Pwification and Characterization of Colicin E,*

(Received for publication, May 24, 1971)

ST.\NLEY A. SCHWARTZ$ 91~~ DONALD R. HELINSKI~

From the Departmen,t ofl Biology, University of California, San Dr’eyo, La Jolla, California 92037

SUMMARY

Colicin Et was purified from mitomycin C-induced Esche-

richia coZi JC411 (ColEI) by salt extraction of extracellular, bound colicin followed by ammonium sulfate fractionation and ion exchange chromatography. The purified prepara- tion was considered homogeneous by several criteria. Gel filtration on Sephadex G-ZOO revealed no additional protein components and only a single band was observed upon cellulose acetate and isoelectric gradient electrophoresis. Sedimentation velocity and equilibrium studies, respectively, indicated the presence of a single molecular species with an S i,, zL1 equal to 3.0 S and a molecular weight of 56,000. Double diffusion experiments using antisera prepared against purified colicin E1 yielded a single precipitin line in agar gel.

Colicin E1 is a simple protein and no other moieties could be detected in association with the molecule. The isoelectric point (PI) of colicin El, as determined by electrofocusing, is 9.05, and amino acid analysis revealed a high lysine and arginine content. A single NH2-terminal amino acid, methionine, was recovered in approximately equimolar yield per mole of protein. Circular dichroism spectra of purified colicins E1, E?, and E3 indicated a greater a-helix content in colicin E1 when compared to the other two colicins. Finally, high concentrations of purified colicin El were active against sensitive, immune, and resistant strains; however, relatively lower concentrations exhibited a normal activity spectrum.

Early in the st’udy of bacteriocins, it was recognized that these bactericidal agents were distinct from other antibiotics. In con- trast to their low molecular weight counterparts, bacteriocins are proteins that exhibit a high degree of antibiotic specificity. Few hacteriocins have been sufficiently purified to permit rigorous physical and chemical characterization; however, among the various purified preparatiom5 an extensive variation in structure aud composition has been noted. A characteristic of all bac- teriocins is that they are prima~rily, if not exclusively, protein in nature.

* This research was supported by United States Public Health Prr~ ice Research Grant AI-Oil94 and National Science Founda- tion Grant GB-11818.

$ Recipient of United States Public Health Service Fellowship FI-G&t-21,263. Present address, Ijepartmcnt of Biochemistry, Albert, Einstein College of ;\Icdicine, Bronx, New York 10461.

4 Iynitcd States Public Health Service Research Career De- velopment AlI-ardee (No. KOI-Gh107821).

Colicins are bacteriocins produced by certain strains of Entero- bacteriaceae and are active against the same or closely related members of this fam,ly. The formidable task of classifying colicins was undertaken by Fredericq (I, 2) ut,ilizing the proper- ties of specific adsorption and immunity. Single step mutants resistant to several colicins could bc isolated from a population of sensitive cells. Thesr mutants retained their sensitivity to most other colicins, prompting the conclusion that the mutation irr- volved the loss of a specific common receptor for a partirular class of colicins. Coliclns with similar adsorption properties could be further subdivided on the basis of their immunity spec- ificity. Colicinogenic organisms adsorb a hornologous cohcin but remain unaffected by it. This property is referred to as ‘Limmunity.” Sensitivity to other colicins, including those which share the receptor, is retained. As an example of the claas,fica- tion scheme, resistance to colicins El, Ez, and Ea can be acquired by a single step mutation. They can be differentiated, however, by the lack of cross-immunity of a strain producing any one of them.

The following studies were undertaken to purify colicin E1 to a state which would permit physical and chemical chnracterizat,ion. Herschman and Helinski (3) have shown that colicins Eg-I’9 and E3-CA38 are simple proteins which possess several common phys- ical and chemical characteristics. Colicins E1, E?, and Ea re- portedly share a similar binding site on the cell wall (I, 4) al- t.hough their modes of bacteroidal action differ (5-7). Thus, a comparison of the physical and chemical properties of these pro- teins could provide a structural basis for their biological specific- ities as well as an insight into the evolutionary development and relatedness of the genetic factors responsible for their production.

EXPERIRIENTAL PROCEDURE

Culture Condilions and Induction of Colicin E,

L broth contains (per liter of distilled water) : tryptone (Difco), 10 g; yeast extract (Difco), 5 g; NaCl, 5 g; glucose, 1 g; it, is ad- justed to pH 7.0 with 10 N NaOH. Escherichia co& JC411 (ColEI), obtained by conjugal transfer of the ColEI factor from E. coli K-30 to JC411, was inoculated from a nutrient agar slant into 40 ml of L broth and grown overnight at 37” with agitation to late log phase. The characteristics of this strain have been described previously (8). This start,er culture was added to 40 liters of sterile I, broth cont,aining 4 ml of SAG471 antifoani (Union Carbide Corporation) and incubated at 37” in a model F-130 Fermacell Fermentor (Xew Brunswick Scientific Com- pany) to a concentration of 5 X IO* cells per ml. -1ir sparging was set to a flow rate of 4 cubic feet per min and agitation wah maint,ained at 250 rpm.

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Colicin synthesis was induced by adding a solution of mitomy- tin C (Kyowa Hakko Company, Lt~d., Tokyo) in 0.9% NaCl ;olution to yield a final concentration of 0.1 pg per ml of culture. The induced culture was incubated for a,n additional 12 hours at 37” and a sample was removed for colicin assay. Harvesting of the cell mass was accomphshed in a refrigerated Sharples model A4S-16 continuous flow centrifuge fitted with a plastic bowl liner.

Colicin Assay and Speci$c Activity

Colicin activity and prot,ein concentration jvere determined as previously described (3). The specific activity of colicin prep- arations was defined as the number of units of colicin activity per mg of p~otcill.

