Biochemical Studies on Ricin. IV. Amino Acid Analysis - Ishiguro - Agr. Biol. Chem 35 (1971)

8/13/2019 Biochemical Studies on Ricin. IV. Amino Acid Analysis - Ishiguro - Agr. Biol. Chem 35 (1971)

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The Journal of Biochemistry, Vol. 55, No. 6, 1964

Biochemical Studies on Ricin

I. Purification of Ricin

By MASATSUNE ISHIGURO, TAKAO TAKAHASHI, GUNKI FUNATSU,KATSUYA HAYASHI and MASARU FUNATSU

(From the Laboratory of Biochemistry, Faculty of Agriculture,Kyushu University, Fukuoka)

(Received for publication, November 29, 1963)

Since O s b o r n e et al. (l) isolated a toxic

protein, ricin, from castor bean, many studiesof this protein have been made. Partiallypurified ricin preparation or a crystalline ricinpreparation, which was obtained by K a b a tet al. (2), Kunitz and McDonald (3),and M o u 16 (4) separately, had high hemag-glutinating or proteolytic activity in additionto toxicity, and these activities were thoughtto be intrinsic properties of ricin itself.

As in the case of Cl. botulinum (5) or Habusnake venom (6), however, trace amounts ofimpurities which have physiological activitycan often bring about symptoms differentfrom those brought about by the toxin.Therefore, when the study of the primarymechanism of a toxin is undertaken, theexperiments must be carried out with purepreparations. Thus ricin C1, purified byhydroxylapatite column chromatography ofcrystalline ricin which had been obtainedusing the method of K u n i t z and M c-Donald (3), was shown by Funatsu (7)to have neither proteolytic nor hemaggluti

nating activities. In this connection, it wassuggested that these activities were not essential for the toxic action of ricin. Nevertheless,the toxicity of this ricin Ct was not enhancedby this purification procedure. In addition,it was revealed that ricin Ct was not yetchemically homogeneous.* This indicatedthat ricin Ct contained other proteins besidesproteinase(s) [EC 3.4.4. group] and hemagglutinin or was degraded by contaminating proteinase(s) during purification.

* Unpublished experiments .

Consequently, our attempt to obtain an

improved and reproducible method of purification led to the conclusion that ion-exchangecellulose column chromatography was effectivefor the purpose. This paper deals mainlywith the purification of ricin by cellulosederivative column chromatography and itscrystallization.

EXPERIMENTAL

Protein Concentration-Protein concentration was

determined either by the spectrophotometric method

at 280 m (1.156 O.D./l mg. protein/ml.) with a

Beckman DU spectrophotometer or by the nitrogen

content using the micro-Kjeldahl apparatus.

Toxicity-Pure-bred mice (ddN-CF#i and CH) of

both sexes weighing 20-25g. were used to determine

the lethal toxicity throughout this study. A sample

solution diluted with physiological saline was injected

intraperitoneally into the mice and the results were

observed at intervals of 24 hours. The minimun lethal

dose at 48 hours (MLD48) was adopted as a measure

of toxicity and was expressed as g. of ricin nitrogen

per gram body weight of a mouse. We used eightmice for one dose and set always a control experi

ment using a mouse injected with a volume of 0.5-

0.75 ml. of saline solution.

Proteolytic Activity-Proteolytic activity in castor

bean, as already reported (8), has its optimum action

at pH values 3.0 and 6.0, so that activity was meas

ured at both these pHs by a slight modification of

H a g i h a r a 's method (9) as follows: a mixture con

taining 1 ml. of a sample solution, 2 ml. of a 1.2%

aqueous solution of casein and 2 ml. of 0.1 M buffer

solution (hydrochloric acid-potassium biphthalate buffer

for pH 3.0 and phosphate buffer for pH 6.0) was

incubated at 37G for 24 hours. The reaction was

stopped by adding 5 ml. of 20% trichloroacetic acid

587


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588 M. ISHIGURO, T. TAKAHASHI, G. FUNATSU, K. HAYASHI and M. FUNATSU

and the precipitate was removed by filtration throughToyo Roshi filter paper No. 4. To 2 ml. of thefiltrate, 5 ml. of 0.4 M sodium carbonate solution and

1 ml. of a fifth fold diluted phenol reagent wereadded, and incubated at 30C for 30 minutes. Opticaldensity was measured at 660 mp with a Hitachi photo-electric spectrophotometer, type EPO-B. Proteolyticactivity was represented as pg. tyrosine liberated after24 hours' incubation at 37C.

