Proc. Natl. Acad. Sci. USA 75 (1978) 4067

Correction. In the article "Membrane-bound neuraminidasefrom calf brain: Regulation of oligosialoganglioside degradationby membrane fluidity and membrane components" by K.Sandoff and B. Pallmann, which appeared in the January 1978issue of Proc. Natl. Acad. Sci. USA (75,122-126), an undetectedprinter's error occurred in the second paragraph of Results.Line 18 should read: "centrations of 0.1 as well as 2.0 mM. Onthe contrary, the deg-"

Also, the description of the incubation mixture in the legendsof Figs. 1 and 4 is slightly in error. The buffer used throughoutin this paper was 0.15 M sodium acetate that had been titratedto pH 4.2 with acetic acid.

Correction. In the article "The disulfide bond connecting thechains of ricin" by Douglas A. Lappi, Wolfgang Kapmeyer,Janice M. Beglau, and Nathan 0. Kaplan, which appeared inthe March 1978 issue of Proc. Natl. Acad. Sci. USA (75,1096-1100), the authors request that the following paragraphbe added on p. 1097, preceding the paragraph entitled Mo-lecular Sieve Chromatography:Recombination of Separated A and B Chains. Both chains

were dialyzed separately against 10 mM phosphate buffer (pH8.0) with 10 mM lactose. The solutions of both chains were

mixed in equimolar amounts, giving a final protein concen-

tration of 0.5 mg/ml. This mixture was then made 0.6 mM inoxidized glutathione and 0.2mM in reduced glutathione. Afterincubation for 24 hr at 22°, the recombination was completeas judged by NaDodSO4/polyacrylamide electrophoresis.

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Proc. Natl. Acad. Sci. USAVol. 75, No. 3, pp. 1096-1100, March 1978Biochemistry

The disulfide bond connecting the chains of ricin(A-B chain complex/reduction of ricin/protein synthesis inhibition/ultracentrifugation/ricin purification)

DOUGLAS A. LAPPI, WOLFGANG KAPMEYER, JANICE M. BEGLAU, AND NATHAN 0. KAPLANDepartment of Chemistry, University of California, San Diego, La Jolla, California 92093

Contributed by Nathan 0. Kaplan, November 28, 1977

ABSTRACT Studies on the disulfide bond connecting thetwo polypeptide chains of ricin are reported. Reduction of thisbon in the native protein requires approximately 50-fold moremercaptoethanol than the reduction of the bond in the proteindenatured by sodium dodecyl sulfate. An improved procedurefor the formation of this disulfide bond from recombined chainsis reported. A and B chains spontaneously and rapidly reasso-ciate into a stable complex with a sedimentation velocity similarto that of native oxidized ricin before the disulfide bond re-forms. The mixture of both chains also behaves on Bio-Gel P-100like native oxidized ricin. However, the complex formed by thetwo chains, assayed before the disulfide bond can reform, andreduced ricin, carboxymethylated to prevent reoxidation, showsa significant decrease in toxicity to mice and a decrease inability to inhibit protein synthesis in HeLa cells in culture.

Ricin, the toxic protein from the beans of Ricinus communis,consists of two polypeptides connected by one disulfide bridge(1). The larger polypeptide with a molecular weight of 35,000,the B chain, binds to cell surface receptors with an associationconstant close to that of intact ricin (2). The smaller polypeptidewith a molecular weight of 30,000, the A chain, inhibits cell-freeprotein synthesis by catalytically inactivating the 60S ribosomalsubunit (3). Lin et al. (4, 5) reported that ricin is able to preventdevelopment of ascites tumors in mice, and ricin has been usedin the treatment of uterine cancer (6). In our efforts to evaluatericin as an anti-tumor agent, we have undertaken studies tounderstand the mechanism of action in greater detail. We haveexamined the disulfide bond connecting the two chains withrespect to their role in binding the two chains together and thetoxicological activity of the ricin. Our results indicate that (i)the disulfide is necessary for inhibiting protein synthesis inHeLa cells in culture and for toxicity in mice; (ii) the disulfidebond connecting the chains in the native protein is approxi-mately 50-fold more resistant to the reduction by chemicalreagents than the disulfide bond in the denatured toxin; (iii)upon reduction and carboxymethylation, the two chains remainbound together; and (iv) when mixed together, purified A andB chains form a stable complex with a sedimentation velocitysimilar to that of nonreduced ricin. The complex has reducedtoxicity. In addition, we report a procedure for the purificationof ricin.

