biochemical studies on ricin ii. m. w. and physiochemical properties - ishiguro - j biochemistry 56...

8/13/2019 Biochemical Studies on Ricin II. M. W. and Physiochemical Properties - Ishiguro - J Biochemistry 56 (1964)

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The Journal of Biochemistry, Vol. 56, No. 4, 1964

Biochemical Studies on RicinII. Molecular Weight and Some PhysicochemicalProperties of Crystalline Ricin D

By MASATSUNE ISHIGURO, TAKAO TAKAHASHI, KATSUYA HAYASHIand MASARU FUNATSU

(From the Laboratory of Biochemistory, Faculty of Agriculture,Kyushu University, Fukuoka)(Received for publication, March 21, 1964)

A procedure of the purification of ricinby cellulose-derivatives column-chromatogra-phies and subsequent crystallization has beenreported previously (1). By the crystallization a protein component which was intimately accompanied by ricin was removedresulting in a remarkable increase in toxicity.In addition, the crystalline ricin obtainedwas revealed to be homogeneous either physicochemically, i. e. ultracentrifugally and electro phoretically, or physiologically. The mini-mum lethal dose of the crystalline ricin was0.001 g. ricin nitrogen per gram of bodyweight of mouse. There was no evidencethat the crystalline ricin contains hemagglutinating and proteolytic activities. A non-specific protein coagulating activity containedin crude ricin was finally eliminated by thecrystallization. Consequently, ricin thus obtained in a crystalline form was concludedto be the principle of castor bean toxin andreferred to as ricin D.

This paper deals mainly with some physicochemical properties and molecular weightof ricin D.

EXPERIMENTALRicin Preparation -Ricin D prepared by the pro

cedure described in the previous paper (1) was employ-ed after recrystallization throughout this study. Theminimum lethal dose of this preparation was 0.001g.ricin nitrogen per gram of body weight of mouse.

Sedimentation Constant-Ricin D was dissolved inacetate buffer solution (=0.1) of pH 5.0 and dialyzedagainst the same buffer solution for at least 48 hoursat 4C. Removing insoluble materials by centrifugation, a clear supernatant was brought to the sedimen-

tation measurement with a Spinco model E ultra-centrifuge at 20C. All runs were done at a speedof 56,100r.p.m. The concentration of protein usedwas 0.78 per cent.

Determination of Molecular Weight - Molecularweight of ricin D was determined by A r c h i b a l d' smethod (2). A 0.7 per cent solution of ricin D, whichwas previously dialyzed against 0.IM phosphate buffersolution of pH 6.0, was used for experiment. Determinations of concentration gradients at the meniscusand bottom of the cell, (dc/dx)m and (de/dxb, andof an initial concentraton, c0, were conducted in anusual standard cell and in a synthetic boundary cellrespectively. The speed of rotor was maintained at12,590r.p.m. Molecular weight was calculated accord-ing to the equations, (la) and (lb), described byArchibald (2).

Here M is the molecular weight, R the gas constant,T the absolute temperature, V partial specific volumeof solute, p the density of solution, cu the angularvelocity, c the solute concentration, and x the di-stance measured from the center of rotation to the topor bottom of the liquid column in the cell, where thevalues of c and dc/dx must be determined. Thesubscripts, m and b, refer to the position of themeniscus and bottom of the cell, respectively.Measurement of Electrophoretic Mobility and Isoelectric Point of Ricin D-The mobility of ricin D wasmeasured by Hitachi HTD-1 (Tiselius type) eleetro

phoretic apparatus under the conditions shown inTable I.Measurement of Ultraviolet Absorption Spectrum ofRicin D-Measurement of ultraviolet absorption spectrum of ricin D was conducted at pH 5.0 with a

Beckman DU spectrophotometer. Protein concentration usd was 0.083 per cent.325


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326 M. FUNATSUTABLE I

Summary of Electrophoretic Spreading Experiments of Ricin D

RESULTS AND DISCUSSIONSedimentation constant, s20, of ricin D

was calculated to be 4.64x 10-13 at 20C.From the data obtained with Archibald'smethod, (dc/dx)m/cmxm, and (dc/dx)b/Cbxb,were calculated and plotted as a function ofrunning time as shown in Fig. 1, where thebest straight line was drawn. The values ofthe intercepts of these lines at zero timewere 1.28 and 1.21 for meniscus and bottomrespectively. From these values and usingR=8.3144x l07 dyne/cm2, T=281.4,(02=1.7382X106, V=0.74, and p=1.0, molecular weights,Mm and Mb, were determined to be 6-17X104 and 5.83 X 104. Average molecular weightwas, therefore, 6.00 x 104. As has been previously reported (3), although the molecularweight of ricin C1 was determined to be3.10x104 by the light scattering measurement, it was found that ricin C1 tended toassociate producing dimer in its concentratedsolution. Therefore, it may be reasonable topostulate that the value of molecular weight

FIG. 1. Determination of molecular weightof ricin D by Archibald's method.(J at meniscus, L at bottom,of ricin D in question is that of a particle weight of ricin D-dimer. This will be studied in detail in the followinIG. 2. pH-Mobility curve for ricin D.

of ricin D in question is that of a particleweight of ricin D-dimer. This will be studiedin detail in the following paper.The electrophoretic mobility of ricin Dhas been determined at the pH range from3.6 to 9.0. The mobilities estimated are summarized in table I, and plotted against pHin Fig. 2. In these experiments, a symmetrical peak was obtained, and there was noindication of the presence of other protein.The isoelectric point of ricin D was thusfound to be pH 5.9. It is of great interestthat ricin D moved to anode surprisinglyfaster at pH 3.6 than did at pH 5.0. This


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Crystalline ricin D 327seems to indicate that a remarkable changeof charge density occurs on the surface ofthe protein particle at the pH range below5.0. Ultraviolet absorption spectrum of ricinD was shown in Fig. 3. The maximum andminimum absorptions were observed at 280and 250 mp respectively.FIG. 3. Ultraviolet absorption spectrum of ricin D. Protein concentration : 0. 083 per cent.

SUMMARYInvestigation of molecular weight andsome physicochemical properties of ricin Dwas carried out. Sedimentation constant, s20,was calculated to be 4.64x 10-13 in acetatebuffer solution (p=0.1) of pH 5.0. Molecularweight of ricin D was measured by Arch i-b a l d's method and calculated to be 6.00 x104. Electrophoretic mobilities were measuredat pH values 3.6, 5.0, 6.0, 8.0, and 9.0 respectively and the isoelectric point of ricin Dwas found to be pH 5.9. Ultraviolet absorption spectrum of ricin D was consistent with

that of a common protein.REFERENCES

(1) Ishiguro, M., Takahashi, T., Funatsu, G., Hayashi, K., and Funatsu, M., J. Biochem., 55,587, (1964)(2) Archibald, W.J., J. Phys. Colloid. Chem., 51,1204 (1947)(3) Funatsu, M., and Funatsu, G., TanpakushitsuKakusan Koso (in Japanese), 5, 451 (1960)IG. 3. Ultraviolet absorption spectrum of

ricin D. Protein concentration : 0. 083 per cent.

biochemical studies on ricin ii. m. w. and physiochemical properties - ishiguro - j biochemistry 56...

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