Hemagglutinating Activity-Hemagglutinating activ

ity by was assayed ty the method described by T a k a-

h a s h i et al. (10). The hemagglutinating potency was

expressed as the minimum number of g. of protein

nitrogen regarded to give definite macroscopic evidence

of agglutination when 0.5 ml. test solution was incub

ated at 37C for 1 hour with 0.2 ml. washed rabbiterythrocytes.

Non-specificProtein Coagulating Activity-Since thenon-specific protein coagulating factor has not beenstudied in detail, the activity was temporarily measured by the increase in turbidity caused by coagulation. Turbidity was measured spectrophotometricallyat 660 mp after the sample solution was incubatedwith the substrate, casein in this case, at 37C for24 hours.

tained by saturation of the supernatant withammonium sulfate. The yield of crude ricinwas 4.6 per cent by weight.

Purification of Crude Ricin by Ion-exchangeCellulose Column Chromatography-Ion-exchangecellulose column chromatography was per-formed by the method of Sober et al. (11)with DEAE- and CM-cellulose. Approximately 100 mg. of crude ricin which hadbeen dialyzed against 0.005 M phosphatebuffer, pH 7.0, was applied to DEAE-cellulosecolumn (1.7 x 30 cm.) previously equilibratedwith the same buffer as that used for dialysis.The column was developed by stepwise

elution with the phosphate buffer solutionsof the following concentrations and pHs :0.005 M, pH 7.0; 0.01 M, pH 7.0; 0.02 M, pH6.0 and 0.05 M, pH 6.0.

Ultracentrifugation and Electrophoresis-Spinco ultra-centrifuge Model E and Hitachi electrophoretic ap

paratus (Tiselius type) were used for determination ofthe purity of crystalline ricin. Ricin was dissolved in0.1 M acetate buffer, pH 5.0 and acetate buffer containing sodium chloride (p=0.3), pH 3.6, and run at56,100 r.p.m. at 20C, and at 52 volt and 4.5 mA ofcurrent at 6C, respectively.

RESULTS AND DISCUSSION

Separationof CrudeRicin-The experimentswere carried out at 15C except mentioned.Defatted castor bean meal was obtained bymechanical removal of fat from shelled castor

bean (Ricinus sanguineusL.) after homogenization in ice-cold petroleum ether. The extraction and separation of the crude ricin wereperformed by a slight modifications of themethods of K u n i t z and M c D o n a l d (3)and M o u. l e (4). The defatted meal wasthen suspended in water and adjusted to pH3.8 with dilute hydrochloric acid. After stir-ring, the suspension was filtered and thefiltrate was saturated with sodium chloride.The resulting precipitate was dissolved inwater and any insoluble material was removedby centrifugation. The crude ricin was ob-

FIG. 1. Chromatogram of crude ricin on acolumn (1.7 X 30 cm.) of DEAEcellulose. Stepwise

elution was carried out with (1): pH 7.0, 0.005Mphosphate buffer, (2) : pH 7.0, 0.01 M, (3) : pH6.0, 0.02M, (4) : pH 6.0, 0.05M. Flow rate :60 ml. per hour. Total recovery of protein fromthe column was 100 per cent estimated by opticaldensity at 280 my.