Immunochemical Analysis

-4n emulsion of 1.0 ml of a l.OyO (w/v) solution of purified col- icin E1 in 0.01 RI potassium phosphate buffer, pH 7.0, plus an equal volume of Freund’s complete adjuvant (Difco) was in- jcctcd into the toe pads of a female, white New Zealand rabbit weighing approximately 2 to 3 kg. Six weeks after the primary immunizat’ion, 1.0 ml of a O.3L70 colicin solution in 0.01 Iw potas- sium phosphate buffer, pH 7.0, was injected into the ear vein of t,he rabbit. Approximately 50 ml of blood were collected from the ear vein 10 days after the secondary booster, allowed to clot at room temperature for 30 mm, and transferred to 4” to permit the clot mass to contract overnight. The serum was decanted and clarified by centrifuging at 1290 x g in n refrigerated cen- trifuge. Separate l-ml aliquots \T-ere frozen at -20” until ready for use. Immunochemical homogeneity of purified colicin El was determined by the micromodification (9) of the Ouchterlony double immunodiffusion procedure (10).

Electrophoresis

Cellulose Acetate Gel Electrophoresis-Samples of colicin E1 in electrode buffer were applied as narrow zones to strips of cellulose acetate (Sepwaphore 111, Celman Instruments, Inc.) which had been soaked in buffer to restore the hydrated colloidal structure. After electrophoresis for various times at, a constant current den- sity of 1 ma per strip, protein bands were fixed and stained with a 0.57; solution of Ponceau S in 57, t,richloroacetic acid. Several washings in SC/o acetic acid served to decolorize the electrophero- gram. The strips were rendered t,ransparent by t,reating with a 5% solution of acetic acid in methanol, followed by drying on a glass plate at 60” for 15 min. The cleared gels were scanned in a Joyce-Loebel recording densitometer.

Isoelectric Focusing with Carrier Ampholytes--The technique of Vesterberg and Svensson (11) which fractionates high molecular ampholytes on the basis of their relabive po,+itions in a stabilized natural pH gradient was used as an additional test of homoge- neity and t,o determine the isoelectric point of c&cm E1. A 110.ml ampholine electrofocusmg column (LKB-Produkter, Stockholm) was prepared according to the mstructlons supplied with the inst,rument. In view of the cat1on.c electrophoretic be- havior of colicin E1 in several buffer ,systems, clectrofocusng was attempted in a pH 8 to 10 lineal gr:ad;cnt gcncrnted with LKB ampholinc carrier ampholyte No. 8156. A .>tepwise sucrose gradient stabilized the liquid column and the system was main- tained at’ 0” by circulating ice watrr through the cooling jackets. With the cathode at the t,op of t,he column, a continuous total power consumption of approximately 1 watt was sustained. Us- ing a constant voltage power supply, the potential was gradually

raised to compensate for increased resistsuce. Equilibrium was achieved in 48 hours, with the system stabilized at 700 volts, 1.5 ma. After closing the c~cntral electrode chamber, l-ml frac- tions were collected and analyzed for colicin activity and absorb- ance at 280 nm. The p1-I of selected fractions was determined with a Radiometer titrator type TTTlC equipped with a type PHX630T scale expander.

Ultracentrijugal Analyses

Sedimentation Velocity-Protein solutions of varying concen- trations were prepared in 0.1 M potassium phosphate buffer, pH 7.0. Samples were placed in analytical cells fitted with a 12.mm, 4” sector Kel-F centerpiece and quartz windows and sedimented at’ 59,780 rpm in an AN-H rotor using a Beckman model E am- lytical ultracentrifuge equipped with an RTIC t,emperaturc con- trol unit set to 20”. The rate of change of the concentration di+ tribution within the cell was determined with schlieren opttes and recorded on metallographic film lilntes (Eastman Kodak Corn- pany) at 8-min intervals.

Determination of Diffusion C’oeficient-The diffusion coefficient for colicin El was determined by the method of Ehrenberg (12). A double sector, synthetic boundary, capillary type cell was filled with solutions of colicin E1 in 0.1 RI potassium phosphate buffer, pH 7.0, and the samples lx-ere centrifuged at 11,272 rpm using an AN-H rotor in the analytical ultracentrifuge maintained at 20”. Upon attaining running speed, photographs were taken every 8 min with the schliereii optical system. Using a photo- graphic enlarger, the filrn plates j\-ere projected at known magni- fication onto graph paper and trared. A calibrated planimeter was used to measure the areas under the inner and outer bound- aries of the schlieren line. The total area was averaged from these values. The height of the peak was obtained by averaging the distance between the outer and inrier edges of the schlieren curve.

sedimentation Equilibrium--~olutioll columns of 3 mm were prepared using the six-channel centerpiece described by Yphamis (13). To attain equilibrium, solutions of colicin in 0.1 M potas- sium phosphate buffer, pH 7.0, were centrifuged in the analytical ultracentrifuge for 20 hours at 16,200 rpm in an SN-D rotor (the heavier rotor was used to maintain inertial stability at lore speeds). The time and speed required to attain equihbrium have been shown to depend upon t.he column height and the mo- lecular weight of the solute (13). Upon reaching equilibrium, the resulting concentration gradient was recorded photographi- cally with schlieren optics. TVith the aid of a microcompnrator the coordinates of 25 to 30 arbitrary points along the refractive index curve were determined relative to the base line. These data were converted to the molecular weight of solute at each point and extrapolated to yield a molecular weight for short spans throughout the gradient by means of a computer program based on the relationships described by ,Johnson, Kraus, and Scatchard (14). An average molecular Tvcight value for the solute ~‘a.5 also obtained.