MATERIALS AND METHODSRicinus communis beans were purchased from Geo. W. ParkSeed Co., Greenwood, SC. Mercaptoethanol (lot 76C-0049),reduced glutathione (lot 36C-0046), dithioerythritol (lot36C-0081), and iodoacetamide were purchased from SigmaChemical Co., St. Louis, MO. All other reagents were fromstandard commercial sources.

Preparation of Ricin. All steps were performed at 40 unlessotherwise noted. Decorticated Ricinus communis beans (185g) were stirred overnight in 500 ml of 5% acetic acid and ho-mogenized in a Waring blender. Additional 5% acetic acid wasadded to make a smooth suspension. This material was centri-fuged at 10,400 X g for 20 min. The oil was scooped off andammonium sulfate was added to the supernatant to 80% satu-ration. The precipitate was collected by centrifugation, redis-solved in distilled water, and dialyzed against distilled waterfor 18 hr. The resulting yellow solution was passed through aDEAE-Sephadex column (3.5 X 23 cm) equilibrated with 5mMsodium acetate (pH 5.8). Ricin did not bind to this ion ex-changer and appeared in the void volume in a colorless solutioncontaminated with material that had properties similar toagglutinin and heterogeneous low molecular weight material,as judged by sodium dodecyl sulfate (NaDodSO4) gel electro-phoresis. This solution was treated with ammonium sulfate andthe precipitate was redissolved in a minimum volume of 10mMphosphate buffer (pH 7.4). Four hundred milligrams of thissolution (approximately one-third of the total) was applied toa Sephadex G-100 column (5.5 X 55 cm) equilibrated in a 10mM phosphate buffer (pH 7.4). Two sharp peaks, one with theproperties of agglutinin, the other, ricin, were eluted, along witha third broad peak. A total yield of 800mg of ricin was obtained.When injected intraperitoneally, 300 ng of the purified ricinwas toxic to Swiss mice within 72 hr.A and B chains were purified by a modification of the

method of Olsnes and Pihl (3).Reduction of Disulfide Connecting the Chains of Ricin.

Ricin was reduced in the native state and denatured state withthe different reducing agents, 2-mercaptoethanol, dithioer-ythritol, and glutathione. For the reduction in the native state,to 100 ,l of a ricin solution (3.8 mg/ml) in 0.4 M phosphatebuffer (pH 7.4) was added 2-mercaptoethanol to the concen-trations indicated in the figure legends. The mixture was in-cubated at room temperature (210) for 20-30 min and appliedto a Sephadex G-10 column (1.1 X 12 cm) to remove the mer-captoethanol. The peak fraction from this column was made1% (wt/vol) in NaDodSO4. This was followed by NaDodSO4gel electrophoresis to determine the extent of reduction. Thetime between application to the Sephadex G-10 column andaddition of NaDodSO4 was less than 12 min. The same proce-dure was used to reduce native ricin with dithioerythritol andglutathione. The reduction of the denatured enzyme was fol-lowed directly by electrophoresis, omitting the Sephadexchromatography.Carboxamidomethylation of Ricin. Ricin (2 mg) was dis-

solved in 0.6 ml of 20 mM phosphate buffer (pH 7.5), 10 mMlactose, 0.5 M galactose, and 5% mercaptoethanol. The mixturewas incubated for 16 hr at room temperature. The excessmercaptoethanol was removed by dialysis twice against 1 liter

Abbreviation: NaDodSO4, sodium dodecyl sulfate.