As shown in Fig. 1, five peaks, R-I (41.2%),R-II (17.6%), R-III (18.6%), R-IV (11.8%)and R-V (13.2%), were obtained. After pool-ing each fraction, the physiological activities

were measured (Table I).Most of the toxicity was contained in


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Crystalline Ricin. I 589

TABLE I

Physiological Activities of Fractions Obtained by DEAF-cellulose Column

Chromatography of Crude Ricin

1) Methods and representation of these activities were described in the text.

the first three peaks, but these fractions couldhardly be distinguished by physiological activities. Fraction R-III is apparently differentfrom fractions R-I and R-II. However, R-Iand R-II could be same, because, in general,the trailing part of a peak could make the

second peak by a change of buffer concentration in stepwise elution system. Sincefraction R-I which was the main componentpassed through the DEAE-cellulose withoutadsorption under this condition, further purification was required. For this purpose, CM-cellulose column chromatography was used.The fraction R-I was precipitated by saturating with ammonium sulfate. The resultingprecipitate was dissolved in water and dialyzed against water and finally against 0.005M phosphate buffer of pH 6.5. After removalof the insoluble material by centrifugation,the supernatant was applied to a CM-cellulosecolumn (1.7 x 30 cm.) which was previouslyequilibrated with 0.005M phosphate buffer,pH 6,5 and eluted stepwise with 0.005M, 0.01M, 0.02M and 0.05M phosphate buffers,pH 6.5.

As shown in Fig. 2, four peaks, R-I-1(14.2%), R-I-2 (9.0%), R-I-3 (23.7%) and R-I-4(24.9%), were obtained. From the determi-nation of physiological activities of eachfraction (Table II), it was revealed thatfraction R-I-4 had the highest toxicity, mini-

FIG. 2. CM-cellulose column chromatogramof fraction R-I. Stepwise elution was carried outwith 0.005 M, 0.01 M, 0.02 M, and 0.05 M phos

phate buffers of pH 6.5. Column : 1.7 x 30 cm. ;flow rate : 40 ml. per hour. Total recovery ofprotein from this column was 76 per cent byoptical density at 280 my.

mum hemagglutinating and no proteolyticactivities. Rechromatography of fractionR-I-4 gave an evidence that this fraction washomogeneous chromatographically and wasnot the secondary product by chromatography(Fig. 3). The toxicity of fraction R-I-4 wasthree times higher than that of ricin Ct(Table III).

Crystallization of Fraction R-I-4-The frac


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TABLE 11

Physiological Activities of Fractions Obtained by CM-cellulose Column

Chromatography of Fraction R-I.

1) Though we could not detect any proteolytic activity by H a g i h a r a's method, there occurredvisibly strong coagulation in each fraction.

FIG. 3. Rechromatogram of fraction R-I-4CM-cellulose column (1.7 x 22 cm.). Stepwise elution was carried out under the same conditionsas seen in Fig. 2.

TABLE III

Comparison of Tooxiity of Riein Preparations

tion which was rechromatographed on CM-cellulose column was pooled, and saturatedwith ammonium sulfate. The resulting precipitate was dissolved in water and dialyzedagainst tap water for a day, against distilledwater for three days, and then against 0.005

M phosphate buffer, pH 6.5. After two orthree days in the last step, small crystals wereobserved in a dialysis bag (Visking tube #18/32). The inner parts were then placed in arefrigerator until crystallization was completed (for about one week). Recrystallizationwas carried out as follows : The crystalswere centrifuged in the cold and washedseveral times with a small amount of ice-cold0.005M phosphate buffer, pH 6.5. The crystals were then suspended in a small amountof water and dissolved by adjusting the mixture to pH 3-4 with one drop of 0.1 N hydrochloric acid. After removal of the insoluble material by centrifugation, the super-natant was treated to yield crystals by thesame dialysis method as described above(Fig. 4). The yield of crystalline ricin from100 g. of defatted castor bean meal was 210mg. (0.21%).

Purity of Crystalline Ricin-The crystallinericin obtained from fraction R-I-4 was refer-red to as ricin D. Studies of the homogeneityof ricin D revealed that it was homogeneousultracentrifugally (Fig. 5) and electrophoreti


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Crystalline Ricin. I 591

FIG. 4. Photograph of crystalline ricin obtained from fraction R-I-4 by dialysismethod.

