Agr.

Biol.

Chem.,

38

(1),

65`70,

1974

An

Improved

Method

of

the

Purification

of

Ricin

Kenji HARA,Masatsune ISHIGURO, Gunki FUNATSU and Masaru FUNATSU Laboratoryof Biochemistry, Facultyof Agriculture , Kyushu University, Fukuoka812,Japan ReceivedMay29, 1973
An by improved method through at of ricin point and ORD and of the pH purified behaved 7.34. of the purification G-75 at of pH ricin 8.0, D was investigated by either . Ricin was purified column 8 .5. The . the

gel-filtration

Sephadex 6.5 or ricin

followed

CM-cellulose at gel disc pH

chromatography homogeneity The purified

DEAE-cellulose was criticized also purified was of in

column by

chromatography

polyacrylamide ampholine

electrophoresis , indicating electrophoretical of to the be purified 235 nm

homogeneous Ricin By the thus

electrophoresis with rotatory ricin D in

isoelectric migration ricin, -138

identical optical a0 and

toxicity.

measurement parameters,

dispersion evaluated

constant, c, -66 , respectively.

Moffitt-Yang

b0,

were

In a previous DEAE-cellulose

paper,1) column

ricin was separated chromatography

by at

tion with DEAE-cellulose at pH 7.0. Based on these observations, an improved method for the purification of ricin D was sought and it was found that ricin D could be purified by a simplified method by utiliz ing gel-filtration through Sephadex G-75 and CMor DEAE-cellulose column chromato graphy. This paper is to describe a simplified method for the purification of ricin D and some phy sico-chemical properties of the ricin obtained by this method. MATERIALS AND METHODS

pH 7.0 as a non-adsorbed fraction from he magglutinin. This fraction was applied onto a CM-cellulose column equilibrated at pH 6.5 and ricin was obtained as a fraction eluted with 0.02M purified through phosphate buffer at pH 6.5. Finally, ricin D was obtained by gel-filtration Sephadex G-75 equilibrated at pH 8.0.

As described previously, a slight modification had been introduced to the original procedure for the purification of ricin D when deionized water is employed. however, some dif in separating hemag In later experiments, ficulty was experienced

glutinin from ricin by DEAF-cellulose column chromatography at pH 7.0. Also, gel-filt ration through Sephadex G-75 was proved more efficient than DEAE- or CM-cellulose to separate ricin from hemagglutinin, nonspecific protein coagulating factor teases in crude ricin. In addition, served that ricin could be retained matographed employing originally
vious Agr. Biochemical paper) Biol. M. Chem.,

The crude ricin was prepared from castor beans (Ricinus cornmunis L., large grain type) imported from Thailand without selection as to color. All experimen tal methods were same as described in the previous paper2) unless otherwise specified. Gel-filtration through Sephadex G-75. Gel-filtra tion was carried out through Sephadex G-75 (obtained from Pharmacia Co.) with borate buffer (0.05M sodium borate-0.1M HCl) of pH 8.0. The crude ricin was concentrated and dialyzed against the above buffer in the cold. After removal of insoluble material by centrifugation, the supernatant solution was applied onto a Sephadex G-75 column and developed with the same buffer. CM-cellulose column chromatography. A column of CM-cellulose was prepared and equilibrated with

and pro it was ob and chro at pH 8.5

with Tris-HCl obtained

DEAF-cellulose

buffer although ricin was as a non-adsorbed frac

on G. 729 Ricin. Funatsu Part and M. V. (Pre

Studies Ishiguro, 35,

Funatsu,

(1971).

66

K.

HARA,

M.

ISHIGURO,

G.

FUNATSU

and

M.

