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Biochemical properties of ricin inimmature castor seedSrinivas V. S. Chakravartula a & Nagaraj Guttarla ba Natural Products and Medicinal Chemistry Laboratory, IslandVitamins - Venus Pharmaceuticals Inc. , Farmingdale, NewYork–11735, USAb Division of Biochemistry , Directorate of Oilseeds Research ,Rajendranagar, Hyderabad–500 030, IndiaPublished online: 19 May 2008.

To cite this article: Srinivas V. S. Chakravartula & Nagaraj Guttarla (2008) Biochemical propertiesof ricin in immature castor seed, Natural Product Research: Formerly Natural Product Letters, 22:7,600-606, DOI: 10.1080/14786410701602593

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Natural Product Research, Vol. 22, No. 7, 10 May 2008, 600–606

Biochemical properties of ricin in immature castor seed

SRINIVAS V. S. CHAKRAVARTULA*y and NAGARAJ GUTTARLAz

yNatural Products and Medicinal Chemistry Laboratory,Island Vitamins - Venus Pharmaceuticals Inc., Farmingdale, New York – 11735, USA

zDivision of Biochemistry, Directorate of Oilseeds Research, Rajendranagar,Hyderabad – 500 030, India

(Received 29 May 2007; in final form 28 July 2007)

The biochemical properties of ricin at different stages of seed i.e. from immature to mature seedwere studied. Hemagglutination, SDS-PAGE and UV-spectrometry studies showed totalabsence of RCA protein in the immature seed. Interestingly, ricin extract on SDS-PAGEshowed only one protein band with a molecular weight of 29,000 dalton corresponding to themolecular weight of A chain of ricin. Our results have shown that at immature seed level onlythe toxic moiety of ricin (A chain) is being synthesized first and gradually the RCA and B chainof ricin.

Keywords: Castor seeds; Ricin, Ricinus communis agglutinin; Hemagglutination; Gelchromatography; SDS-PAGE

1. Introduction

Ricin a toxic protein is found in castor oil plant (Ricinus communis, L) seeds. Ricin iscomposed of two poylpeptide chains (A & B chain) connected by a single disulphurbond [1]. It has got interesting antiviral activity [2–10]. Toxicity was believed to resultfrom the observed ability of the ricin preparation to agglutinate red blood cells ofmammals [11,12]. More recent studies established that ricin preparations were a mixtureof potent cytotoxins RCA (R. communis agglutinin) [12]. Both the toxin proteins aresynthesized in the endosperm cells of maturing seed, and are stored in an organellecalled the protein body, a vacuole compartment [14,15]. Ricin is classified as ribosomeinactivating protein (RIP-II) whose member disrupts ribosomal architecture andtherefore stops protein synthesis [15,16]. RCA is nontoxic but agglutinates red bloodcells [17,18].

One common factor in most of the work reported in the literature involving ricin andRCA toxins is the lack of information related to their biochemical properties atdifferent stages during the seed germination and their mode of formation in differentcultivars of castor seed. Knowledge of these factors may contribute to a better

*Corresponding author. Tel.: 631-249-4140. Fax: þ16312494125. Email: srinivas@venuspharmaceuticals.com

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online � 2008 Taylor & Francis

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DOI: 10.1080/14786410701602593

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understanding of ricin levels and its toxicity. The present article talks about these

factors.

2. Results and discussion

The crude ricin extract from immature and mature seeds were used to perform the

hemagglutination assay in order to confirm the presence of RCA protein, although RIA

method (Radio Immuno Assay) has been reported to confirm the presence of ricin in all

the ricin extracts [19]. As we obtained minute quantities of the extract from immature

seed we limited with agglutination test.Agglutination assay is an indirect method available for confirming the presence of

ricin in the extract because ricin binds to RCA protein before separation [1] and thiswas confirmed by chromatographic experiments discussed in this study.

The ricin extract from the immature seed did not show any signs of agglutination

(figure 1) while in the normal seed agglutination was positive (unpublished results).

Thus it is clear that the RCA protein is totally absent in the immature seed, which

further revealed that the synthesis of RCA did not take place at the immature stage or

A B B′ C C′ D D′

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Figure 1. Hemagglutination of ricin extract from immature seed.