1096

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Natl. Acad. Sci. USA 75 (1978) 1097

C D E F

:-he->

-EE-t-w-t^

A B C D E F G H I

FIG. 1. (Upper) Gel electrophoresis of native ricin reduced with mercaptoethanol. Samples were run in duplicate. Mercaptoethanol con-

centrations were (A) 596, (B) 2.5%, (C) 1%, (D) 0.5%, (E) 0.25%, and (F) 0.1%. (Lower) Denatured ricin reduced with mercaptoethanol. Mercap-toethanol concentrations were (A) 5%, (B) 2.5%, (C) 1%, (D) 0.5%, (E) 0.25%, (F) 0.1%, (G) 0.05%, (H) 0.025%, (I) 0.01%, and (J) 0.005%.

of 20 mM sodium phosphate/10 mM lactose/0.1 mM EDTAat pH 7.4 for 6 hr. This dialysis and the following modificationwere performed under nitrogen to prevent any reoxidation byoxygen. To the reduced ricin 17.5 mg of iodoacetamide, dis-solved in 35 Atl of dry methanol, was added and the reaction wasallowed to proceed for 6 hr at room temperature under nitro-gen. Hydrolyzed iodoacetamide was removed by dialysisagainst 20 mM phosphate/10 mM lactose/0.1 mM EDTA atpH 7.5. A complete carboxamidomethylation was achieved byrepeating the reduction with mercaptoethanol and the reactionwith iodoacetamide.

Molecular Sieve Chromatography. Ricin, reduced ricin, andthe separated chains were chromatographed on a Bio-Gel P-100mesh column (1.6 X 140 cm). The column was equilibratedwith 20 mM phosphate, 50 mM NaCl, 10 mM lactose, and0.02% NaN3. The flow rate was 4.5 ml/hr. The reduced ricinwas chromatographed in the same buffer containing additional0.1% mercaptoethanol. The samples contained 1.5 mg of pro-

tein in 0.5 ml of buffer, 20% sucrose was added to increase thedensity and the samples were underlayered on top of the col-umn. Fractions of 2.25 ml were collected and the absorbancewas read at 280 nm at a Zeiss spectrophotometer. The voidvolume of this column was 103 ml, determined with dextranblue; the salt volume was 340 ml, determined with reduceddithiobisnitrobenzoate.

Ultracentrifugation. Sedimentation coefficients were de-termined with a Beckman Model E ultracentrifuge equippedwith a photoelectric scanner (7). Protein concentrations of ap-proximately 0.5 mg/ml were used.

Toxicity for Mice. Toxicity was determined by the methodof Olsnes et al. (8).

Cells. HeLa cells, obtained from John Holland of the Uni-versity of California, San Diego, were maintained in a mixtureof Dulbecco's modified Eagle's medium and Ham's nutrientmixture F-12 (1:1) containing 15 fnM N-2-hydroxyethyl-piperazine-N-2-ethanesulfonate (Hepes), 100 units of penicillin

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Biochemistry: Lappi et al.

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Proc. Nati. Acad. Sci. USA 75 (1978)

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FIG. 2. Reduction of denatured ricin with dithioerythritol. Concentrations were (A) 0.5 M, (B) 0.25 M, (C) 0.1 M, (D) 50 mM, (E) 25 mM,(F) 10 mM, (G) 5 mM, (H) 2.5 mM, (I) 1 mM, (J) 0.5 mM, (K) 0.25 mM, and (L) 0.1 mM.

per ml, 100 tig of streptomycin per ml, 25 ,gg of ampicillin perml, and supplemented with 2.5% of fetal calf serum (Gibco) and5% horse serum (Gibco). The cells were grown in Falcon tissueculture dishes in a 5% C02/95% air atmosphere at 37O.Amino Acid Incorporation. Our procedure is a modification

of that used by Venter and Kaplan (9). Protein synthesis wasmeasured as a function of the incorporation of 3H-labeledamino acid mixture (New England Nuclear, NET-250) intotrichloroacetic acid-precipitatable protein. The cells werewashed with phosphate-buffered saline (free of Ca2+ andMg2+). Whatman GF/A filters were used, and radioactivity wasmeasured in Aquasol (New England Nuclear, NEF-934).

