Bioscience Reports, Vol. 6, No.

12, 1986

Dye Affinity Chromatography of Ricin Subunits

Simonetta Sperti, Lucio Montanaro and Fioretta Rambelli
Received January 10, 1987

KEY WORDS: ricin; A chain; blue-Sepharose; blue dextran-Sepharose; poly(U)-Sepharose; ribosomeinactivating proteins.

A rapid and simple method for the isolation of the two chains of ricin is described. The method involves two chromatographic runs, the first on blue-Sepharose and the second on blue dextran-Sepharose. It is moreover shown that the A chain of ricin, but not the B chain, binds to poly(U)-Sepharose.

INTRODUCTION A large number of proteins of plant origin, named ribosome-inactivating proteins (RIPs) (Barbieri and Stirpe, 1982; Roberts and Selitrennikoff, 1986; Stirpe and Barbieri, 1986), irreversibly inactivate the 60S ribosomal subunit by a yet unknown mechanism of action. Ricin belongs to the family of double chain RIPs and consists of an A chain, which catalytically inactivates ribosomes, and a B chain, which binds to D-galactose containing receptors of the cell surface allowing the entry of the toxin. The two chains of ricin are held together by a disulphide bridge and by hydrophobic forces. These play a dominant role in keeping the chains associated after reduction of the disulphide bond (Lewis and Youle, 1986) hinder the isolation of the two subunits of ricin. Appukuttan and Bachhawat (1979) reported that ricin A chain, but not the intact toxin or the B chain, gives a red shift of Cibacron blue in solution. Since dye affinity chromatography on blue-Sepharose (in which Cibacron blue is directly bound to the agarose matrix) or on blue dextran-Sepharose (in which the same chromophore is linked to the matrix through a dextran spacer) is widely used in the purification of
Dipartimento di Patologia Sperimentale, Universit/l di Bologna, Via S. Giacomo, 14, 1-40126 Bologna, Italy.
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0144-8463/86/1200-1035505,00/0 ~:~1986PlenumPublishing Corporation

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Sperti, Montanaro and Rambelli

proteins, it was envisaged that chromatography of reduced ricin on dye columns might prove a useful technique in the separation of its constituent chains. Many methods have already been described for their isolation, but none of them is really satisfactory, as proved by the many modifications suggested. The basic procedures involve anion and cation exchange chromatographies (Olsnes, 1978) or aff chromatography on Sepharose 4B followed by ion exchange chromatography (Yoshitake et al., 1979). The A chain has also been obtained by repeated elution from Sepharose 4B (Lewis and Youle, 1986). Single step procedures involve chromatography at acid pH in the presence of urea (Giirtler and Horstmann, 1973) or chromatography on guar gum columns (Appukuttan and Bachhawat, 1979). In these last studies the isolated A chain was not tested for its biological activity. The results reported in the present paper show that a single chromatographic run on dye affinity columns is insufficient to yield both pure A and B chains. However combination of affinity chromatography on blue-Sepharose and on blue dextranSepharose yields pure B chain in the first step and fully active pure A chain in the second. It is moreover shown that the purified A chain binds to poly(U)-Sepharose and is eluted from blue dextran-Sepharose by poly(U).