Amino Acid Analysis and Per Cent by Weight Recovery as Amino Acids

Bmino acid analysis was yeriormed similar to t’he method de- scribed by Spackman, Stein, and Moore (15). A sample of lyophilized colicin E, was dried at 100” over PZOS in vacua for 24 hours. The dry protein was accurately weighed and dissolved in a known volume of distilled 5.7 x HCI. Aliquots were sealed in

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evacuated glass tubes and hydrolyzed for 12, 24, or 48 hours at 107”. NC1 was removed by evaporation in an evacuated des- iccator containing NaOH pellets. The hydrolysate was dis- solved in distilled water and lyophilized. After volumetrically redissolving the dry residues in dilutor buffer (0.2 N, sodium cit- rate, pH 2.2), aliquots were applied to a Spinco automatic amino acid analyzer model 120B (Beckman Instruments, Inc.). Cys- teine content was determined by performic acid oxidation (16) of the protein prior to hydrolysis and amino acid analysis. TWO

independent techniques were used to determine the tryptophan content of colicin E1. Using the relationships described by Goodwin and Morton (17), the tyrosine and tryptsphan content was determined from the extinction values at 280.0 and 294.4 nm for a solution of colicin E1 in 0.1 s NaOH. The calorimetric trppt,ophan analysis of Spies and Chambers (18) was also used.

Carbohydrate Analyses

For the analysis of reducing sugars a sample of colicin E, was hydrolyzed for approximat,ely 8 hours in an evacuated tube at 100” with 3 N H2S04. After hydrolysis the preparation was ti- trated to neutrality with 3 N SaOH. An aliquot was removed for asra,y by the method of Park and Johnson (19). A separate sample of colicin E1 was dissolved in 3 N HCl and sealed under vacuum in a glass tube. The protein was hydrolyzed for 2 hours at 100”. When cool, the tube was opened and the acid was re- moved by lyophilization. The residue was dissolved in water and relyophilized to remove any traces of acid. The hydrolysate was redissolved in 1.0 ml of w&er and a 0.5ml aliquot was as- sayed for hexosamines using a modification (20) of the procedure of Elaon and Morgan (21). The remaining 0.5.ml aliquot was analyzed for total carbohydrate content by the indole method (22).

Phosphorus Determination

Colicin prepara.tions were dialyzed exhaustively against dis- tilled water and analyzed for total and inorganic phosphorus by the method of Lowry et al. (23).

Circular Dichroism Measurements

The circular dichroism spectra of purified colicins E1, Ez, and Es were determined at room temperature in 0.1 M potassium phosphate buffer, pH 7.0, with a Durrum-Jasco model J-10 spec- tropola,rimeter, which records the angle of ellipticity, 0, in de- grees. The molecular ellipticity [0] (degrees cm2 per decimole) was calaulat’ed from Equation 1:

e. J!!R w [el = -

10.1.c (1)

where -‘MRW is the mean residue weight, I is the path length, and C is t’he solute concentration in grams per ml.

Colicins Ez and Ea were prepared as previously described (3). Protein concentrations were estimated from the absorbance of solutions at 280 nm using knorvn extinction coefficients. Mean residue weights of 110, 108, and 107 for colicins El, Ez, and Es, respectively, were obtained from total amino acid composition.

NN&erminal Analysis

Quantitative estimation of the KHz-terminal amino acid(s) of colicin E1 was performed using a modification (24) of the dini- trophenyl method of Sanger (25). Approximately 0.2 pmole of

colicin, oxidized with performic acid as descrtbed above, was dis- solved in 0.1 M NaHC03 containing 8 M deionized urea to yield a 0.1 y0 protein solution. After adding 0.1 ml of 1-fluoro-2,4- dinitrobenzene, the solution was incubated for 4 hours with occa- sional stirring. All derivatized protein was precipitated by the addition of 5 ml of 1 Y HCl. The precipitate was sedimented in a clinical cent,rifuge and washed five times with 5 ml of 1 N HCl, followed by five 5-ml acetone washes and three washes with 5 ml of fresh ethyl ether. The precipitate was dried in vacua, quan- titatively transferred to a glass tube, and rinsed in with a total of 5 ml of distilled 5.7 x HCl. The tube was evacuated, sealed, and heated at 105” for 24 hours. The hydrolysate was dried by evaporation over NaOH pellets and the residue was dissolved in 4 ml of distilled water. After adjusting the pH of the solution to 1.5 to 2.0 by adding a few drops of 4.7 N HCl, 2 ml of fresh ether were added and the mixture was vigorously mixed to extract DNP’-amino acids. The phases were separated by centrifuga- tion in a clinical centrifuge and the upper, ether phase was as- pirated off into a conical bottom tube. Ether extraction was re- peated an additional three times. All extracts were combined and dried under a stream of Nz. The dry residue was dissolved in approximately 0.1 ml of acetone and applied to a sheet of Whatman No. 1 filter paper, previously treated with 0.05 M

phthalate buffer, pH 6.0 (26), and dried at room temperature. DNP-amino acids were identified by paper chromatography versus authentic standards (Mann) using the two-dimensional system of Phillips (27), consisting of ascending chromatography in set-butyl alcohol saturated with 0.05 M phthalate buffer, pH 6.0, in the first dimension, followed by descending chromatog- raphy in 1.5 M phosphate buffer, pH 6.0. Resolution of DNP- methionine sulfone from DNP-serine and DNP-threonine was obtained with the fert-amyl alcohol chromatographic system of Blackburn and Lowther (26). To quantitate the recovery of NH*-terminal aanino acids, the resolved spots were cut from the chromatogram and transferred to a test tube. The DNP-amino acid was eluted with 1% NaHC03 for 15 min at room tempera- ture and filtered over glass wool and the absorbance of the extract was determined at 360 nm. After first correcting the optical densities with the appropriate conversion factor (28), recoveries of DNP-amino acids, calculated from the molar extinction coeffi- cient (29, 30), were adjusted for losses by comparing with the re- covery of total protein as determined from the amino acid analy- sis of the DNP-protein hydrolysate. Derivatized residues, such as c-DNP-lysine, 0-DNP-tyrosine, and im-DNP-histidine, which were not resolved on the amino acid analyzer, were normalized to the recovered leucine value. The NHz-terminal residue(s) of unoxidized colicin E, was also determined as described above; however, recoveries were considerably reduced.