FUNATSU

0.005M against

phosphate the same

buffer, buffer, the

pH ricin

6.5. fraction

After

dialysis by onto of the and

was diluted

determined with

by physiological mice (ddN)

an

injection saline of into both mice, The as

of

sample

solution into 20 were lethal of ob dose toxicity body and

obtained applied buffer 0.02

intraperitoneally sexes the weighing results

gel-filtration the column following 0.05M,

through and eluted

Sephadex stepwise

G-75 with 0.005,

was the 0.013,

pure-bred ` 30g. served at and 48hr was After at

injection intervals of was

concentrations: in this order.

24hr. adopted

minimal a measure per for one

(MLD48) expressed of mouse. a control

as tug of ricin We used ten

nitrogen mice using

gram dose

DEAE-cellulose column chromatography. DEAEcellulose column chromatography was carried out with Tris-HCl buffer (0.005M Tris-0.1M HCl) of pH 8.5 and developed with the same buffer and 0.2M NaCl.
Polyacrylamide preparation polyacrylamide of 3.5 tube Protein Black several Ornstein.3) hr approximately (7~0.5cm) band 10 B, in gel followed of 7% was gel disc

weight set with always

experiment of of saline ricin. to of 3`5%) ricin was

a mouse

injected

0.5`0.75ml

solution. Crystallization the previous (protein dialyzed buffer at 4. two one A days week. of procedure.5) concentration successively of pH turbidity and cry 6.5 ricin

containing

0.01, 0.04,
Crystallization was performed aqueous approximately water and 10-6M in the was according solution

electrophoresis. to disc according was with

The electrophoresis to the

ricin in method out for

An was against

subjected gel The prepared

0.005M cupric dialysis

phosphate acetate bag after within about

electrophoresis at using was 250V

carried

containing appeared stallization

a current buffer

of 2.0mA/ (pH with 8.3). Amido dye with

Tris-glycine detected by removing acid by

completed

staining unbound (v/v).

changes

acetic

RESULTSAND DISCUSSION A. Purification of ricin D Separation of crude ricin. Defatted castor bean meal was suspended in water and ad justed to pH 3.8 with dilute hydrochloric acid. After stirring for 3hr, the suspension was filtered and the extraction was repeated. The combined filtrates were saturated with sodium chloride. The resulting precipitate was dis solved in water and the solution was dialyzed against water. The dialysate was adjusted to pH 8.0 with 2% ammonium hydroxide and the resulting precipitate was centrifuged off. To the clear supernatant solution, saturated ammonium sulfate solution was added to give a final saturation of 50% , to precipitate crude ricin.
Gel-filtration crude and 8.0, ricin dialyzed at 4 for material was was through collected, 0.05M After centrifugation, through washed A in ricin The was was typical Fig. 1. separated F-2 and by saturatwith the Sephadex dissolved borate removal the G-75. in buffer, of The water, pH any

Ampholine (2.0mg) 0.8% from ducted for 50 was carrier

electrophoresis. applied ampholytes AB. electrofocusing with After into portion and about fractionation 1.5ml-portions, was each to ampholine (pH

lyophilized electrophoresis medium

ricin in obtained was con 8101, the contents of

5`8)

LKB-Produkter using hr at an 2`3

Electrophoresis column, 1.0W

LKB throughout

electrophoresis. of the column in each at

of the

the

amount spectrophoto

protein metrically by

determined pH value

280nm,

was with

measured a glass

a Hitachi-Horiba 6028-10T at

pH-meter 19.5.

equipped

electrode

Optical persion Model 16. ultraviolet with Phosphate The and (2),4) value the ricin

rotatory measurement ORD/UV-5 For the region D buffer

dispersion. was Recording measurement (600`300nm) solution (0.005M, calculated and the bo by at a pH

Optical performed

rotatory with a

dis Jasco at

Spectropolarimeter in a visible and was of used as near

10mm-cell concentration 6.5) by was Drude's

used 1.9%.

solvent. (1)

of c, was value of Mo a0 is

equation equation

Moffitt-Yang's

where

average

against 3days. by

insoluble ed hadex
molecular length refractive and tively. 1.33 weight concerned index. were used per with In for the residue, 0 the rotation present values of the absorption and work, Mo, 0 n the wavesolvent 212nm n, respec

dialyz a Sep suf gelAs

solution G-75 with

gel-filtrated previously same is 1, buffer.

column the

ficiently filtration shown into the in three

pattern Fig.

presented crude

129.8, and

fractions. toxicity was

fraction collected

highest material

The toxicity of ricin.