MATERIALS AND METHODS Ricin was a generous gift of Professor F. Stirpe. An aliquot of the toxin was labelled with 3H by reductive methylation with [3H]formaldehyde (New England Nuclear Corp., USA) and sodium cyanoborohydride as described by Cox and Greenwell (1980). Before use the radioactive protein (8640 dpm/#g) was mixed with unlabelled toxin to a specific radioactivity of 35-65 dpm/ktg. Blue-dextran 2000, blueSepharose CL-6B, CNBr-activated Sepharose 4B and poly(U)-Sepharose 4B were from Pharmacia, Uppsala, Sweden. The activated Sepharose was coupled with blue dextran according to the instructions of the manufacturer. All other chemicals were reagent grade. Affinity chromatography was performed at room temperature with the gels packed in 0.9 x 1.0 cm or 1.4 x 2.0 cm columns. Details of the single procedures are in the legends to figures. Radioactivity was measured on 0.1 ml of the chromatographic functions (1 ml). Protein synthesis was carried out with a lysate of rabbit reticulocytes as described by Sargiacomo et al. (1983). IDs0 is the amount of inhibitor causing 50 % inhibition in 1 ml of protein synthesizing system and was calculated by the least-squares method applied to the linear regression between fractional activity and log of inhibitor concentration. SDS-polyacrylamide gel electrophoresis was performed with the Laemmli (1970) system on gel slabs in the presence of 2-mercaptoethanol. The molecular weight of ricin was assumed to be 65,000 and those of its A and B chains 32,000 and 34,000 respectively (Olsnes, 1978). E (1%; 280; 1 cm) is 7.65 for the A chain and 14.9 for the B chain (Olsnes, 1978).

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RESULTS AND DISCUSSION

Whole ricin, dissolved in 10 mM Tris-HC1, pH 7.5, containing 0.2 M lactose, does not bind to blue dextran-Sepharose nor to blue-Sepharose. When reduced ricin was applied to blue dextran-Sepharose (Fig. 1, top), a sharp peak containing most of the total radioactivity (86 ~) emerged immediately with the 0.2 M lactose washing, while the residual radioactivity was eluted by 0.1 M NaC1. SDS-polyacrylamide gel electrophoresis showed that the first peak contained both chains of ricin, while the second peak was pure A chain. Rechromatography of the first peak on the same blue dextran-Sepharose column only moderately increased the yield of pure A chain, It is well known (Hughes et al., 1982) that low concentrations of divalent metal ions, particularly Zn 2 +, promote the binding of proteins to immobilized triazine dyes. However attempts to increase the yield of pure A chain by adding Zn 2 + to the sample of reduced ricin prior to adsorption onto the blue dextran-Sepharose column and including the same cation in the chromatographic buffers were without success. In fact, although the distribution of radioactivity between the first and the second peak became 7 0 ~ and 3 0 ~ at l m M Zn 2 and 5 1 ~ and 49~o at 2 m M Zn 2+, SDS-

15
E

10

%
0..

-o 4
~E z z

/I

~1

10

20 30 Fraction number

40

Fig. 1. Elution profile of reduced ricin from blue dextran-Sepharose and from blue-Sepharose. [3H]Ricin (1 mg/ml) was reduced 1 h at 37~ in 1 ml of 10mM Tris, pH 7.5, containing 0.2 M lactose and 5~o 2-mercaptoethanol. A small amount of precipitate formed during reduction and was removed by centrifugation; examined by SDS-polyacrylamide gel electrophoresis it contained approximately equal amounts of A and B chains. Top: 1 ml of supernatant (63,500dpm) was applied to a blue dextran-Sepharose column (1.4 2.0cm; flow rate 45 ml/h). Bottom: 0.63 ml (40,000dpm) was applied to a blue-Sepharose column (1.4 2.0cm; flow rate 50ml/h). Both columns were equilibrated with 0.2 M lactose in 10mM Tris, pH 7.5, containing 1 ~ 2mercaptoethanol and were washed with the same buffer after adsorption of the sample. The arrows indicate where the washing buffer was substituted with 10mM Tris, 0A M NaCI and 0.5 M NaC1 in 10 mM Tris.

