REFERENCES AND NOTES

1. B. J. Mason, The Physics of Clouds (Clarendon,Oxford, 1971), p. 174.

2. K. Sassen, D. O'C. Starr, T. Uttal, J. Atmos. Sci.46, 371 (1989).

3. A. J. Heymsfield and R. M. Sabin, ibid., p. 2252.4. K. Sassen and G. C. Dodd, ibid., p. 3005.5. It is more accurate to refer to these droplets as

haze particles, because at such low temperaturesthey probably remain in environmental equilibriumbelow their critical radii until freezing.

6. K. Sassen, Bull. Am. Meteorol. Soc. 72, 1848(1991).

7. _, K. N. Liou, S. Kinne, M. Griffin, Science227, 411 (1985).

8. We have regularly collected polarization ruby lidardata from cirrus clouds since 1987 in support ofthe Extended Time Observations component ofProject FIRE.

9. Local Project FIRE radiosonde data have provid-ed atmospheric temperature and wind informa-tion, based on the use of a time-weighted averageof high-resolution (-40 m) data obtained at 0030and 0905 UTC on 6 December 1991.

10. K. Sassen, C. J. Grund, J. D. Spinhirne, M. M.Hardesty, J. M. Alvarez, Mon. Weather Rev. 118,2288 (1990).

11. The question of the ambiguity in PDL cloud phasediscrimination deserves discussion. Near-zero 8values can be produced by homogeneousspheres, plate crystals large enough (>250 Apm indiameter) to orient horizontally, and asphericalparticles smaller than the incident wavelength (airmolecules). The rapidly rising turrets (note theabsence of wind shear effects) in Fig. 3 mustcontain relatively small particles, certainly too smallto orient uniformly. On the other hand, laboratoryexperiments with artificially "seeded" supercooledclouds demonstrate that minute frozen dropletsrapidly grow the facets needed to produce opticaldepolarization [K. Sassen and K. N. Liou, J. Atmos.Sci. 36, 852 (1979)]. Lidar observations of aircraftcondensation trails and glaciating clouds (7) showthe same rapid transition in 8 as ice crystals form.Although some ice nucleation probably took placein the cell heads, we conclude that liquid dropletbackscattering predominated.

12. Cloud-forming nuclei derived from at or near theEarth's surface are deficient at cirrus altitudesbecause of sedimentation. Lidar studies of cirrusclouds that straddle high tropopauses have re-vealed unusual cloud properties that probablyreflect the effects of stratospheric-derived CCNs[K. Sassen, Appl. Opt. 30, 3421 (1991)].

13. It is also possible that the sulfuric acid dropletscould freeze directly into ice crystals duringgrowth from tropospheric water vapor.

14. K. Sassen et al., Appl. Opt. 28, 3024 (1989).15. K. Sassen and J. D. Horel, J. Atmos. Sci. 47, 2881

(1 990).16. Preliminary supporting remote and in situ data

from Project FIRE have shown directly the mixingof stratospheric ozone and volcanic aerosols intothe cirrus cloud altitude during this period.

17. For example, Mie scattering theory simulationsreveal that a water droplet of radius 1.3 gmbackscatters -70 times as much light at the0.532-1Lm wavelength as a 0.7-pLm droplet, sug-gesting that, if only submicrometer-sized dropletswere present, these unusual cloud features mighthave gone unnoticed.

18. P. E. Ardanuy and H. L. Kyle, J. Clim. Appl.Meteorol. 25, 505 (1986).

19. K. N. Liou, Mon. Weather Rev. 114, 1167 (1986).20. Support for our FIRE IFO research has come from

National Science Foundation grant ATM-8914348and National Aeronautics and Space Administra-tion grant NAG-1-868 and for PDL developmentfrom Department of Energy grant DE-FGO2ER1059from the Atmospheric Radiation Measurement pro-gram. thank M. T. Davies and B. S. Cho for theircontributions, and various FIRE coinvestigators formaking available their preliminary data.