RESULTS

Puri$cation of Colicin El

Extraction of Co&in-The extracellular nature of colicin El permitted the extraction of active protein from the cell surface without lysis, affording a high initial specific activity. All opera- tions were performed at 4”. The cell mass obtained from 40 liters of culture was transferred to a Waring Blendor to which was added 500 ml of 0.1 3r1 potassium phosphate buffer, pH 7.0, con- taining 1 lt NnCl. The cells were resuspended and mixed with

1 The abbreviations used are: DNP, 2,4-dinitrophenyl-; CM- Sephadex, c,2rbo~ymetIlyl-Sephadex; CD, circular dichroism.

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extractant by running the blender on low speed at 30 volls for 30 min. After centrifuging the suspension at 16,300 X g for 10 min, the supernata.nt was decanted and saved. Three additional 500.ml extractions were carried out and the pooled extracts were adjusted to a total volume of 2 liters and assayed for colicin ac- tivity and protein concentration.

Salt Fractionation-A beaker containing the combined extracts

FIG. 1. DEAE-Sephadex column chromatography of ammo- nium &fate-fractionated colicin El. The column (4.5 X 60 cm) was equilibrated with 0.01 M potassium phosphate, pI1 7.0, made 0.8.5y0 in NaCl. Elution with the same buffer was carried out at 4O and V-ml fractions were collected. Absorbance at 280 mM (m--O) and colicin activity (O-----O) were determined for each fraction.

FRACTION NUMBER

Pro. 2. Elution profile of colicin EI from CM-Sephadcx. The column (2.5 X 40 cm) was equilibrated wit,h 0.05 M sodium borate, pII 9.5, and eluted at 4’ with a 1400-ml linear salt gradient of 0.0 to 0.3 M KC1 in borate buffer. Fractions of approximat,ely 5 ml tvere collected. 0 ~-~- 0, absorbance al, 280 mp; O- - -0, coli- tin activity; q - --O, conductivity.

was placed in an ice bath and 484.6 g of enzyme grade (NH&SO4 (Manrl) were slowly added with stirring to yield a final (NH&SO* concentration of 40% saturation. After continued stirring for 30 min the resulting precipitate was sedimented at 16,300 x g for 10 min at, 0” and discarded. The supernatant was returned to the ice bath and a 40 to 60% fraction was obtained by adding an additional 260.0 g of (NH&S04. The mixture was again stirred for 30 min and centrifuged as above. The precipitate, containing virtually all of the colicin activity, was dissolved in 40 ml of 0.1 M potassium phosphate buffer, pH 7.0, and dialyzed against 4 liters of this buffer at 4“ for 12 hours.

DEAIGXephadex Column Chromatography-The dialyzed (NH&SO4 fraction in a volume of 64 ml was added to a DEAE- Sephadex A-50 (Pharmacia) column, 4.5 x 60 cm, equilibrated with 0.1 M potassium phosphate buffer, pH 7.0, containing 0.8597, NaCl. After rinsing with three lo-ml aliquots of the equilibra- tion buffer, chromatographic separation was obtained by eluting the column with 1 liter of the equilibration buffer. A distinct yellow band was retained at the top of the column, leaving the resulting eluate colorless. Fractions of 15 ml were collected and analyzed for absorbance at 280 nm. Selected fractions were as- sayed for colicin activity. Fig. 1 is a typical elution profile from DUE-Sephadex. Fractions 11 to 18 were pooled, assayed, ex- haustively dialyzed against distilled water, and lyophilized.

CM-Sephadex Column Chromatography-The lyophilized pro- tein from the DEAE-Sephadex column was dissolved in 30 ml of 0.05 M sodium borate, pH 9.5, and applied to a CM-Sephadex C-50 (l’harmacia) column, 2.5 X 40 cm, prepared in the same buffer. After three 5-ml rinses with column buffer, elution was obtained with a 1400-ml linear salt gradient consisting of 700 ml of 0.05 M sodium borate, pH 9.5, in the mixing chamber and 700 ml of borate buffer containing 0.3 M KC1 in the reservoir. The 5-ml fractions collected were examined for absorbance at 280 nm and colicin activity. The salt gradient was followed by measur- ing the conductivity of the fractions with a Radiometer type CDM 2d conductance meter. AS shown in Fig. 2, colicin activity elutes as a single, homogeneous peak corresponding to a concen- tration of approximately 0.07 M KC1 in borate buffer. Fractions 128 to 140 were pooled and exhaustively dialyzed against dis- tilled water, and the protein solution was lyophilized. The re- sults of the purification procedure are summarized in Table I.

Immurwdiffusion Studies

Antibody wells were filled with undiluted antiserum prepared as described and a 0.1 y0 solution of colicin El in 0.1 M potassium phosphate buffer, pH 7.0, containing 0.15 M NaCl was added to the circumferential wells. The immune reactants were permitted to diffuse toward each other at room temperature for 24 hours. The resulting reactions are shown in Fig. 3. Immunochemical homogeneity of colicin E, was demonstrated by the appearance of

step VChlle Activity Protein Specific activity Recovery Purification __c______

ml units mg zmits/mg %

Pooled NaCl extracts 2 x lo” 1 .o x 108 6000 1.7 x 104 100 40-60yc (NHa)&Oa fraction.. . . . . . . 64 I .3 x 108 1.500 8.7 x 104 130 5.1 Pooled DEAE-Sephadex peak fractions. 109 4.5 x 107 305 1.5 x 10” 45 8 .6 Pooled CM-Sephades peak fractions. 6t2 2.5 x 107 I74 1.5 x 105 25 X.6

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FIG. 3 (left). Double immunodiffusion analysis of colicin El in agar. mologous antiserum (Ab El) was undiluted.