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Sperti, Montanaro and Rambelli

p o l y a c r y l a m i d e gel electrophoresis showed the presence of the A a n d B chains in b o t h peaks. The elution profile of r e d u c e d ricin from b l u e - S e p h a r o s e is quite different (Fig. 1, b o t t o m ) . The r a d i o a c t i v i t y was a l m o s t equally d i s t r i b u t e d between two peaks. The first p e a k , which e m e r g e d with the 0.2 M lactose washing, was slightly r e t a r d e d a n d the second p e a k r e q u i r e d 0.5 M NaC1 for elution. S D S - p o l y a c r y l a m i d e gel electrophoresis s h o w e d t h a t all the fractions of the first p e a k c o n t a i n e d p u r e B chain, while the second p e a k was m o s t l y A chain with a small a m o u n t of B chain. F i g u r e 2 shows the c o m b i n e d p r o c e d u r e for the purification of b o t h chains of ricin. R e d u c e d ricin (5 mg) was first a p p l i e d to the b l u e - S e p h a r o s e c o l u m n yielding p u r e B
P e a k II

Peak

IV

I" ,r

'-r" o9

2
Q. "10 Peak I

Zlt
20 4O Fraction n u m b e r

O3

--

10

20

Fig. 2. Two step procedure for the purification of ricin A chain. [3H]Ricin (5 mg) was reduced 1 h at 37~ in 5 ml of 10 mM Tris, pH 7.5, containing 0.2 M lactose and 5 ~ 2-mercaptoethanol. Left: After centrifugation, the supernatant (176,250 dpm) was applied to a blue-Sepharose column (1.4 x 2.0 cm) equilibrated with 0.2 M lactose in 10 mM Tris, pH 7.5, and 1 ~ 2-mercaptoethanol. A first peak (I) was eluted with the 0.2 M lactose buffer and a second peak (II) with 1 M NaC1 in 10raM Tris, pH 7.5. Fractions 36--39 of peak II were pooled (1.16 A2so) and dialysed at 4~ against 2 x 1 1 of 10 mM Tris, pH 7.5, containing 1 ~ 2-mercaptoethanol. Right: a sample of the dialysate (0.90 A2s0)was applied to a blue dextran-Sepharose column (1.4 x 2.0 cm) equilibrated with the same buffer. From this column a small amount of radioactivity was eluted by 0.2 M lactose in 10 mM Tris, pH 7.5, and 1 ~ 2mercaptoethanol (III) and a sharp peak by 0.1 M NaCI in 10 mM Tris. In the insert, SDS-polyacrylamide gel electrophoresis of: a, reduced ricin; b, peak I; c, peak II; d, peak IV. The pooled fractions of peak I, freed from 2-mercaptoethanol and lactose by dialysis against 10 mM Tris, contained 3.49 A2so, equivalent to 2.34 mg of pure B chain (yield 89~). In peak IV (fractions 17-19) from the blue dextran-Sepharose column 0.61 A2s o were present, equivalent to 0.8 mg of pure A chain (overall yield 55 ~). SH, 2-mercaptoethanol.

Dye Affinity Chromatography of Ricin Subunits

1039

chain in peak I. Peak II was eluted with 1 M NaCI to obtain a sharp peak. After dialysis against 10 mM Tris-HC1, pH 7.5, containing 1 ~o 2-mercaptoethanol it was applied to the blue dextran-Sepharose column, yielding pure A chain in peak IV. The isolated A chain maintained full inhibitory activity on protein synthesis. The IDso of reduced ricin was 3.95 ng (0.06 pmol) and that of the A chain was 1.61 ng (0.05 pmot). The method described is convenient and rapid, each chromatographic run requiring not more than 30-45 min. It must be pointed out that: (i) reduction of ricin (1 mg/ml) in the presence of lactose minimizes aggregation and precipitation of the B chain; (ii) lactose is necessary to elute the B chain from both dye-Sepharose columns; (iii) in step two the sample is applied to the blue dextran-Sepharose column in the absence of lactose, since dialysis against 0.2 M lactose or addition of solid lactose to peak II causes precipitation of the A chain; and (iv) the volume of the eluents in the second chromatographic run must be kept as low as possible to minimize loss of the A chain. It has been recently reported (Sperti et al., 1987) that gelonin, a single chain RIP, which, like the A chain of ricin, inactivates the 60S subunit of ribosomes by a yet unknown mechanism of action (Stirpe et al., 1980), and alpha-sarcin, which inactivates the 60S subunit acting as a RNAse (Endo and Wool, 1982), bind to poly(U)-Sepharose and adsorb to blue dextran-Sepharose columns from which they are eluted by low concentrations of polynucleotides. The presence of a polynucleotide site on both proteins has been suggested, possibly related to a similar mechanism of action. Gelonin and alpha-sarcin are however basic proteins and in their interaction with polyanionic molecules a charge effect cannot be excluded. The A chain of ricin is not a basic protein (pI 7.5, Olsnes, 1978). As shown in Fig. 3, it binds to poly(U)-Sepharose (left) and it is effectively desorbed from blue dextranSepharose by poly(U) at the concentration of 100#g/ml (right). On the contrary, poly(U), even in the presence of 100 mM NaC1, did not significantly displace the A chain from blue-Sepharose (not shown). The different effectiveness of poly(U) in eluting the A chain from the two dye affinity columns is probably related to the stronger binding of the protein to blueSepharose, of which advantage is taken in the above described method for the

