An Improved Method of the Purification of Ricin D - Hara - Agr Biol Chem 38 (1974)

8/13/2019 An Improved Method of the Purification of Ricin D - Hara - Agr Biol Chem 38 (1974)

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Agr. Biol. Chem., 38 (1), 65`70, 1974

An Improved Method of the Purification of Ricin D

Kenji HARA,Masatsune ISHIGURO, unki FUNATSUand Masaru FUNATSU

Laboratoryof Biochemistry, aculty f Agriculture KyushuUniversity,Fukuoka 12,Japan

ReceivedMay29, 1973

An improved method of the purification of ricin D was investigated . Ricin was purified

by gel-filtration through Sephadex G-75 at pH 8.0, followed by either CM-cellulose column

chromatography at pH 6.5 or DEAE-cellulose column chromatography at pH 8.5. Thehomogeneity of the purified ricin was criticized by polyacrylamide gel disc electrophoresis .The purified ricin behaved homogeneous also in ampholine electrophoresis , indicating the

isoelectric point of 7.34. Ricin thus purified was identical with ricin D in electrophoreticalmigration and toxicity. By the measurement of optical rotatory dispersion of the purified

ricin, ORD constant, c, Moffitt-Yang parameters, a0 and b0, were evaluated to be 235 nm,-138 and -66 , respectively.

In a previous paper,1) ricin was separated byDEAE-cellulose column chromatography atpH 7.0 as a non-adsorbed fraction from hemagglutinin. This fraction was applied ontoa CM-cellulose column equilibrated at pH 6.5and ricin was obtained as a fraction eluted with0.02M phosphate buffer at pH 6.5. Finally,purified ricin D was obtained by gel-filtrationthrough Sephadex G-75 equilibrated at pH 8.0.As described previously, a slight modificationhad been introduced to the original procedurefor the purification of ricin D when deionizedwater is employed.

In later experiments, however, some difficulty was experienced in separating hemag

glutinin from ricin by DEAF-cellulose columnchromatography at pH 7.0. Also, gel-filtration through Sephadex G-75 was provedmore efficient than DEAE- or CM-cellulose toseparate ricin from hemagglutinin, non-specific protein coagulating factor and proteases in crude ricin. In addition, it was observed that ricin could be retained and chromatographed with DEAF-cellulose at pH 8.5employing Tris-HCl buffer although ricin wasoriginally obtained as a non-adsorbed frac

tion with DEAE-cellulose at pH 7.0.Based on these observations, an improved

method for the purification of ricin D wassought and it was found that ricin D couldbe purified by a simplified method by utilizing gel-filtration through Sephadex G-75 andCM- or DEAE-cellulose column chromatography.

This paper is to describe a simplified methodfor the purification of ricin D and some physico-chemical properties of the ricin obtainedby this method.

MATERIALS AND METHODS

The crude ricin was prepared from castor beans

(Ricinus cornmunis L., large grain type) imported fromThailand without selection as to color. All experimental methods were same as described in the previouspaper2) unless otherwise specified.

Gel-filtration through Sephadex G-75. Gel-filtration was carried out through Sephadex G-75 (obtainedfrom Pharmacia Co.) with borate buffer (0.05M sodiumborate-0.1M HCl) of pH 8.0. The crude ricin wasconcentrated and dialyzed against the above buffer inthe cold. After removal of insoluble material bycentrifugation, the supernatant solution was appliedonto a Sephadex G-75 column and developed with thesame buffer.

CM-cellulose column chromatography. A columnof CM-cellulose was prepared and equilibrated with

Biochemical Studies on Ricin. Part V. (Pre

vious paper) M. Ishiguro, G. Funatsu and M. Funatsu,

Agr. Biol. Chem., 35, 729 (1971).


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66 K. HARA, M. ISHIGURO, G. FUNATSU and M. FUNATSU

0.005M phosphate buffer, pH 6.5. After dialysis

against the same buffer, the ricin fraction obtained by

gel-filtration through Sephadex G-75 was applied ontothe column and eluted stepwise with the buffer of thefollowing concentrations: 0.005, 0.013, 0.02 and

0.05M, in this order.