Colicin El @I) concentration was 1.0 mg per ml and the ho- The reaction was stopped after 24 hours at room temperat,ure in a water-saturated atmos-

phere by soaking the gel in 0.15 M NaCl to dilute soluble reactan&. FIG. 4 (rigkl). Cellulose acetate electropherograms of colicin El. Solut,ions of colicin El (1 mg per ml) in t,ray buffer were applied

to the cellulose acetate strips and electrophoresed at room temperat,ure nnder t,he following condit,ions: A, 30 min at pH 4.5; B, 45 min at pH 8.6. -4 constant current of 1 ma per strip was maiotaincd. The gels were stained wit,h O.V$ Ponceau S.

a single precipitin line when reacted in double diffusion experi- ments with antisera prepared against the purified protein.

Electrophoresis

CeZZuZose Acetafe EZectrophwesis-Fig. 4 shows the stujncd electropherograms of colicin E, obtained by electrophoresis of the active protein from CM-Sephadex chromatography at two dif- ferent p1-I values, using either pH 4.5 /3-alanine-acetic acid buffer (31), or a 0.05 M sodium barbital buffer, pH 8.6. Colicin E1 migrates as a single band toward the cathode in bo& systems. h\icrodensitometer tracings of the resolved bands 0\-1 cellulose acetate, using a Joyce-Loebel double beam recording micro- densitometer, revealed only a single component even I&en thr protein concentration was increased 5-fold to 5 mg pry ml.

Isoelectric Focusing-The isoelectr,c fractionation profi]r of 7 mg of purified colicin E1 in a pH 8 to 10 linear gradletlt 1s shown ill Fig. 5. A high degree of homogeneity is suggehted by the sharp single peak at pH 9.03. From the results of several experi- ments, the average isoelectric point (PI) of colicin E1 equal\ 9.05, indicating a relatively basic protein.

Gel Piltrcrtion

Colicin El WS~S excluded in the void volume in preliminary gel

filtration experiments with Sepha&x G-100. Using the ]es>s densely cross-linked Sephadex G-200, the active prot,ein was in-

cluded in the inner volume of the column, affording resolution of any macromolecular components. A column, 2 x 40 cm, equili- brated with 0.1 M potassium phosphate buffer, pH ‘7.0, jvas charged with 5 ml of column buffer containing 5y0 sucrose and 20 mg of lyophilized colicin El. The dense protein solution was layered under the eluant through a thin tube attached to a Crough-Keyes pipette. To elute the sample, the column wa.s corlnected t,o a Mariotte flask filled with 0.1 M potassium phos- phate buffer, pH 7.0. Fig. 6 shows the elution profiles of blue de&an and colicin E, from Sephadex G-200. No other protein components were resolved from the sample, which eluted as a single symmetrical peak behind t’he void volume. In additim to size homogeneity, these observations irldicute that colicin E1 has a high molecular weight or exists as an extended molecule in solu- tion (or both). Fractions 35 to 41 were pooled and found t,o have t.he same specZic activity as the active fract,ions from (XI-Se+- dcx chrornat,ography.

Bydrodynamic PToperties

8edimentation Ve&&ly Crllra~en~ri~?~g&ion-~-ar~ous concen- trations of colicin EI were prepared in 0.1 M potassium phosphate buffer, pII 7.0, and centrifuged at 59,180 rpm at 20”. ‘I’he sedi- rnerltation pattern for a 9.23 rng per ml solution of co&cm El is shown in Rg. 7. Homogeneity of the preparation on the basis of hydrodynamic criteria is suggested liy the single, symmetrical

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> e*......... IO 20 30 40 50 60 70 80 90 100

FRACTION NUMBER

FIG. 5. Electrofocusing of purified colicin El with carrier ampholyt,es. The experimental procedure is described in the text. a-0, absorbance at 280 rnp; O- - -0, colicin activity; O-O, PH.

16

FRACTION NUMBER

FIG. 6. Gel filtration of purified cohcin El on a Sephadex O-200 column. Blue dextrnn 2000 in the equilibration buffer, 0.01 M pot,assium phosphate, pH 7.0, was used to monitor the column, 2 x 40 cm, for irreglllarit’ies and to determine the void volume. Coliciu E, (20 mg of lyophilized protein) was eluted wit,h equili- bration buffer and 2.ml fractions were collected.

peak. To determine the sedimentation coefhcicnt at iufinite dilution (s&J the sedimentation coefficientIs obtained for solu- tions of varying concentrations were plotted and extrapolated to zero concentration (Fig. 8). From these results, colicin E, was calculated to have an s$,~ equal to 3.0 X lo-l3 set, corresponding to a minimum molecular weight of 23,300 for an unhydrated sphere. Gel filtration data implied, however, that cohrin El was much larger than 23,300. To resolve the anomaly, addltional experiments were undertaken to determine those hydrodynamic parameters necessary to define the molecular weight.

Determination o.j Diffusion Coeficient-The drffusion constant was measured as a function of the spread of an artificial boundary formed between the protein solution and solvent. Solutions of varying concentrations of cohcin E, were centrifuged at 11,272 rpm in a double sector, capJlary type synthetic boundary cell. Temperature was maintained at 20”. The diffusion of the pro- tein solution into the solvent layer was followed by observing the spread of the schliereu peak. The results of these analyses are

FIG. 7. Sedimentation of purified colicin EI in the analytical ultracentrifuge. A 9.23 mg per ml of solution of colicin E1 was prepared in 0.01 M potassium phosphate, pH 7.0, and centrifuged at, 20”. The pattern was recorded at a bar angle of 55’ 152 min after attaining 59,780 rpm. Sedimentation was from left to right.