The lethal toxicity of ricin

active

precipitated

Biochemical

Studies

on

Ricin.

Part

67

FIG.

2.

Column

Chromatogram

of

F-2

on

CM-

cellulose. Column flow FIG. Sephadex Column flow rate, 1. Gel-filtration G-75. size, 4.3~15cm; total fraction recovery volume, of protein, 9.8ml; 99%. Pattern of Crude Ricin through (S-1, rate, 33% size, 2.8~26.0cm; total 38%). fraction recovery volume, of protein, 6.2ml; 71

120ml/hr; ; S-2,

slightly

more

acidic

than column

ricin

D. chromatography.

120ml/hr;

DEAF-cellulose
ing The that over, pitate sulfate. solution toxicity of the by fraction fraction saturating of with fraction F-3 F-3 it was did with solid F-1 not not ammonium was detected. give solid any ammonium very sulfate. low and More preci

The was tion ed

precipitate dissolved was water in

obtained deionized successively for pH of the 3days 8.5,

from water

fraction and against the

F-2 solu

dialyzed at 4

deioniz 0.005M 2days. by cen

and at

against 4 for

Tris-HC1 After trifugation, CM-cellulose precipitate dissolved was water phosphate After trifugation, a CM-cellulose the was 0.02 in was and to this same removal the in column obtained deionized successively for 3days pH of 6.5, and at chromatography. from water fraction and against against 4 material was previously as that out used applied equilibrat for with buffer, in fractions conditions papers,, to was but, slightly as shown ricin Fig. 2, dialysis. 0.005, pH pro for the F-2 The was onto a

buffer, removal

insoluble

material solution column was

dialyzed

applied

DEAE-cellulose with out 0.01, A typical in Fig. the stepwise 0.04 and same

previously Elution same NaCl buffer in this is Fig. 3,

equilibrated was carried

buffer. the

solution deionized 0.005M 2days. by cen onto

with 0.2M

dialyzed at 4

containing order. presented

column 3. As

chromatogram shown in

buffer,

insoluble

supernatant column buffer

ed with Elution 0.013, 6.5, tein S-1 similar fraction The than Fig.

carried and order. 0.05M

stepwise phosphate shown two

As into As

separated S-2). those S-2

(fraction were II D. lower in ch is puri

Column size, rate, 63% 2.4~30cm; total 24%). fraction recovery volume, of protein, 10.0ml; 95 flow 120ml/hr; ; D-2, FIG. 3. Column Chromatogram of F-2 on DEAEcellulose.

these

described should of fraction fraction S-1 was If of fraction

in previous correspond S-1 S-2, not fraction S-1 was

toxicity that 2, of

fraction

homogeneous S-1 may found

romatographically. fied, higher. the toxicity The

fraction S-1

become to be

(D-1,

68

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

fraction F-2 was separated into two fractions (fraction D-1 and D-2), as in the case of CMcellulose column chromatography. From the chromatograms shown in Figs. 2 and 3, and the polyacrylamide gel disc electrophoretic pattern (Fig. 5), it was assumed that 0.01 M fraction (fraction D-1) corresponds to frac tion S-2, and 0.04 M fraction (fraction D-2) to fraction S-1. The fraction D-1 was col lected and rechromatographed on a DEAEcellulose column under the same conditions as above, and it was found that fraction D-1 behaved homogeneously on DEAE-cellulose column chromatography, as obviously seen in Fig. 4. The yield of the purified ricin from 100 g of defatted castor bean meal was 1.2g (1.2%), which is about 6times higher than that pre viously reported.