Well A (1 to 7 top to bottom) :Antiserumþ SRBC (Standard); Well B, B’ (1 to 7 - Ricin standardþ SRBC)C & D (1 to 7 top to bottom - Ricin extractþSRBC without antiserum) Well C’ & D’ are duplicates of ricinextract. Well (8) in all contain blank (saline solutionþ SRBC)

Well no. 1: 1 : 100 Well no. 2: 1 : 200Well no. 3: 1 : 400 Well no. 4: 1 : 800Well no. 5: 1 : 1600 Well no. 6: 1 : 3200Well no. 7: 1 : 6400 Well no. 8: Blank

Biochemical properties of ricin in immature castor seed 601

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the activity did not develop at that stage. For further confirmation, ricin extracts fromimmature and mature seeds were passed on the gel column.

The ricin extract from immature seed showed a sharp peak (figure 2a). Ricin from the

normal seed (mature) separated quickly into two fractions, i.e. peak (1) correspondingto RCA protein and the second, peak (2) to ricin (figure 2b). RCA being a larger protein

got separated first followed by ricin.These results suggest that there is a significantdifference in ricin synthesis with respect to seed maturity. To understand this in detail

the above fractions were pooled, concentrated and loaded on SDS-PAGE in order toknow the purity and molecular weights using standard protein markers along with ricin

from the normal seed.Electrophoresis studies of ricin fractions of immature and mature seeds from the

sephadex columns (figure 2) showed single protein band with and without betamercaptoethanol while ricin from the mature and over mature seeds had separated into

two polypeptides (A & B chain) with beta mercaptoethanol having molecular weights of29,000 and 28,500 daltons (figure 3b) which are in agreement with earlier reports [1–11]

while ricin from the immature seed had separated into a single polypeptide which isnothing but A chain with a molecular weight of 29,000 daltons, suggesting the absence

of sulphur bonds (figure 3a). The RCA protein was totally absent, this wasconfirmed from the hemagglutination assay (figure 1) and also from gel chromato-

graphy (figure 2a). The fraction obtained from the gel column was also found to be pureand homogenous. These studies for the first time revealed a low molecular weight

protein of ricin obtained from the immature seed and it does not belong to the RCAprotein mostly the A chain of the ricin molecule. Ricin as a whole A & B chain once

purified does not show agglutination [1,2] and [8,10] while in wild castor seed at allstages of seed maturity (immature to over mature) contained RCA protein and the toxic

protein ricin (A & B chains) [14]. Our studies in DCH 32, castor variety revealeda different picture. This might be due to the rapid enzymatic hydrolysis reactions, which

are active at the early stages of the seed development that might have resulted in thedegradation of RCA protein at the initial stages. It is also possible that the degradation

of RCA may be due to the stress conditions that prevail during the initial stages of seed

(a) (b)

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Absorbance

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0.7

0 20 40 60 80 100 120 140 160 180

0.2

0.1

0.3

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0.5

0.6

0.7

0.2

0.1

0.3

0.4

Fractions0 20 40 60 80 100 120 140 160

Fractions

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Figure 2. Purification of ricin from immature seed (a) and mature seed (b) on sephadex G-75 separated on acolumn (2.0� 90 cm) with 50mM Tris-Cl buffer, pH 7.5 with flow rate of 60mLh�1. Fractions collected 3mL.

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formation [1,14] [20,21] or possibly may be due to genetic variation in the hybrid varietyof castor DCH-32 (used in this study) which might have influenced the growthconditions and the biosynthetic pathway of ricin synthesis at initial stages of seedgermination while in the same variety at the later stages (mature and over mature) boththe proteins (RCA and ricin A & B subunits) were observed (figure 3b), thereforedifferent ricin preparations may behave differently with respect to its castor cultivars,their toxic properties and the structural compositions in different parts of the world.

The A subunit or the toxic moiety of ricin showed its strong presence and did notshow any signs of degradations as evident from the electrophoresis (figure 3a) whichfurther reveals that the A subunit is resistant from enzymatic cleavages. The earlysynthesis of A subunit of ricin in immature seed may also be possibly to protect theplant itself from pests attack.

Based on the above studies it is evident that the RCA protein is not being synthesizedat the immature levels, only A chain of ricin with a molecular weight of 29,000 daltonsis been observed first. The driving factor in this study is we can straight way isolate theA subunit of ricin followed by it’s purification which is thousand times easier thanpurifying ricin from a normal seed as it involves separation from RCA initially anda series of purification steps.

Several researchers have reported and continuously being studied about the A chainof ricin, which is being used as immunotoxin for therapeutic purposes like in thetreatment of leukemia or blood cancer, and also for HIV [22]. With these observationsone can possibly get the A subunit of ricin straight away from immature seed forpharmaceutical applications.