CONCENTRATION ~mqlmll

Fro. 8. Sedimentation coefficient as a function of concentra- Con of colicin El. The values have been extrapolated to yield t,he sedimentation coefficient at infinite dilution.

5,

1 -9

8 IO

CONCENTRATION imp/ml)

! i

FIG. 9. Dependence of the diffusion coefficient on the concen- tration of colicin Et. The values have been extrapolated to yield the diffusion coefficient at infinite dilution.

shown in Fig. 9. Extrapolation of the diffusion constant to in- finite dilution (D.$,,,) yields a value of 4.2 x lo-' cm2 see-l.

Partial Speci$c Volume-One additional property, the partial specific volume (o), was needed to provide a reliable estimate of the molecular weight of colicin El. From pycnometric measure- ments using a Mettler model M5 microanalytical balance and a 5.0-cm3 glass pycnometer, ii was found to equal 0.725 cm3 g-l.

Together, the assembled hydrodynamic data define the mo- lecular weight, *U, of colicin Er as determined from Equation 2:

&I = RTs

D(1 - BP)

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6324

.4mino acid ___.

Colicin EI“ Colicin Id (‘olicin JSa”

Lysine. (-

Histidine

ilrgininc 27 31 Cysteine 1C 1 Sspartic acid. 52 78 Threonine. 25” 21 Scrinc 31d 39 Glutamic acid. Proline Glycine. Alanine. Valine Nethionine Isoleucine. Leucine. Tyrosine. Phenylalanine Tryptophan.

72

7 28 82 23e

4 27e

43 12 11

3’ 1

.53 30 59

56 3G

7 20

24 7

17

10 -

where M is the molecular weight, R is the gas constant, 1’ equals the absolute temperature, s is the sedimentation coefficient, L) is the diffusion coefficient, ii equals the partial specific volume, and p equals the solvent density.

Substituting the appropriate values in the above equal,ion yields a molecular weight of 63,013 for colicin E,.

Sedimentation Equilibrium Ultracentrifugation-Another tech- nique based upon the attainment of an equilibrium state, sedi- mentation equilibrium ultracentrifugation, was also used to deter- mine the molecular weight of colicin El. The use of a Yphantis six-chamber centerpiece permitted the simultaneous analysis of three different samples. Colicin solutions in 0.1 M potassium phosphate buffer, pH 7.0, were centrifuged for 20 hours at 16,200 rpm to establish an equilibrium concentration gradient. The computerized results obtained as described under “Experimental Procedure” gave the molecular weight of the polymeric solute for several overlapping intervals along the gradient. Colicin E1 was homogeneous with regard to molecular weight by this test. In addition, no concentration-dependent aggregation or dissociation was observed as evidenced by comparable molecular weight,s at both ends of the gradient, which corresponds to regions of rela-- tively high and low polymeric solute concentrations. The data obtained from sedimentation equilibrium ultracentrifrlg:~t;ion yielded an average molecular weight, of 56,000 + 700 for colicirr El.

Amino Acid Analysis

A sample of lyophilized colicin E1 from CM-Sephadex was dricatl in an Abderhalden drying apparatus and accurately lveighed.

TABLE II

Amino acid composition of colicins El, E2, und H’:a

a Average values for 12., 24., and &hour hydrolysates (cxc~pt

as noted) of two separately purified samples calculated as residues per 55,000 daltons rounded off to the nearest whole nllmhcr.

h From Nerschman and Helinski (3).

c After oxidation to eysteic acid by performic acid. d Values determined by extrapolation to zero time of h,ydrr~l,ysk. 8 Values obtained from M-hour hydrolysis only.

f Determined from the average of spektrophotornet ric and chemical analyses.

The water-free protein was hydrolyzed in 5.7 x KC1 for 12, 24, or 48 hours, and analyzed for amino acid composition as dcscrihed. Separate samples were oxidized with performic acid prior to hi-= drolysis to convert all cysteine residues to the stable cy&ic acid derivative. Table 11 shows the average values of amino acids for two separately purified samples of colicin El for severs1 times of hydrolysis together with the amino acid compositions of coli- tins I& and ES for comparison. From the quantitative a&J-ses, 96% of the dry weight of colicin E, was recoverable as amino acids, indicating that colicin El is a simple protein. Tests for total carbohydrates, reducing sugars, and hexosamines indicated that less than I y0 of the dry weight of colicin E1 could he at- tributed to these moieties. Similarly, less than 0.1 mole of or- ganically bound phosphorus was associated with each mole of protein, lending further support to the conclusion that colicin El is a simple protein.

Extinction Coe$cient

An aliquot of exhaustively dialyzed colicin E1 in distilled water was transferred to a tared weighing bottle, lyophilized, and dried in an hbderhalden drying apparatus. A separate aliquot was mixed with an equal volurne of 0.02 M potassium phosphate buffer, p1-I 7.0, and the ultraviolet absorption spectrum was de- termined in a Unicam SP800 recording spectrophotometer. The absorption spectrum obtained was characteristic for proteins with a maximurn at 279 rim. Dry weight analysis of colicin El in the original solution together with the absorbance of the neu- tral solution at 280 nm yielded arl extinction coefficient for a 1 ‘;;, neutral solution (Ei%) equal to 7.36. This value was routinely used for spcctrophotolnetrically det,errnining the concentr:\tion of colicin E, solutions.

Circular Dichroism Spectra

The CD spectra for approximately equimolar residue concell- trations of purified colicirw E, (1.79 x 1OF decimole per crn3)> E, (1.88 x 1OF decimole per cm”), and Es (1.93 X low5 dt~cGrnole per cd) in 0.1 M potassium phosphate buffer, pH 7.0, art’ showvrl in E’ig. 10. Although all three curves seem to be a composite of contributions frorn oc-helix, ,f3 structure, and random coil, when evaluated by the method of Greenfield and Fasman (32)) the CD spectrum ol colicin E, appears to be considerably more influcrlced ljy a-helix than the spectra of colicins & or Ea. CD mt-‘:~sure- rnents of these latter two colicins are suggestive of a si$rlificarrt dcgret: of random coil conformafion.