17 March 1992; accepted 1 June 1992

State-to-State Rates for the D + H2(v =1, j= 1)HD(v', j) + H Reaction:

Predictions and Measurements

Daniel Neuhauser,* Richard S. Judson, Donald J. Kouri,tDavid E. Adelman, Neil E. Shafer, Dahv A. V. Kliner,f

Richard N. Zaret

A fully quantal wavepacket approach to reactive scattering in which the best available H3potential energy surface was used enabled a comparison with experimentally determinedrates for the D + H2(v = 1, j= 1) HD(v' = 0, 1, 2; j') + H reaction at significantly higher

total energies (1.4 to 2.25 electron volts) than previously possible. The theoretical resultsare obtained over a sufficient range of conditions that a detailed simulation of the exper-

iment was possible, thus making this a definitive comparison of experiment and theory.Good to excellent agreement is found for the vibrational branching ratios and for therotational distributions within each product vibrational level. However, the calculated ro-

tational distributions are slightly hotter than the experimentally measured ones. This smalldiscrepancy is more marked for products for which a larger fraction of the total energy

appears in translation. The most likely explanation for this behavior is that refinements are

needed in the potential energy surface.

Ultimately, the test of any theory is itspredictive power. In chemistry, theoreticalcalculations based on first principles havebeen applied to static properties of mole-cules with great success. However, theoret-ical calculations of dynamical propertieshave met with less success because of thelarge number of quantum mechanical (QM)scattering channels in such processes. Re-cent experimental advances have permittedthe study of the H + H2 reaction and itsisotopic analogs at a level of detail previ-ously not possible (1-9). Correspondingadvances in theoretical techniques haveallowed these new measurements to becompared with exact QM calculations attotal energies up to 1.82 eV (10-22). Cal-culations for a range of total energies havebeen restricted to energies below 1.35 eV(10, 12-14, 18), whereas results for reac-tions in which the H2 was vibrationallyexcited have been obtained only for totalenergies ranging from 0.82 to 1.35 eV (18)and for 1.82 eV (16, 21). The QM calcu-lations (10-14, 18, 20) agree well withintegral cross section measurements (1-3,8) in the total energy regime below 1.5 eV.

D. Neuhauser, James Franck Institute, University ofChicago, Chicago, IL 60637.R. S. Judson, Sandia National Laboratory, Center forComputational Engineering, Livermore, CA 94551-0969.D. J. Kouri, Department of Chemistry and Departmentof Physics, University of Houston, Houston, TX 77204-5641.D. E. Adelman, N. E. Shafer, D. A. V. Kliner, R. N. Zare,Department of Chemistry, Stanford University, Stan-ford, CA 94305-5080.

*Present address: Department of Chemistry, Universi-ty of California, Los Angeles, CA 90024.tTo whom correspondence should be addressed.tPresent address: Department of Chemistry, Univer-sity of Minnesota, Minneapolis, MN 55455.

SCIENCE * VOL. 257 * 24 JULY 1992

Significant differences have been foundbetween experiment (5, 8) and theory (16,21) at the higher total energy of 1.82 eV.Two factors may influence this comparison:(i) the theoretical calculations did not fullysimulate the experimental conditions; and(ii) the experimental results may have beenimpaired to some extent by space-chargeeffects caused by ion production in the use ofDBr as a photolytic source of fast D atoms.Consequently, it was not possible to deter-mine whether the differences between ex-periment and theory were caused by experi-mental error, the incomplete simulation ofthe experiment, the potential energy surface(PES), or the accuracy of the QM calcula-tions. The theoretical predictions were es-sentially unchanged in a subsequent moreaccurate study (22); however, these calcula-tions also did not include a full simulation ofthe experimental conditions. At such highenergies, it may well be that the PES is lessreliable for these comparisons with experi-ment because regions of greater anisotropyand anharmonicity can be sampled, that is,experiments at lower energies may be lesssensitive to inaccuracies in the PES.