SDS-PAGE PROFILE OF RICIN’S

Ricin from immature seed Ricin from normal seed

(a) (b)

205kD

97.5kD

66kD

44kD

29kD

20kD

14kD

29kD

205kD

97.5kD

66kD

44kD

29kD

14kD29.5kD28.5kD

Figure 3. SDS-PAGE profile of purified fractions of ricins from immature seed (a) and mature seed(fraction 2) (b) collected from sephadex columns (figure 2(a) and (b)) separated on 12.5% acryl amide gel atpH 8.8 Tris-Cl buffer under reducing conditions (using betamercaptoethanol) along with std. protein markersleft side decreasing order (2.05 kD, 97.5 kD, 66 kD, 44 kD, 29 kD, 20 kD, 14 kD).

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Structural studies of ricin A subunit can give more detailed information to pin pointits resistance towards enzymatic cleavages for its wider utility. However, quantifying the

yields of A chain of ricin and its toxicity in the immature seed has to be studied in detailfor its wider applications.

3. Experimental

DCH-32, a hybrid castor seed about (50 g each) of different stages namely immature

(20 days prior to maturity), mature (normal) and over mature seeds (15 days aftermaturity) were collected in the month of November, at DOR research farm,

Rajendranagar, Hyderabad, India. The seeds were stored in polythene bags in a deepfreezer at �20�C until analysis.

3.1. Isolation of ricin

Ricin from the castor seed was isolated as per the procedure of [7] with slightmodifications. The outer covering of the seeds (hulls) were removed, the kernel was

powdered in a Knifetec sample mill and completely defatted using ice-cold petroleumether (40–60�C) and diethyl ether. The defatted powder was suspended in double

distilled water and adjusted to pH 4.0 using HCl. The suspension was agitated for 7–8 h.The solution was filtered under vacuum. The yellow supernatant liquid was collected

and the residue was decanted. The supernatant was precipitated by adding solid NaCltill its saturation. The resulting precipitate was dissolved and extensively dialyzed for

72 h in double distilled water and in Tris-Cl buffer at pH 7.5 at 4�C. The insolubleparticles and other impurities separated in dialysis were decanted. The clear protein

solution was precipitated using 2% ammonium hydroxide. The resulting precipitate wasdecanted. The clear protein solution was finally precipitated with saturated ammonium

sulphate salt solution. Crude ricin was separated. The crude ricin was dialyzed asmentioned above and used for further studies.

3.2. Hem agglutination

This test was performed in a 96 well U-bottomed microtitre plate [23] in order tocharacterize the ricin and the RCA proteins. SRBC (sheep red blood cells) was

collected from Veterinary Science College ANGRAU campus, Rajendranagar,Hyderabad. 4% suspension of sheep blood erythrocytes were washed in 0.9% saline

solution. 0.5mL of normal saline solution was added in all the wells. 0.5mL of ricinextracts were added so that each well has half the concentration of the preparations

than the preceding one, but has double the concentration than the following one, laterthe wells were mixed keeping blanks (saline solutionþ SRBC). A total of 0.5mL of

SRBC were added in all the wells along with standard ricin (Sigma Chemicals, USA)and left undisturbed for 9–10 h at room temperature. The least hem agglutination factor

was taken as the end point titer.

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3.3. Preparation of standard (antiserum1SRBC)

The sheep red blood cells were injected male albino mice. The antibodies were collectedafter a week by retro orbital plexus (vein from the ear). The antiserum was then addedto the sheep red blood cells. Agglutination was observed within minutes. This was usedas the standard and diluted as mentioned above.

3.4. Gel chromatography

The ricin fraction was concentrated and applied on a column (2.0� 90 cm) packed withsephadex G-75 (Pharmacia biotech, Sweden. The ricin fractions were eluted withTris-Cl buffer (0.05M) pH 7.5, about 3mL each of ricin fractions was collected andmonitored on UV-spectrometer at 280 nm [25].

3.5. SDS-PAGE electrophoresis

The molecular weight and purity of protein was determined by SDS-PAGE [25]. The gelwas stained with 0.1% coomasie brilliant blue and then destained with 7% acetic acid.The molecular weight standards used in this study were Myosin rabbit muscle(MW 205,000), Phosphorylase (MW 97,400), BSA (MW 66,000), Ova albumin(MW 44,000), Carbonic anhydrase (MW 29,000), Trypsin inhibitor (MW 20,000) and�-lacta albumin (MW 14,200). The protein markers were procured form SigmaChemicals, USA.

Acknowledgements

This work is supported by Indian Council of Agricultural Research, grant to GN andfellowship to CVS. We wish to thank the Director of DOR, for his encouragement andthe plant breeding laboratory for providing the seed material.

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