NJJi?-terminal A nalysis with the Xauger Reagent

As shown in Table III, thr, ether-soluble l)SP-amino acid oh- lairred f’rom t,he hydrolysis ol’ native: colicin El had a mobilir> coincident with that of authentic I INI’-methionine in several solvent, systems. The single residue ext,rncted from prrforrnic acid-oxidized colicin El cochromatographed with DNP-methio- nine sulfone, thus confirming the identity of the NI-IZ-rerminal amino acid. Moreover, approximately 0.7 mole of DKP-meI hi- oninc sulfone was recovered per mole of input protein. Recause OF the relatively low methiolline content of colicin El, it W:IS pos- s~l)le to show that the extraction of the NIIt-terminal DXP- amino acid resulted in the loss of approximately 1 mcthioninr sulfone residue per 55,000 molecular weight when an aliquot of the ether-extracted, oxidized colicin E1 \+x:: acid-hydrolyzpd and applied to the automatic amino acid :m:dyzer. The recovery Ol

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Issue of October 25 1071 7 c S. A. Wwarlz and D. R. Helinski 6325

-15i i we .J4 180 190 200 210 220 250 WAVELENGTH Imp1

FIG. 10. Circular dichroism spectra of purified colicins El, Ez, a.nd Ea. Protein solutions were prepared in 0.01 M potassium phosphate, pH 7.0, and examined at ambient temperature. Resi- due concentrations of colicins EL, Et, and Ea were 1.79 X 10e6, 1.88 X lF, and 1.93 X lo+ decimole per cm3, respectively.

TABLE 111

Identification of NHS-terminal DNP-am&o acid of colicin El by paper chromatography

Samples of native and performic acid-oxidized colicin El were analyzed for NH?-terminal amino acids as described in the text,. Solvents were: S1, set-butanol, pH 6.0 (30); SZ, 1.5 M sodium phos-

phate, pH 6.0 (30); SS, tert-amyl alcohol, pH 6.0 (29).

I RF

s1 SZ SS ~_

DNFresidue from native colicin El 0.22 0.77

DNP-methionine 0.21 0.77

DNP-residue from oxidized colicin El 0.38 0.41 0.43

DNP-methionine sulfone 0.37 0.41 0.43

a single NH2 terminus, methionine, without traces of other com- ponents is additional evidence for the purity of colicin El.

Activity Spectrum of Colicin El

A variety of bacterial strains were selected as indicators of purified colicin E1 activity. Several concentrations of purified colicin were prepared in nutrient broth and 0.02-ml aliquots were spotted on nutrient agar plates, seeded with the respective indi- cator at, an initial concentration of 2 X lo6 cells per ml. It is evident from Table IV that relatively high concentrations of colicin El were active against all of the E. coli strains tested, in- cluding the immune and resistant bacteria. At lower colicin concentrations, however, immune and resistant organisms were unaffected. Also, a Proteus mirabilis strain and an isogenic de- rivative containing the homologous colicinogenic factor were in-

TABLE IV

Range of activity of colicin El Approximately 0.02 ml of colicin solution of the indicated con-

centration was spotted on a nutrient agar plate seeded with the designated strain at a concentration of 2 X 106 cells per ml. All strains are E. coli unless otherwise designated. +, complete

inhibition of growth; -, no inhibition of growth; f, partial inhibition of growth. The characteristics of these strains have been described previously (33, 34). E1r, Ez*, S*, and Azr refer

to resistance to colicin El, colicin Et, streptomycin, and azide, respectirely. CoZEl, CoZEz, and CoZI refer to the possession of colicinogenic factors El, Es, and I, respectively.

Strain

caoo SrE1r. ..........................

JC411. .............................. JC411E2’ ........................

JC411(ColE:1) ....................... JC411 (CoZE2 CoZT) ................... qs ................................... w311o(coZE~). .......................

W3110(CozE2). ....................... W3110(CoZE,)E&~. ..................

W3110(CoZE3 CoZI)E&r ..............

YS40VrAzr ........................... YS40E~‘~Azr. .......................

YS40(CoZE1) .........................

YS40(CoZE2)Azr ......................

Proteus mirobilis (ColEI). ............

Proteus mirabilis .....................

-

-

Effect of colicin EI

1 mg/mi

+

+

+

+

+

+

+

+

+

+

+

+

+

+ -

-

I-

-

10 /%/ml

t -

-

+ + -

+ - -

+ - -

+ - -

sensitive to even high concentrations of colicin El purified from E. coli.

DISCUSSION

The purification of colicin from a complex medium was aided by the binding of the majority of active protein to the surface of the induced bacteria. Since colicin can be extracted and sub- sequently purified without disrupting the cells, only about a lo-fold purification is necessary to obtain a homogeneous prep- aration as determined by chromatographic, electrophoretic, immunochemical, and hydrodynamic criteria. Colicin E1 differs considerably from the other members of its resistance class. Unlike colicins Es and ES, whose isoelectric points Iie close to neutrality (3), colicin El was found to be a relatively basic pro- tein. The pa-dependent elution of colicin El from CM-Sepha- dex was effected only at approximately pH 9.5. Electrophoretic mobility also suggested a positively charged protein. Electro- focusing experiments yielded a value of pH 9.05 for the isoelectric point of colicin E1. Notable differences between the amino acid compositions of colicin El and colicins Ez and Es are evident, with particular regard to the lysine, aspartic acid, glutamic acid, proline, glycine, alanine, leucine, and tryptophan contents. The latter two proteins, however, have been shown to have a high de- gree of similarity in amino acid composition (3). The presence of a single residue of cysteine per molecule of colicin E1, Ez, and

