Crystallization by Centrifugation Cso Separation SIR - Dover1 Pointed out That "Those and the Experiment Took 2.5 Hours

SCIENTIFIC CORRESPONDENCE

Crystallization by centrifugation Cso separation SIR - Dover1 pointed out that "those and the experiment took 2.5 hours. on coal investigating protein structure by X-ray During the removal of the concen- SIR - We reported recently1 that coal crystallography depend on a reliable trate, I observed large crystals; these could be used as a source material to supply of sufficiently large, well-ordered were carefully transferred into a micro- produce a crude fullerene mixture con single crystals on which to work". The fuge tube and stored at 4 oc. Figure 1 sisting of 90% C60 , 10% C70 and trace production of good crystals forms a crit- shows one of the crystals produced with amounts of other fullerenes. The separa ical rate-limiting step in undertaking approximate dimensions of 0.6 X 0.4 x tion of such a mixture into its campo three-dimensional X-ray analysis. 0.2 mm recorded on a Zeiss photomicro- nents is an arduous task, usually carried Methods for the crystallization of pro- scope. I selected one of these for analy- out by chromatography on silica2 or 2 3 teins have been intensively studied • The alumina . We have found that fullerene requirement for new approaches to the mixtures from coal can be purified by problem has led to experiments at zero chromatography on the same coal from gravity in space, high g-force and which it is derived. 2 pressures -4. At normal pressure and We ground and size-separated a sam- gravity, proteins have traditionally been pie of an Australian semi-anthracite, a crystallized by inducing supersaturation / coal from the Yarrabee mine, Queens- using parameters including the addition land, (H/C ratio 0.74, 0/C 0.013) known 4 of precipitant, for example salts or to be a good source of fullerenes . The alcohols, altering the pH towards the ··· fraction that passed through a 125-f.A,m- isoelectric point, slowly changing the mesh sieve but was retained by a 63-~--tm- temperature of the system or concen mesh sieve was separated and extracted tration by evaporation. These crystalliz- in a Soxhlet apparatus for three days ation experiments are performed over with toluene to remove soluble material. a period of time which can be days, A glass column (1.2 x 58 em) was weeks, months or even years. I have packed with the 63-125-~--tm fraction us- devised a new method for rapid crystal- Fig. 2 A 1 ·5o-oscillation photograph of a ing hexane, and a crude fullerene mix- lization by centrifugation. single crystal ofT. reesei aspartic proteinase ture (15 mg) was dissolved in toluene (3 grown by centrifugal ultrafiltration, indicat- I have grown large single crystals of a ing good diffraction to high resolution (2.3 ml) and adsorbed on the head of the newly purified aspartic proteinase iso- A). column. After elution with hexane (800 1 lated from the filamentous fungus ml) at a flow rate of 2 ml min- , 8.7 mg Trichoderma reesei using centrifugal sis at the SERC synchrotron X-ray of solid was obtained from the eluate. ultrafiltration. The experiment rapidly source at Daresbury, UK; the prelimin- This was identified as pure C60 by in 7 creates a protein concentration gradient ary result is shown in Fig. 2. frared spectroscopi- . Further elution resulting in supersaturation and crystal- The rapid production of high-quality with 10% toluene/90% hexane mixtures lization. I have used centricon-10 devices crystals using a low speed centrifuge and (80 ml) did not yield distinctive magenta (Amicon) that allow solute to pass commercially available concentrators solutions of C60 , and further elution was through the 10,000 MW cutoff mem- offers a new approach to the problem of continued with toluene. Initial elution brane while retaining the protein, thus crystallization. The forced increase in with toluene (200 ml fraction) yielded increasing its concentration. The purified concentration of the enzyme under low C60/C70 mixtures enriched in C70 relative enzyme was added to a pre-washed centrifugal force while maintaining the to the crude fullerenes. Further elution centricon-10 device at 0.5 mg ml- 1 in 0.1 starting buffer conditions allows this with toluene (200 ml) produced fractions M sodium acetate at pH 5.6 in a volume parameter to be varied singly and should enriched in C60 , suggesting at least two of 2 mi. The sample was reduced by produce a more controlled approach to different binding sites for C60 . Recovery centrifugation at 3,000g (Sorval RC-5B, supersaturation. The exploration of of fullerene was quantitative within ex SS-34 rotor) for 25 min at 4 octo 0.5 ml other protein systems by the use of small perimental error. The spent coal can be and the spin-through buffer solution was crystal seeds may allow a rapid improve- used for further purification of C60/C70 removed. Another 2 ml of dilute enzyme ment in crystal size. Additionally, in- mixtures. solution was layered carefully onto the formation from screening trials indicat- The ability of coals to separate ful retained concentrated protein solution ing insolubility or the formation of lerenes may derive from the disordered and centrifuged again. This process was microcrystals may provide suitable sheets of aromatic rings that they con repeated until 10 ml of the proteinase conditions for crystallization using this tain. Either the pores formed by these solution had been processed. The final method. sheets may be the right size for separat protein concentration was 20 mg ml-1 Although the success in producing using fullerenes or there could be inter able crystals for X-ray analysis in less actions between the sp2 hybridized than 2.5 hours indicates the potential of structures in coal and the fullerenes. this approach, further investigations are ______needed to explore its general applica- 1. Pang, L s. K., Vassallo. A. M. & Wilson, M. A. Nature M~. ~~~o~~~ 2. Ajie, H. et af. J. phys. Chem. 94, 8630-8633 (1990). JIM E. PITTS 3. Taylor. R., Hare, J.P., Abdui-Sada, A. K. & Kroto, H. W. J. Department of Crystallography, chem. Soc. Chern. Commun. 1423-1425 (1990). Birkbeck College, University of London, 4. Greenwood, P. F., Strachan, M. G., Willett, G. D. & Wilson, M. A. Org. Mass Spectrometry 25, 353-362 Malet Street, London WC1E 7HX, UK (1990) 5. Hare, J. P. et a/. J. chem. Soc. Chern. Commun. 412-443 (1991). 1. Dover, S. D. Nature 354, 388 (1990). 6. Vassallo, A. M., Pang, L. S. K., Cole-Clarke, P. A. & Fig. 1 A crystal of the aspartic proteinase 2. McPherson, A. Preparation and Analysis of Protem Wilson, M. A. J. Am. chem. Soc. 113, 7820-7821 Crystals (Wiley, New York, 1982). (1991). from T. reesei grown in 2.5 hours by centri 3. Delucas, L J. eta/. Science 246, 651-654 (1989). 7. Bethune, D. S. eta/. Chern. Phys. Lett 179, 181-186 fugal ultrafiltration. 4. Visuri, K. eta/. Biolrechnology 8, 547-549 (1990). (1991). 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