RESULTSA previous purification procedure (10) resulted in ricin that washomogeneous as judged by NaDodSO4 gel electrophoresis.However, we observed autoproteolysis in NaDodSO4-con-

A B C D E F

taining buffers (11). When this ricin preparation was treatedwith phenylmethylsulfonyl fluoride, the autoproteolytic activitywas removed but toxicity was retained, suggesting that theproteolytic activity was from copurification of a protease. Thisactivity was removed by chromatography on Sephadex G-100.The purification procedure presented here has the advantageof yielding a large quantity of ricin, also yielding purifiedagglutinin, and removing autoproteolytic activity.

Figs. 1-3 show the reduction of the disulfide bridge con-necting the two chains of ricin by the reducing agents mer-captoethanol, dithioerythritol, and glutathione. At room tem-perature, 5% (0.71 M) mercaptoethanol incubated with ricinfor 30 min was necessary to reduce the disulfide bridge com-pletely (Fig. 1 upper). Concentrations of dithioerythritol upto 0.5 M were unable to reduce the disulfide bridge under theconditions used. Increasing the temperature resulted in in-creased solubility of dithioerythritol to 1 M. However, even at

G H I J K L

C-- w......w

_.. _-I_% lw-.z4* =

FIG. 3. Reduction of denatured ricin by glutathione. Glutathione concentrations were (A) 0.25 M, (B) 0.1 M, (C) 50 mM, (D) 25 mM, (E)10 mM, (F) 5 mM, (G) 2.5 mM, (H) 1 mM, (I) 0.5 mM, (J) 0.25 mM, (K) 0.1 mM, and (L) 0.05 mM.

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1098 Biochemistry: Lappi et al.

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Proc. Natl. Acad. Sci. USA 75 (1978) 1099

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10 30 50 70 90 110Minutes

FIG. 4. Ultracentrifugation of ricin (3; slope 3.87 X 10-4), Achain (v; slope 2.67 X 10-4), B chain (0; slope 2.77 X 10-4), and A andB chain (A; slope 3.89 X 10-4). Ultracentrifugation was in 10 mMphosphate buffer (pH 8.0)/10 mM lactose. Photoelectric scans at 280nm were taken upon reaching speed and at 8-min intervals thereafter.Temperature was 14.40. The speed was 59,780 rpm.

this concentration, ricin was not completely reduced. We werealso unable to reduce the disulfide bridge completely with upto 0.25 M glutathione at room temperature.The denatured enzyme was reduced by much lower con-

centrations of reducing agent. For instance, at room tempera-ture for 30 min in 1% NaDodSO4, 0.1% (14 mM) mercapto-ethanol was sufficient to reduce denatured ricin completely(Fig. 1 lower). This is a decrease in the requirement for re-ducing agent to 2% of that of the native toxin. Similarly, therequirement for total reduction by dithioerythritol is 10 mMand for glutathione, 0.25 M (Figs. 2 and 3).The differences in the susceptibilities of the disulfide con-

necting the two chains to reduction in the native and denaturedenzyme are an indication of a disulfide that is buried in theinterior of the protein. Though dithioerythritol is consideredto be a more effective reducing agent due to its ability to forman intramolecular disulfide bond to complete reduction, it isrelatively less effective than mercaptoethanol in reduction ofnative ricin. A 50-fold excess of the amount of mercaptoethanolrequired to reduce the denatured ricin can reduce the nativeenzyme. If dithioerythritol was as efficient in reducing nativericin, one would expect 0.5 M dithioerythritol to reduce thenative enzyme completely. However, this concentration doesnot completely reduce native ricin. This may be due to the in-creased size of the dithioerythritol which makes it unable todiffuse into the area containing the disulfide in the nativeprotein. With respect to the mechanism of action of the toxin,if complete reduction is required for toxicity, the difficulty inreducing this protein may suggest a specific process for changein structure followed by an enzymatic or nonenzymatic re-duction. The difficulty in reduction is paralleled by a similarresistance of the native protein to proteolytic attack found inour laboratory as well as by others (1).The procedure presented here for the reformation of the