DEAE-cellulose column chromatography. DEAE-cellulose column chromatography was carried out withTris-HCl buffer (0.005M Tris-0.1M HCl) of pH 8.5 anddeveloped with the same buffer containing 0.01, 0.04,and 0.2M NaCl.

Polyacrylamide gel disc electrophoresis. The ricin

preparation was subjected to disc electrophoresis in

polyacrylamide gel prepared according to the method

of Ornstein.3) The electrophoresis was carried out for

3.5 hr approximately at 250V with a current of 2.0mA/

tube (7~0.5cm) using Tris-glycine buffer (pH 8.3).

Protein band in gel was detected by staining with Amido

Black 10 B, followed by removing unbound dye with

several changes of 7 acetic acid (v/v).

Ampholine electrophoresis. A lyophilized ricin

(2.0mg) was applied to ampholine electrophoresis in

0.8 carrier ampholytes (pH 5`8) medium obtained

from LKB-Produkter AB. Electrophoresis was con

ducted using an electrofocusing column, LKB 8101,

for 50 hr at 2`3 with about 1.0W throughout the

electrophoresis. After fractionation of the contents

of the column into 1.5ml-portions, the amount of

protein in each portion was determined spectrophoto

metrically at 280nm, and each pH value was measured

by a Hitachi-Horiba pH-meter equipped with a glass

electrode 6028-10T at 19.5.

Optical rotatory dispersion. Optical rotatory dis

persion measurement was performed with a Jasco

Model ORD/UV-5 Recording Spectropolarimeter at

16. For the measurement in a visible and near

ultraviolet region (600`300nm) 10mm-cell was used

with ricin D solution at a concentration of 1.9 .Phosphate buffer (0.005M, pH 6.5) was used as solvent.

The value of c, was calculated by Drude's equation (1)

and the value of a0 and bo by Moffitt-Yang's equation

(2),4) where Mo is the average

molecular weight per residue, 0 the absorption wave-

length concerned with the rotation and n the solvent

refractive index. In the present work, 129.8, 212nm

and 1.33 were used for values of Mo, 0 and n, respec

tively.

The toxicity of ricin. The lethal toxicity of ricin

was determined by an injection of a sample solution

diluted with physiological saline intraperitoneally into

pure-bred mice (ddN) of both sexes weighing 20

` 30g. After injection into mice, the results were ob

served at intervals of 24hr. The minimal lethal dose

at 48hr (MLD48) was adopted as a measure of toxicity

and was expressed as tug of ricin nitrogen per gram body

weight of mouse. We used ten mice for one dose and

set always a control experiment using a mouse injected

with 0.5`0.75ml of saline solution.

Crystallization of ricin. Crystallization of ricin

was performed according to the previous procedure.5)

An aqueous solution of ricin (protein concentration

was approximately 3`5 ) was dialyzed successively

against water and 0.005M phosphate buffer of pH 6.5

containing 10-6M cupric acetate at 4. A turbidityappeared in the dialysis bag within two days and cry

stallization was completed after about one week.

RESULTSAND DISCUSSION

A. Purification of ricin DSeparation of crude ricin. Defatted castor

bean meal was suspended in water and adjusted to pH 3.8 with dilute hydrochloric acid.After stirring for 3hr, the suspension was

filtered and the extraction was repeated. Thecombined filtrates were saturated with sodiumchloride. The resulting precipitate was dissolved in water and the solution was dialyzedagainst water. The dialysate was adjusted topH 8.0 with 2 ammonium hydroxide and theresulting precipitate was centrifuged off. Tothe clear supernatant solution, saturatedammonium sulfate solution was added to givea final saturation of 50 to precipitate crude

ricin.Gel-filtration through Sephadex G-75. The

crude ricin was collected, dissolved in water,

and dialyzed against 0.05M borate buffer, pH

8.0, at 4 for 3days. After removal of any

insoluble material by centrifugation, the dialyz

ed solution was gel-filtrated through a Sep

hadex G-75 column previously washed suf

ficiently with the same buffer. A typical gel-

filtration pattern is presented in Fig. 1. As

shown in Fig. 1, crude ricin was separated

into three fractions. The fraction F-2 with

the highest toxicity was collected and the

active material was precipitated by saturat-


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Biochemical Studies on Ricin. Part V 67

FIG. 1. Gel-filtration Pattern of Crude Ricin through

Sephadex G-75.

