectric point of EGF was determined by the isoelectric focusing technique. The experiment was performed with a 1.2-mg sample in a pH gradient of 3 to 5 at 5”. The result, as shown in Fig. 4, indicated that the isoelectric point was at pH 4.60. The sharp, symmetrical boundaries that were observed suggested a high degree of sample purity.

Gel Electrophoresis--Electrophoresis of EGF on polyacrylamide gels revealed a single protein band, shown in Fig. 5. Occa- sionally, faint traces of faster migrating bands were detected in EGF preparations. These bands could be minimized by pre- liminary electrophoresis of the resolving gels in buffer to wash out residual, unreacted persulfate (33). Such bands might also be due to deamidation of side chain carboxyl amides, as reported by Lewis et al. (34).

Circular Dichroism-The circular dichroic spectrum of EGF is shown in Fig. 6. The dominant feature of the spectrum is the strong negative dichroic extremum at 200 nm having an ellip- ticity of approximately -12,700 deg cm2 per decimole. This dichroic band has been assigned to a r - 7r* transition of the peptide bond (35) and is a characteristic property of nonhelical polypeptides and proteins (36). The n - 7r* peptide bond transition which is characteristic of a helical structures (35) is absent in FGF spectra. With a computer-assisted curve-fitting

5931

0.7 - 4’

/ I’ - 5.0

-. I

0.6- ,’

2 *a’ 0.5- #‘PH 4.60 - 4.5

zs / t N

0.4- t

,/*

I- /-- *-- /

- 4.0 f

\, i i

FIG. 4. (left). Isoelectric focusing of epidermal growth factor. The pH range of the ampholyte solution was from pH 3 to 5. The solid line shows the absorbance at 280 nm of the fractions (2 ml) obtained from the isoelectric focusing column. The dashed line represents the pH gradient developed during the experiment.

FIG. 5. (right). Electrophoresis of epidermal growth factor (40 pg) on a polyacrylamide gel at pH 9.5. The cathode is at the top of the gel.

4 I I

200 220 240

WAVELENGTH (m)r)

FIG. 6. Circular dichroic spectrum of epidermal growth factor (15 pg per ml) in distilled water.

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program3 based on the polylysine homopolymer system of Greenfield and Fasman (I@, EGF is principally nonhelical with a random coil content of 74’7; and p helical content of 257;. Or11v trace Q helical contributions exist under the exnerimental conditions. The main error in curve fitting is centered at the weak positive dichroic band at 231 nm which has no counterpart in the model system used in the curve-fitting procedures. Never- theless, it seems clear that, EGF is largely nonhelical in its sec- ondarx stIruct,ure; alt,hough a significant proportion of the mole cule is apparently the 6 conformation.

T.\B I,,.; I ‘1 mine acid conzposition of epidernml growlh~ facfol

I Apparent residues per mole of EGF”

Amino acid Time of hydrolysis

4s hrs 72 hrs 96 hrs I- I

lverage Or Assumed

“d;:z- ~composition I

Lysine Histidine. Arginine Aspartic acid Threonine Serine Glutamic acid. Proline Glycine Alanine. Half-cystine Valinc.. Methionine Isolelrcine Leucine Tyrosine Phenylalanine. Tryptophan.

0 0.95 3.T9 7.4 2.04 5.76 3.18 2.00 G. 14 0 4.95 l.iO 0.75 1.74 3.85 4.55 0

0 0.94 3.72 7.26 1.90 5.10 3.24 1.99 5.89 0 5.44 1.79 0.88 1.71 3.82 4.34 0

0 0 0.97 0.95 3.77 3.82 7.28 7.24 1.86 1.83 4.78 4.38 3.12 3.16 2.67 2.19 5.70 G.22 0 0 5.09 4.90 1.46 1 ,42 0.85 0.93 1.64 1.74 3.80 3.83 4.51 4.55 0 0

0 0 0.95 3.78 7.31 2.11c G.lGC 3.18 2.21 5.99 0 5.09 1.59 0.85 1.71 3.82 4.49 0 1.95e

n’umber of Polypepfide Chains-The possibility that EGF might be composed of more than one polypeptide chain was esamilled. Kative EGF was dissolved in 0.1 M Tris buffer, pH 8.0, containing 0.01 M dithiothreitol (Calbiochem) and 6 M gua- nidirle hydrochloride (Mat,heson, recrystallized from an ethanol solution that had been filtered through activated charcoal) The mixture was allotted to stand at room temperature for 4 hours, and then applied to a previously calibrated column of l%io-Gel P-10. The elution buffer was 0.01 M sodium acetate, ptI 5.9, containing 0.1 JI sodium chloride and 0.01 M dithio- threitol. The reduced EGF had essentially the same elution volllrne as ulrreduced native EGF in nonreducing buffers with the same column, indicative of a single polypeptide chain. The Fractions containing the reduced EGF from the Uio-Gel column lvere combined, dialyzed against water, and taken to dryness by air-dr!-ing. The reoxidized EGF possessed approximately the same biological activity when injected into newborn mice ac- cording to the conditions of the bioassay in viva (1).

