Preliminary Crystallographic Data for a New Crystalline Form of Abrin*

SUMMARY A new crystalline form of abrin has been obtained from the most toxic constituent of the seed extract of Abrus precatorius. An x-ray diffraction study shows that the toxin crystallizes in a monoclinic unit cell of probable symmetry P21 and parameters (I = 113 A, b = 72 A, c = 71 A, and /3 = 103”. The asymmetric unit contains 2 protein molecules of molecular weight 63,800 and has a solvent content of approximately 45 % by volume. of protection various peptide

Rincc the first reports that abrin, present iu the seeds of Abrus precatorius, affords therapeutic protection against various tumors in rats and mice (1, 2), this toxic plant protein has been studied intensively iu several laboratories. ' It has been shown that abrin prevents peptide chain elongation (3), and thus inhibits protein synthesis in vitro (4) as well as in. vivo (5). Athough the mechanism of this toxic effect is not well understood, it has been attributed by Lin et al. (6) to the degradation of polyribosomes of liver or tumor cells. Olsncs and Pihl (4,7) have studied the properties, including inhibition of protein synthesis and binding to specific sites on the cell surface, of their abrin preparation and of subunits which they prepared from it.2 Recently we have developed a procedure by which two major toxic proteins have been isolated from the seeds of A. precalorius and purified to homogeneity by chromatography on I>EAE-* This research was supported by the United States Atomic Energy Commission under contract with the Union Carbide Corporation.
1 The equally toxic protein, ricin, present in the seeds of Ricinus commu?Lis, has several biological properties closely resembling those of abrin. Studies on this toxin have also been carried out by several groups, including our own.
2 On the basis of studies in a cell-free system of the biological functions of t.wo subunits isolated by 2-mercaptoethanol treatment of their abrin preparation, Olsnes and Pihl (4, 7) have shown that the inhibition of protein synthesis is exclusively associated with one of the subunits, while the ot,her subunit is mainly responsible for the binding of abrin to specific sites on the cell surface (7). The functions of the two subunits are thus analogous to those reported for the subunits of ricin (8,9) and of diphtheria toxin (see Ref. 7,8, and 10, and references cited therein).
It is of particular interest that reduced abrin exhibits less toxicity itL viva than the native protein (4, 7), a phenomenon also reported for ricin (7, 11, 12).
Sephades A-50 and CM-and DEAE-cellulose (13). Although the molecular weights of these two abrins are similar (60,100 for one species and 63,800 for the other, as determined by sedimentatiou equilibrium), they bchavc quite differcutly in several aspects (13). Our results strongly suggest that the abrin preparat.ions previously described by Lin et al. (14) and by Olsnes aud Pihl (7) arc not identical.
In fact, one of our abrins (hereafter designated abrin A) possesses properties similar to those of the preparation of Lin el al. (14), and the other of molecular weight 63,800 (hereafter designated abrin C) closely resembles that of Olsnes and Pihl (7).
Abrin A, the less toxic of the two, and the more positively charged fraction obtained from the DEAE-cellulose chromatography, has been crystallized in the orthorhombic space group P21212, with unit-cell parameters a = 75, b = 270, and c = 70 A, as communicated earlier (15). We now report the crystallization of the more toxic abrin C and preliminary x-ray data for these crystals.

MATERIALS AND METHODS
The abrin C preparation used was obtained from seeds of A. precalorius and purified by successive chromatography on DEAE-Sephadex A-50 and CM-and DEAE-cellulose (13). Crystallization was by the free interface diffusion technique described by Salemme (1F). Crystals were grown at 37" in Pyrex tubes (5 X 30 mm) by layering 50 ~1 of protein solution (22 mg per ml) over 100 ~1 of unbuffered 7OY,-saturated ammonium sulfate solution.
The x-ray diffraction patterns were recorded at room temperature (24") with nickel-filtered CuKol radiation from an Elliott rotating-anode generator operated at 40 kv and 40 ma. A Nonius precession camera with a crystal-to-film distance of 75 mm and a 0.25.mm collimator was used.

AND DISCUSSION
Unlike abrin A, which gives rise to crystals up to 1 mm long within 2 days under analogous treatments (15), crystals of abrin C required up to 2 mont,hs to grow to a maximum length of 0.5 mm. Although most of the crystals were poorly shaped, good rod-shaped specimens, elongated along the c axis, could be selected for x-ray diffraction.
The volume of the unit cell is 563,000 A3. The only observed systematic absences were Ok0 for k odd, indicating the probable space group to be P2,. As shown in Fig. 1, diffraction patterns extended to 3-A spacings, indicating that a high resolution study could be similar to our abrin C and to the preparation of Olsnes and Pihl (7) in its behavior in Sepharose 43 chromatography and in its clectrophoresis pattern on sodium dodecyl sulfate polyacrylamide gel. The molecular weight as measured by gel filtration was approximately 260,000, corresponding to a tetramerit species of a monomer with molecular weight 65,000. The crystals were orthorhombic with space group 1'2I212I and unitcell parameters a = 138, b = 142, and c = 178 A. The authors rcll concluded that the asymmetric unit contained one tetramcr. The asymmetric unit of abrin C crystals contains only 2 (monomer) molecules, rather than a tetramer (as above). The resolution of the data for abrin C crystals was found to be much superior to that obtained with crystals of abrin A (see above). Therefore, of all three crystal forms of "abrin" reported to date, that described hrrc appears to be the most favorable for s-ray structural investigation.
Aclznowlcdgmenfs--We are indebted to Drs. J. IV. Longworth and R. 13. Setlow for their interest in our work, and for reading the manuscript before it was submitted for publication. However, the crystals seemed somewhat sensitive to x-radiation; after 30 hours of exposure considerable weakening of the diffraction pattern was observed.
The number of protein molecules per unit cell is 2n, where n, thr number of molecules per asymmetric unit,, can be estimated by comparing values of VM (defined as the ratio of cryst,al volume to protein weight), based on various assumed values of n, with the normal range of values (I .68 to 3.53 A3 per dalton) compiled by Matthews (17) for crystals of globular proteins. For n = 1 and 3, the VW values are 4.41 and 1.47 A3 per dalton, respectively, which lie outside the normal range. For n = 2, Ii.+, = 2.21 A3 per dalton, which is close to the most commonly observed value. We conclude that) the abrin C crystals contain 2 molecules per asymmetric unit, as do t,he arthorhombic crystals of abrin A. The partial specific volume of abrin C is 0.731 cm3 per g, as calculated from the amino acid composition. With the assumption that n = 2, t,herefore, the solvent content of the crystal is 45% by volume, which is not very different from 43%, the most frequent value (17). Recently