E2 O.

10 Fraction number

\..,.
10

Fig. 3. Affinity ofricin A chain for poly(U). Left: Binding to poly(U)-Sepharose. [SH]A chain (223 #g) or [SH] B chain (225 #g) in 1 ml of 10 mM Tris, pH 7.5, were applied to poly(U)-Sepharose columns (0.9 x 1.0 cm) equilibrated with the same buffer. The B chain ( - - / k - - ) was eluted by 0.2 M lactose in 10 m M Tris, while the A chain ( - - O - - ) remained bound and was eluted by 1 M NaC1 in 10mM Tris. Right: De sorption by poly(U) from blue dextran-Sepharose. [SH]A chain (240 #g) in 1.4 ml of 10 m M Tris, pH 7.5, was applied to a blue dextran-Sepharose column (0.9 1.0cm) equilibrated with the same buffer. The protein was quantitatively eluted by poly(U) (100#g/ml).

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Sperti, Montanaro and Rambelli

purification of the two chains of ricin. Taken together the data of Fig. 3 indicate an interaction between the A chain of ricin and poly(U). The search for specific ligands more efficient in displacing the A chain from dye affinity columns and the extension of the investigation to other RIPs might help to find out the yet unknown substrate of these enzymatic proteins.

REFERENCES
Appukuttan, P. S. and Bachhawat, B. K. (1979). Biochim. Biophys. Acta 580:10-14. Barbieri, L. and Stirpe, F. (1982). Cancer Surv. 1:489-520. Cox, R. A. and Greenwell, P. (1980). Biochem. J. 186:861-872. Endo, Y. and Wool, I. G. (1982). J. Biol. Chem. 257:9054-9060. GiJrtler, L. G. and Horstmann, H. J. (1973). Biochim. Biophys. Acta 295:582-594. Hughes, O., Lowe, C. R. and Sherwood, R. F. (1982). Biochim. Biophys. Acta 700:90-100. Lewis, M. S. and Youle, R. J. (1986). J. Biol. Chem. 261:11571-11577. Laemmli, U. K. (1970). Nature 227:68~685. Olsnes, S. (1978). In: Methods in Enzymology (Ginsburg, V., Ed.), Vol. 50, pp. 330-335, Academic Press, New York. Roberts, W. K. and Selitrennikoff, C. P. (1986). Biosci. Rep. 6:19-29. Sargiacomo, M., Barbieri, L., Stirpe, F. and Tomasi, M. (1983). FEBS Lett. 157:113-118. Sperti, S., Montanaro, L., Rambelli, F. and Zamboni, M. (1986). Biosci. Rep. 6:901-908. Stirpe, F. and Barbieri, L. (1986). FEBS Lett. 195:1-8. Stirpe, F., Olsnes, S. and Pihl, A. (1980). J. Biol. Chem. 255:6947~5953. Yoshitake, S., Watanabe, K. and Funatsu, G. (1979). A#ric. Biol. Chem. 63:2193-2195.