disulfide bridge between the A and B chains of ricin with oxi-dized glutathione has the advantage of being faster than theprevious method of Olsnes et al. (8). They used air oxidationof the sulfhydryl groups, which can require 3 days for totaloxidation because it is dependent on divalent metal ions tocatalyze the reaction. The reoxidation with glutathione allowsmore extensive reoxidation even after storage of the relativelyunstable A chain for several weeks.

1 Opg 1OOpg ling 1Ong 1OOng lug 1OgAmount of ricin or A and B chain added to cells

FIG. 5. The effect of ricin (@) and A and B chains (0) on proteinsynthesis in HeLa cells. HeLa cells were plated at a concentration of2 X 105 cells/35-mm tissue culture dish in a mixture of modifiedEagle's medium and F-12 medium plus 2.5% fetal calf serum and 5%horse serum and incubated overnight. They were washed twice withphosphate-buffered saline, and 0.9 ml of serum-free F-12 was addedto each dish. One-tenth milliliter of the appropriate toxin concen-tration was added to duplicate dishes and samples were incubatedat 370 for 2 hr. Then 1 1sCi of 3H-labeled amino acid mixture wasadded for an additional 1 hr. Cells were then washed with phos-phate-buffered saline, fixed with 10% cold trichloroacetic acid, solu-bilized in 1% NaDodSO4 for 30 min, transferred to tubes containing12.5% trichloroacetic acid, and incubated on ice for 30 min. The pro-tein precipitate was then transferred onto filters and washed twicewith 10% cold trichloroacetic acid and radioactivity was deter-mined.

It became apparent during the purification of the A and Bchains that the chains still bind to each other after reduction.To investigate this, we mixed the purified chains in equimolaramounts and observed their behavior in the analytical ultra-centrifuge in sedimentation velocity experiments. The A chainand B chain have sedimentation coefficients (S20,w) of 3.17 and3.28, respectively, and native ricin has a sedimentation coef-ficient of 4.66. The A and B chain centrifuged together im-mediately after mixing have a sedimentation coefficient of 4.63(Fig. 4), indistinguishable from native ricin.

In previous experiments with a more aged preparation of Achain, the two chains sedimented with S20,w of 4.46, and nodisulfide bond had reformed after centrifugation. Recombi.-nation experiments showed that only 60% of this material wascapable of forming disulfide bonds. Whether the lower S2Owvalue is due to a small amount of free A and B chains or acomplex with a shape different from complexes formed froma fresher preparation, in which almost total disulfide refor-mation can occur, has not been determined.

In another experiment, ricin reduced by mercaptoethanolwas chromatographed over a molecular sieve column in thepresence of 0.1% mercaptoethanol to prevent reoxidation. Thereduced ricin chains together behaved like native ricin, witha partition coefficient (12) Kav = 0.101 for both. The partitioncoefficient for B chain was 0.266. No peaks corresponding toA or B chain were seen when reduced ricin chains were chro-matographed together. These data indicate that the A and Bchains form a complex rapidly, that this complex is stable, andthat little dissociation occurs under the conditions we haveused.We have examined the toxicity of the complex formed by

the A and B chains of ricin in mice and the ability to inhibitprotein synthesis in HeLa cells in culture. In these experiments,A and B chains were added together and were allowed to formthe noncovalent complex in a period of 30 min. The results ofexperiments with intact HeLa cells are seen in Fig. 5. Ricin itself

Biochemistry: Lappi et al.