Column size, 4.3~15cm; fraction volume, 9.8ml;

flow rate, 120ml/hr; total recovery of protein, 99 .

ing solution with solid ammonium sulfate.

The toxicity of fraction F-1 was very low and

that of fraction F-3 was not detected. More

over, the fraction F-3 did not give any preci

pitate by saturating it with solid ammonium

sulfate.

CM-cellulose column chromatography. The

precipitate obtained from fraction F-2 wasdissolved in deionized water and the solution

was dialyzed successively against deionized

water at 4 for 3days and against 0.005M

phosphate buffer, pH 6.5, at 4 for 2days.After removal of insoluble material by cen

trifugation, the supernatant was applied onto

a CM-cellulose column previously equilibrat

ed with the same buffer as that used for dialysis.

Elution was carried out stepwise with 0.005,

0.013, 0.02 and 0.05M phosphate buffer, pH

6.5, in this order. As shown in Fig. 2, pro

tein was separated into two fractions (fraction

S-1 and S-2). As these conditions were

similar to those described in previous papers,, II

fraction S-2 should correspond to ricin D.

The toxicity of fraction S-1 was slightly lower

than that of fraction S-2, but, as shown in

Fig. 2, fraction S-1 was not homogeneous ch

romatographically. If fraction S-1 is puri

fied, the toxicity of fraction S-1 may become

higher. The fraction S-1 was found to be

FIG. 2. Column Chromatogram of F-2 on CM-

cellulose.

Column size, 2.8~26.0cm; fraction volume, 6.2ml;

flow rate, 120ml/hr; total recovery of protein, 71

(S-1, 33 ; S-2, 38 ).

slightly more acidic than ricin D.

DEAF-cellulose column chromatography.

The precipitate obtained from fraction F-2

was dissolved in deionized water and the solu

tion was dialyzed successively against deioniz

ed water at 4 for 3days and against 0.005M

Tris-HC1 buffer, pH 8.5, at 4 for 2days.

After removal of insoluble material by cen

trifugation, the dialyzed solution was appliedonto a DEAE-cellulose column previously

equilibrated with the same buffer. Elution

was carried out stepwise with the same buffer

containing 0.01, 0.04 and 0.2M NaCl in this

order. A typical column chromatogram is

presented in Fig. 3. As shown in Fig. 3,

FIG. 3. Column Chromatogram of F-2 on DEAE-

cellulose.

Column size, 2.4~30cm; fraction volume, 10.0ml;

flow rate, 120ml/hr; total recovery of protein, 95

(D-1, 63 ; D-2, 24 ).


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fraction F-2 was separated into two fractions(fraction D-1 and D-2), as in the case of CM-cellulose column chromatography. From the

chromatograms shown in Figs. 2 and 3, andthe polyacrylamide gel disc electrophoreticpattern (Fig. 5), it was assumed that 0.01 Mfraction (fraction D-1) corresponds to fraction S-2, and 0.04 M fraction (fraction D-2)to fraction S-1. The fraction D-1 was collected and rechromatographed on a DEAE-cellulose column under the same conditions asabove, and it was found that fraction D-1behaved homogeneously on DEAE-cellulose

column chromatography, as obviously seen inFig. 4. The yield of the purified ricin from 100g of defatted castor bean meal was 1.2g (1.2 ),which is about 6times higher than that previously reported.

FIG. 4. Column Chromatogram of D-1 on DEAE-cellulose.

Column size, 2.4~24cm; fraction volume, 7.3ml;flow rate, 120ml/hr.

B. Homogeneity and physical properties of

purified ricinPolyacrylamide gel disc electrophoresis.