Ea suggests that the proteins are composed of either a single poly- peptide chain or nonidentical subunits. Recoveries of approxi- mately equimolar amounts of only one NHz-terminal amino acid,

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6326 Vol. 246, x-0. 20

Characteristic

Ns,ture of active molecule

Molecular weight

0 ~20,w Isoelectric point Amino acid

composition

Cyst,eirres per moleciile

NHZ~terminal amino acid(s)

Immrlrlologv

Cclicin El

Simple pro- tein

:,6,000

3.0 s 9.05

Rich in charged amino acids

Met

Colicin W

Simple pr+ kill

60,000

4.0 s 7.11-7.63 Rich in

chargctl amino acids

1

n From Hcrschmarr and Helinski (X).

methionine, per mole of col,cLn El are corisistent with the former

alternative. The absence of other NHf-terminal amino acids supports t,hc homogeneity of the cohcin I$ preparations.

CD measurements also clearly iiidiceted the structural diffcr- ences between colicin E1 and cohcins Ea and Ea. The analysis of Greenfield and Fasman (32) of the ellipticity at 208 nm of colicins

Ei, E,, and El indicates that these proteins contain 41, 21, and 25% cr-helix, respectively. Although the spectra of colicins EZ :and E3 are sirnilar, the shift between them may be due to a slightly higher random coil content of colicin Ez.

During the characterization of colicin E,, it was noted that the sedimentation coefficient at, infinite dilution, 3.0 S, corresponded to an unhydrated hard spkiere wit’h a minimum molecular weight of 23,300, conflicting with size estimates from both gel filtration data and sedimentation equilibrium studies. Colicin El was ex- cluded in the void volume of Sephadex G-100. This gel report- edly can fractionat,e globular proteins in the molecular weight range of 4,000 to 150,000, excluding larger species. Sedimenta- tion equilibrium studies revealed t,hat the molecular weight of colicin Ei was 56,000. Since the sedimentation constant is a re- flection of both the size and shape of a molecule, the translational diffusion coefficient and the partial spec;fic volume were also determiiied to provide a confirmation of t,he molecular weight and information on the configuration of colicin El. Together t,hese hydrodynamic data yielded a molecular weight estim:it,e of 63,060, in general agreement. with t,he equihbriuni findings. Furthermore, from the calculated frictional ratio (//Jnlin = 2.02), the axial ratio of unhydrated colicin E1 was deterrnincd to be 20 for a prolat,e ellipsoid with major and minor axes of 19 A and 370 A, respectively, usin, 0‘ a graphical representation (35) of I’errin’s function (36). These findings, together with the gel filtration data, are suggestive of extensive axial dissymmetry of the colicin Ei molecule, which contrasts with the hydrodynamic behavior of colicins ES and Es (3). It is of interest that, purified colicins I, and Ih similarly possess unusually high axial ratios (37).

At relatively low concentrations (10 /.4g per ml), colicin El, aS expected, was active against neither immune nor resistant orga-

nisnis. However, concentrations of 1 mg per ml were capable of killing all of the E. coli strains t,hat were examined, including both immune and resistant, bacteria. Immunity breakdown has been previously observed when colicinogcnic cells were treated with relatively high concent,rati.ons of homologous coliciu (4, 38). Several explanations for this phenomenon have been suggested (38, 39). Iligkr colicin multiplicities may overcome a finite num- ber of imniunity substances present within the colicinogenic cells, or t,he combined effects of a local action at ruarly receptor sites may in summation lead to cell death. Alternat~ively, irrinnmity may reduce the number of lethal receptors without affecting the total binding capacity, a higher colicin concentration thereby in- creasing the probability of a killing particle finding an effective receptor site.

Contrary to the results obtained with the Group 1 colicins (38), colicin El at high concentrations was even effective on re- sistant cells. These cells have supposedly lost their specific re- ceptors and consequently cannot bind colicin (4, 40). The lack of sensitivity of P. wrirabilis strains to colicin El indicates the specific nature of colicin activity even under conditions of re- sistance breAkdown using high colicin concentrations. Conse- quently, cert,airi cases of resistance may riot’ result in the complete loss of receptors, kiut,, rather, may affect the ability to bind colicin. ITigh colicin concentrations may thus shift the equilibrium to favor the bound state. In contrast to Nomura’s model of colicin action, specific binding may not be a necessary prerequisite to the lethal effect. The receptor site may merely orient tkie colicin molecde to its biochemical target, thus increaking tkie pI9bdb~bty

of a lethal event. Fredericq’s (1, 2) original classification of the group E colicins

was krased on a common biological functiori, binding to the same receptor site. Studies by 11111 and IIolla~~d (51) indicate that the adsorption site for colicin Ei is dist,iuct front tkiat for colicins Eg arid I!$, althugh the receptors for ii11 three colicins seem t,o pOS-

sess some common components. Tkie physicochernical data pre- sent,ed here further suggest that,, although colicins Es and Ep are similar (3), colicin Er is quite distinct from other members of it.5 resistance class. A coniparison of some of the properties of the E colicins is sumniarized in Table V. Conformational and pri- mary st,ructure analyses may reveal the structural basis for the functional similarit,ies and specificities anmng tile group E coli- curs and whctkier these are tkie results of aucestral homology or convergence from separate origins.

i2ckrL~wledgvnents-The authors wish to thank gratefully l’ro- fessors Russell F. Doolittle and 8. .J. Singer for their advice and mauy stiniulating discussions and D. Hopkins for assistance on a corrrput,er program for analysis of sedimentation equilik)rium data.

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Stanley A. Schwartz and Donald R. Helinski1Purification and Characterization of Colicin E

1971, 246:6318-6327.J. Biol. Chem. 

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