1100 Biochemistry: Lappi et al.

inhibited protein synthesis by 50% at 0.7 ng, while the A andB chain complex inhibited the protein synthesis by 50% at 47ng. This is a decrease in toxicity to 1/67 that with ricin. In mice,the dosage toxic to 50% of the experimental animals (LD5o) ofthe A and B chain complex was 1.75 jig; the LD50 of ricin andof ricin reconstituted from separated A and B chain with thedisulfide bond reformed is 200 ng (8). This is a decrease intoxicity to 1/8.75 that with the A and B chain complex. Thedifference in relative toxicities between the two assays may bedue to incubation time in the assays. In the tissue culture assay,

the A and B complex is exposed to cells for a total of 3 hr. Ap-parently during that time little reformation of the disulfidebond linking the two chains can take place. We may assume

further reoxidation in the peritoneal cavity of the mouse duringthe 3 days after which toxicity was determined.We have also modified ricin with iodoacetamide. Gel elec-

trophoresis shows that less than 10% of whole native ricin re-

mains after modification. Thus, at least one cysteine of the twothat form the disulfide bond connecting the two chains has beenmodified. The modified ricin had a sedimentation velocitysimilar to that of native ricin. However, this material had re-

duced toxicity to mice and reduced ability to inhibit proteinsynthesis in HeLa cells similar to the A and B chain complex,while its activity inhibiting protein synthesis in vitro was similarto that of native ricin.From these data, we conclude that an intact disulfide bridge

between the A and B chains of ricin is necessary for toxocity.However, the reason for this requirement is not simply to holdthe two chains together, since upon reduction, the two chainsremain bound together. The requirement for this intact disul-fide bond may involve some still unknown process on the cellsurface for penetration of the A chain into the cytoplasm. Thisrequirement is in contrast to the results of Li and coworkers with

human somatotropin (13). In that hormone, a disulfide bondconnects two peptide fragments. A recombinant hormone,carboxamidomethylated to prevent disulfide reformation, hadfull biological activity.

This work was supported in part by grants from the AmericanCancer Society (BC-60-R) and the U.S. Public Health Service (CA11683).

1. Olsnes, S., Refsnes, K., Christensen, T. & Pihl, A. (1975) Biochim.Biophys. Acta 405, 1-10.

2. Sandvig, K., Olsnes, S. & Pihl, A. (1976) J. Biol. Chem. 251,3977-3984.

3. Olsnes, S. & Pihl, A. (1973) Biochemistry 12,3121-3126.4. Lin, J.-Y., Lin, L.-T., Chen, C.-C., Tseng, K.-Y. & Tung, T.-C.

(1970) J. Formosan Med. Assoc. 69,53-57.5. Lin, J.-Y., Tseng, K.-Y., Chen, C.-C., Liu, L.-T. & Tung, T.-C.

(1970) Nature 227,292-293.6. Tung, T.-C., Hsu, C.-T. & Lin, J.-Y. (1971) J. Formosan Med.

Assoc. 70, 569-574.7. Schachman, H. K. & Edelstein, S. J. (1973) in Methods in En-

zymology, eds. Hirs, C. H. W. & Timasheff, S. N., (AcademicPress, New York, Vol. 27, pp. 3-59.

8. Olsnes, S., Pappenheimer, A. M. & Meren, R. (1974) J. Immunol.113,842-847.

9. Venter, B. R. & Kaplan, N. 0. (1976) Cancer Res. 36, 4590-4594.

10. Lin, Y.-Y. & Tung, T.-C. (1972) J. Chinese Biochem. Soc., 1,1-20.

11. Kapmeyer, W. & You, K.-S. (1977) Fed. Proc. Fed. Am. Soc. Exp.Biol. 36, 670.

12. Pharmacia Fine Chemicals (1975) Sephadex, Gel Filtration inTheory and Practice (Pharmacia Fine Chemicals, Uppsala,Sweden), Vol. 14, pp. 1-64.

13. Li, C. H., Bewley, T. A., Blake, J. & Hayashida, T. (1977) Proc.Nati. Acad. Sci. USA 74, 1016-1019.

Proc. Natl. Acad. Sci. USA 75 (1978)