Homogeneity of the protein fractions obtained

during the purification was examined with disc

electrophoresis at pH 8.3. Figure 5 shows

disc electrophoretic patterns of the protein

fractions. As seen in this figure, crude ricin

contains mainly three fractions, of which a

protein fraction corresponding to the up

permost band was apparently removed by gelfiltration through Sephadex G-75. The frac-

FIG. 5. Analytical Disc Electrophoresis of theVarious Fractions and Ricin D.(1) Crude ricin (2) Fraction F-2 (3) Fraction S-2. (4) Fraction D-1 (5) Ricin D.

tion F-2 from a Sephadex G-75 column.contains mostly two protein fractions. The-

fraction S-2 and D-1 gave each single bandwhich coincided in migration with that ofricin D. Thus, ricin D could be purified byeither CM- or DEAE-cellulose chromatography from fraction F-2. It should be mentioned, however, that the DEAE-cellulosechromatography yielded a better recovery ofricin D.

Ampholine electrophoresis. The fractionD-1 was subjected to ampholine electrophoresis for the determination of isoelectric point.A single peak was obtained at pH 7.34 on apH-slope drawn by dotted line in Fig. 6.The experimental result obtained indicatesthat the purified ricin is a homogeneous andbasic protein with an isoelectric point of7.34. This electrophoretical behavior of thepurified ricin was not in accord with that ofricin D previously obtained.5) The isoelectric point of ricin D had previously been.reported to be pH 5.9 in a Hitachi HTD-1electrophoretic apparatus (Tiselius type).Since ricin D was crystallized by the dialysis,method using 0.005M phosphate buffer, pH


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Biochemical Studies on Ricin. Part V 69

FIG. 6. Ampholine Electrophoresis of Fraction D-1.

6.5, containing 10-6M Cu2+ ion and had afaint metallic blue color, this crystalline ricinmight contain Cult ion. The difference inisoelectric point observed might be caused bythe change in surface charge of the ricin protein due to the binding of copper.

Optical rotatory dispersion. The ORD constant, Ac, of the purified ricin was estimatedfrom the slope which was obtained from theYang's plot shown in Fig. 7. The Moffitt-

FIG. 7. Yang's Plot of the Purified Ricin.

Protein concentration: 1.9 in 0.005M phosphate

buffer, pH 6.5, 13.0.

Yang's plot of the purified ricin for the cal

culating the Moffitt-Yang parameters, a0,

and b0, is shown in Fig. 8. In this plot, the

FIG. 8. Moffitt-Yang's Plot of the Purified Ricin.

Protein concentration: 1.9 in 0.005M phosphate

buffer, pH 6.5, 13.0.

FIG. 9. Crystals Obtained from the Purified Ricin.

values of a0 is given from the intercept and the

value of b0, from the slope. The values of

,c0, and b, were 235nm, -138 and -66,

respectively. The -helix content of the

purified ricin was calculated to be around

10.5 from the values of c, and b0.

The toxicity of ricin. The toxicity of the

purified ricin (fraction D-1), the minimumlethal dose, in terms of MLD48, was 0.001g

ricin nitrogen per gram body weight of mouse

when it was injected intraperitoneally into

mice. This value is the same as that of ricin

D reported previously.1)

Crystallization of ricin. The crystal ob

tained from the purified ricin was identical to

the crystalline ricin D in form (Fig. 9).


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REFERENCES

1) M. Ishiguro, G. Funatsu and M. Funatsu, Agr.Biol. Chem., 35, 724 (1971)

. 2) M. Ishiguro, T. Takahashi, G. Funatsu, K. Haya

shi and M. Funatsu, J. Biochem., 55, 587 (1964).3) L. Ornstein, Ann. N.Y. Acad. Sci., 121, 321 (1964).4) W. Moffitt and J. T. Yang, Proc. Natl. Acad. Sci.

U.S., 42, 596 (1956).5) M. Ishiguro, T. Takahashi, K. Hayashi and M.

Funatsu, J. Biochem., 56, 325 (1964).

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An Improved Method of the Purification of Ricin D - Hara - Agr Biol Chem 38 (1974)