FIG. 5. Ultracentrifugation of ricin D. Photographs from right to left were taken at inter-vals of 8 minu._s. Protein concmtration was 0.8 per cent in 0.1 M acetate buffer, pH 5.0.The speed of rotor was 6,100r.p.m.

FIG. 6. Electrophoretic patterns of ricin D.The experiment was conducted in acetate buffer,pH 3.6, containing sodium chloride (p=0.3), at6C. 52 volt and 4.5 mA of current were supplied.Protein concentration was 0.64 per cent. Photo-graphs were taken at (1) 908, (2) 1,440, and (3)1,800 seconds.

cally (Fig. 6).It was found by us* that ricin Ct con

tained neither proteolytic nor hemagglutinating activity, but did contain non-specificprotein coagulating activity. This activityseems to be a useful criterion for the purityof ricin. As shown in Table IV, fractionR-I-4 contained non-specific protein coagulating activity. However this could be completely removed by the crystallization procedure.

* Unpublished experiments .


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TABLE I V

Measurement of Nonspecife Protein Cocgulcting Aetiaity of Ricin

D and its Mother Liquor

Measurement of non-specific protein coagulating activity was carried out as follows: Reaction mix

ture containing I ml. of 1 per cent aqueous casein solution of pH 6.0 as substrate, 2 ml. of 0.1 M phos

phate buffer of pH 6.0 and 1 ml. of each sample solution was incubated at 37C. At 24 and 48 hours,the increase in turbidity caused by coagulation was measured at 660 mli.Control contained only casein.

From these results, it was concluded thatricin D was a purer toxic protein than anyother ricin preparations previously reported(Table III).

SUMMARY

1. The purification of ricin was accom

plished by column chromatography on DEAEand CM-cellulose and subsequent crystalliza

tion. The crystalline ricin thus obtained was

referred to as ricin D.

2. Ricin D was found to be homo

geneous electrophoretically and ultracentri

fugally.

3. Ricin D was also found to be homo

geneous physiologially, showing only toxicity

and neither hemagglutination nor proteolysis

nor non-specific protein coagulation.

4. The toxicity of ricin D, in terms of

MLD4S , was 0.001 g. ricin nitrogen per gram

body weight of a mouse, which was about

ten-fold higher than those of the previously

reported preparations.

We wish to thank Dr. B. Nuki and Dr. S. Ueki,

Department of Pharmacology, School of Medicine,

Kyushu University, for their kind instructions andhelps in carrying out toxicity experiments on mice.

REFERENCES

(1) Osborne, T., Mendel, L., and Harris, J., Am.J. Physiol., 14, 258 (1905

)(2) Kabat, E., Heidelberger, M., and Bezer, A.,J. Biol. Chem., 168, 629 (1947

)(3) Kunitz, M., and McDonald, M., J. Gen. Physiol., 32, 25 (1948)

(4) Le Breton, E., and Moule, Y., Compt. rend., 225,152 (1947); Bull, soc. chim. biol., 31, 94 (1949):Moule, Y., Arch. Sci. Physiol., 5, 277 (1951);Bull. soc. chim. biol., 33, 1461, 1467 (1951)

(5) Lamanna, C., Proc. Soc. Exptl. Biol. Med., 69, 332 (1948)

(6) Maeno, H., J. Biochem. (Tokyo), 52, 343 (1962)(7) Funatsu, G., J. Agr. Chem. Soc. Japan, 34, 139

(1960)(8) Funatsu, G., and Funatsu, M., J. Agr. Chem.

Soc. Japan, 33, 461 (1959)(9) Hagihara, B., Koso Kenkyu Ho , ed. byAkabori, S., Asakura Shoten, Tokyo, Vol. H,

p. 237 (1956)(10) Takahashi, T., Funatsu, G., and Funatsu, M,,

J. Biochem. (Tokyo), 51, 288 (1962)(11) Peterson, E., and Sober, H., J. Am. Chem. Soc.,

78, 751 (1956)

Biochemical Studies on Ricin. IV. Amino Acid Analysis - Ishiguro - Agr. Biol. Chem 35 (1971)

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