In :L separate experiment, S-aminoethylated EGF that had twen dialyzed against n-ater to remove escess reagents was apl)lied to a column of Bio-Gel P-10, and eluted with 0.2 x acetic acid. The sample eluted as a smooth, symmetrical peak. The amino acid compositions of the X-aminoethylated EGF were compared before and after gel filtration, and were found t,o be itlel~tical. Each sample contained 6 X-aminoethglcysteine rrsitlrles. The rest of the amino acid composition for both -aml)les was identical with the amino acid composition of native I<GF (described elsewhere in this communication). These re- .-lilts are consistent, with a single chain polppeptide structure for EGB.

Chemical Properlies

Chwlical Colnposition-The amino acid composition of EGF RX. eznmilred, and the results of timed hydrolyses are shown in ‘I’:~blr I. EGF is characterized by t,he absence of 3 specific anlillo acid residues: lysille, alanine, and phenylalanine. Amino acid arlalysis of S~anliiloetllSlated EGF confirmed the presence of 6 Iralf-cystine residues. No free sulfhydryl groups were detected with the spectrol)hotometric method of Ellman (31), illdiwtillg the prol-able e&tence of three disulfide bridges within the Illolrcufe. No detectable hesosamilles or neutral sugars wre observed.

.I nlinimum molecular weight of 6045 was calculated on the basis of 1 llistidine residue and 3 glutamic acid residues per mole 01’ ool>-kwptide. This calculation is consistetlt with the molecular n-eigllt values obtained from sedirnentatiou equilibrium studies :111d gel filtration through columns of I%io-Gel P-10.

;lnrino-ternlinal Residue Detenni~zation-Scid hydrolysis of the :Illilillothjazolillorle derivative obt,ained by Edman degra- dation of EGF yielded aspartic acid in 25I:; yield. Under these conditions asparagine would be converted to aspartic acid. An

: TI. Davies and W. Mitchell, Vanderbilt University, nnpllb- lished computer program.

Total. Minimum rnolecu-

lar weight. --

1 4 7 2 (i 3 2 G 0 G” 2 1 2 4 5 0 2

53

~ GO45

a Calctilated on the basis of the average obtained by assuming 1 histidine and 3 glutamic acid residues per mole of EGF.

b Average of two determinations. c E:xtrapolated to zero time. d Assumed cornposition based on no detectable sulfhydryl in

the native molecule (see text). e Determined by the spectrophot,ometric method of Goodwin

and Morton (20).

examination of the l)hell?-lthioh-dantoirl derivative by gas chromatography indicated that the amino- terminal residue was indeed asparagine, in al)prosimately 50?; yield. This finding of asparagine was reconfirmed by thin layer chromatography.

Wgestion of either native EGF or S-arniuoethvlated EGF with leucine aminopeptidase did not result in the liberation of any amino acids.

Cathepain C Digestion---Native EGF, ill the presence of p- mercaptoethanol, was treated with cathepsin C, a dipeptidyl aminopeptidase. The l>roduct of the reaction was examined by several methods. First, an aliquot of the mixture was an- alyzed directly on the amino acid analyzer with standard citrate elution buffers on the acidic-neutral colunu~. A single peak was eluted near the buffer change indicative of a single amino-ter- millal hydrolysis product. Second, higll molecular weight products were removed from the reaction mixture by ultra- filtration and t,he ultrafiltrate subjected to acid hydrolysis or leucine aminopeptidase digestion. Acid hydrol!-sis yielded aspartic acid a11d seritle. The products of leucine aminopep- tidase digestion were then analyzed on an amino acid analyzer with a litllium buffer system which allows the resolution of nsparagirre and aspartic acid. Asparagine and serine in equiva-

lent molar quantities and trace amounts of aspartic acid were

the only amino acids observed. During this entire sequence of reactiotls, 110 other dilleptide or amino acid reaction products

were observed. These findings supl,ort the amino-terminal

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residue studies which indicated that EGF had a single amino ter- minus consisting of asparagine, as well as a penultimate serine.