FIG. 5. Analytical Disc Electrophoresis Various Fractions and Ricin D.

of

the S-2.

(1) Crude ricin (2) Fraction F-2 (3) Fraction (4) Fraction D-1 (5) Ricin D.

tion F-2 from a Sephadex G-75 column. contains mostly two protein fractions. Thefraction S-2 and D-1 gave each single band which coincided in migration with that of ricin D. Thus, ricin D could be purified by either CM- or DEAE-cellulose chromato graphy from fraction F-2. It should be men tioned, however, that the DEAE-cellulose chromatography yielded a better recovery of ricin D.
FIG. 4. Column Chromatogram of D-1 on DEAEcellulose. Column size, 2.4~24cm; flow rate, 120ml/hr. fraction volume, 7.3ml;

B.

Homogeneity purified ricin

and

physical

properties

of

Polyacrylamide Homogeneity during the of the

gel protein was pH

disc

electrophoresis. fractions obtained with 5 the crude of to removed G-75. The which the by up gel disc shows protein ricin a

purification at

examined Figure of figure,

electrophoresis disc fractions. contains protein permost filtration electrophoretic As mainly fraction band through

8.3. patterns

seen

in three

this

fractions,

corresponding was apparently Sephadex

frac-

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

Biochemical

Studies

on Ricin.

Part V

69

FIG. Protein buffer,

8.

Moffitt-Yang's concentration: pH 6.5, 13.0.

Plot 1.9%

of in

the

Purified

Ricin. phosphate

0.005M

FIG. 6. 6.5,

Ampholine Electrophoresis of Fraction D-1. 10-6M Cu2+ ion and had a

containing

faint metallic might contain

blue color, this crystalline ricin Cult ion. The difference in

isoelectric point observed might be caused by the change in surface charge of the ricin pro tein due to the binding of copper. Optical rotatory dispersion. The ORD con

stant, Ac, of the purified ricin was estimated from the slope which was obtained from the Yang's plot shown in Fig. 7. The MoffittFIG. 9. Crystals Obtained from the Purified Ricin.

values value ,c a0,

of of and

a0 is b0, b,

given from were

from the

the slope.

intercept The -138 content to be b0. toxicity the was weight

and values and of

the of -66, the

235nm,

respectively. purified 10.5% The purified lethal ricin

FIG. Protein buffer, 7. Yang's Plot of 1.9% 13.0. the in Purified 0.005M Ricin. phosphate

The -helix ricin was the of calculated

around

from toxicity ricin dose, nitrogen it was

values ricin.

of c, and The D-1), of MLD48, body

of minimum

the

(fraction in terms per gram

0.001g of mouse into

when mice. D

injected value is the

intraperitoneally same as that of

concentration: pH 6.5,

This

ricin

reported

previously.1)

Yang's culating and b0,

plot the is

of

the

purified

ricin

for

the

cal a0,

Crystallization tained the from crystalline the

of purified D

ricin. ricin in form

The was (Fig.

crystal identical 9).

ob to

Moffitt-Yang in Fig. 8.

parameters, In this plot,

shown

the

ricin

70

K.

HARA,

M.

ISHIGURO,

G.

FUNATSU

and

M.

FUNATSU

REFERENCES 1) . 2) M. Ishiguro, G. Funatsu and M. Funatsu, Agr. Biol. Chem., 35, 724 (1971) M. Ishiguro, T. Takahashi, G. Funatsu, K. Haya

3) 4) 5)

shi and M. Funatsu, J. Biochem., 55, 587 (1964). L. Ornstein, Ann. N.Y. Acad. Sci., 121, 321 (1964). W. Moffitt and J. T. Yang, Proc. Natl. Acad. Sci. U.S., 42, 596 (1956). M. Ishiguro, T. Takahashi, K. Hayashi and M. Funatsu, J. Biochem., 56, 325 (1964).