Carboxyl-terminal Residue Deternzination-Digestion of EGF with carboxypeptidase B liberated 1 mole of arginine per mole of polypeptide. EGF was quite sensitive to the action of car- boxypeptidase B, since this result could be obtained in a 15-min incubation at room temperature with a substrate to enzyme ratio of 400: 1. Changing this ratio to 40: 1 under the same experimental conditions also liberated 1 mole of leucine per mole of polypeptide, even ill the presence of fi-phenylpropionate. Extending the incubation to longer reaction times or the addition of soybean trypsin inhibitor (Sigma) to the reaction mixture did not change these values, or result in the significant appearance of additional amino acids. In separate experiments, carboxy- peptidase A digestion of native EGF did not liberate any de- tecbable amino acids. These results are consistent with a single carboxyl terminus having a sequence of -Leu-Arg.

librium, gel filtration, isoelectric focusing, and gel electrophoresis. EGF was also characterized by the absence of 3 specific amino acid residues (lysine, alanine, and phenylalanine) as well as the absence of hexosamines and neutral sugars. The secondary structure of this polypeptide is predominantly nonhelical as judged by a comparison of experimentally det,ermined circular dichroic data on EGF with model polypeptides.

nISCUSSION

The characteristic properties of EGF are summarized in Table II. In this communication we have reported the molec- ular weight of EGF to be approximately 6,100. This value is the result of sedimentation equilibrium studies, gel filtration on Bio-Gel P-10, and minimum molecular weight calculations based on amino acid analysis. This number differs from the estimate of 14,600 as originally reported in 1962 (1). However, the 1962 estimate was calculated from the amino acid composition on the basis of 1 alanine residue and 10 leucine residues per mole of polypeptide. In comparison, the amino acid analysis of EGF reported in Table I does not reveal the presence of any detectable alanine, suggesting that trace contaminants may have been present in the earlier preparations of EGF. In the present study, the removal of possible trace contaminants was facilitated by the observation that EGF exhibits a weak adsorption phenome- non with Sephadex G-i5, but not with Bio-Gel l’-10. This behavior was therefore utilized as an additional purification treatment for EGF. The resultant material showed a high degree of homogeneity when examined by sedimentatioil equi-

EGF appears to be a riugle chain polypeptide. This coil- elusion is based on four different kinds of experimental evidence. First, gel filtration of reduced EGF on calibrated columns of Rio-Gel P-10 in dithiothreit,ol-containing buffers indicates that. it has the same molecular size as the unreduced, native polypep tide. Second, the amino acid composition of S-aminoethylated EGF that has been dialyzed or passed through gel filtrat,ion columns is identical with native EGF. The 6 half-cystine residues (as S-aminoethylcystine) are still present, indicating that a small peptide fragment has not been lost. Third, only 1 amino-terminal residue (asparagine) and 1 carboxyl-terminal residue (arginine) have been detected. Fourth, reduced EGF that has been oxidized by air-drying retains full biological ac- tivity.

An investigation of the amino-terminal residue of EGF re- vealed the presence of asparagine. However, the yields were lower than might be expected. One possible explanation for this observation is that a portion of the arnino terminus may be blocked, i.e. by an acetyl group, but direct evidence for this has

not been obtained. Another explanation is that a portion of the asparagine may have undergone cyclization with the libera- tion of ammonia, thereby interfering with its detection (37). Similar low yields of amino-terminal aspartic acid have heel1 obtained with other protein polypeptides (38).

TABLE II

In crude homogenates of the mouse submaxillary gland, EGF has been isolated as a component of a high molecular weight complex, having a molecular weight of approximately 74,000 (9). The complex can be reversibly dissociated into EGF and an EGF-binding protein (mol wt, 29,000), the latter an arginine esterase. Thus the finding that EGF has a carboxyl-terminal arginine residue suggests that it may be generated from a pre- cursor protein by the possible proteolytic action of the EGF- binding esterase.

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Molecular weight as determined by Sedimentation equilibrium 6400 Gel filtration (Bio-Gel P-10) 7000 Amino acid composition 6045

Sedimentation coefficient (~20,~)~ 1.25 S Partial specific volume (8) in cm3/g , 0.69 Extinction coefficient (E::_ at 280 nm) Isoelectric point i :g4.,, Conformation Rlaj or, Nonhelical

Minor, p strrlcture Trace, O( helix

No. of polypeptide chains One ilmino terminus Asparagine Carboxyl terminus .4rginine Disulfide bonds I 3 Missing amino acids Lys, Ala, Phe Hexosamine content None detected Xeutral sugar content None detected Antigenicitya Ant,igenic

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John M. Taylor, William M. Mitchell and Stanley CohenEpidermal Growth Factor: PHYSICAL AND CHEMICAL PROPERTIES

1972, 247:5928-5934.J. Biol. Chem. 

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