Existing noniterative time-independentquantal scattering methods rapidly becomecomputationally intensive at higher totalenergies because of the increase in the num-ber of quantum channels (10-22). We re-port here theoretical results obtained with anewly developed wavepacket approach (21,23-26) to exact scattering dynamics thatextends the predictive power of theory tohigher energies and more complex scatteringsystems; consequently, a full simulation ofthe experimental conditions was feasible.We also report new state-to-state integralcross section measurements of the D + H2 (v

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Vlbrational level v'Fig. 1. Comparison of experimental and theoreti-cal relative vibrational population distributions forthe reaction D + H2(v= 1,j= 1) - HD(v', j') +H at E,. 1.4 eV. The experimental (9) data(squares connected by solid lines) are recordedby using a variable-wavelength photolysis source,and the theoretical calculations (26) (circles con-nected by dashed lines) are obtained with thewavepacket approach. The experimental errorbars represent one standard deviation.

= 1, j = 1) reaction (where v is thevibrational quantum number and j is therotational quantum number) in which DIwas used as the fast D atom photolyticprecursor, thus significantly reducing thespace-charge effects associated with the DBrprecursor. Comparison of the new experi-mental results with the calculations of thewavepacket approach yields a definitive testof theory at higher total energies than pre-viously possible.A combination of laser techniques has

enabled true state-to-state experiments for theH + H2 reaction family to be carried out. TheD+H2(v= 1,j= l) ->HD(v' =0, 1,2;j')+ H reaction rates have been measured, inwhich the H2 reagent was prepared in the (v= 1, j = 1) state by stimulated Ramanpumping (7-9), and fast D atoms were pro-duced by laser photolysis of DBr or Dl. Inthese experiments the DBr(DI) and H2 weremixed with He in a reservoir and then flowedinto a high-vacuum chamber through a pulsedsupersonic nozzle. The same laser both gener-ated the fast D atoms needed to initiate thereaction and ionized the nascent HD productthrough (2 + 1) resonance-enhanced mul-tiphoton ionization (REMPI) (27), in whicha two-photon transition connects the X stateto the E, F state of HD that is subsequentlyionized by a one-photon transition. TheREMPI detection scheme has been calibratedagainst an effusive high-temperature nozzle(27) such that relative quantum-state popula-tions can be obtained from the measured ionsignals. A shuttered time-of-flight mass spec-trometer was used to detect the HD+ ions (3,28). Because the same laser effected the pho-tolysis and ionization steps, Er,, varied as afunction of product state detected. By replac-ing the DBr precursor (Erei = 0.95 to 1.07eV) used in the earlier experiments with DI,higher values ofEre, are obtained 11.38 to 1.54eV for HD(v' = 0), 1.31 to 1.43 eV for

520

Fig. 2. Comparison of ex-perimental and theoretical 0.20 Arelative rotational populationdistributions for the reactionD + H2(v = 1,1 = 1) -_HD(v', j') + H for a vari- 0.10able-wavelength photolysissource: (A) HD(v' = 0, j') atEei 1.5eV; (B) HD(v' = 1, 0.00 ._j') at Erei = 1.4 eV; and (C)HD(v' = 2, j') at Er., -- 1.3leV. The experimental data 0.14(8, 9) are represented by Bsquares connected by solidlines; the error bars are one f 0.10standard deviation. The the- .oretical calculations (26) :are represented by circles ' 0.06connected by dashed lines.Dotted lines connect thepopulations of levels adja- 0.02cent to a level for which thepopulation was not mea-sured. 0.14 C

0.10

0.06

0.020

HD(v' =1), and 1.26 to 1.32 eV for HD(v'= 2)]. At these relative collision energies andwith 0.52 eV in the H2 reagent vibration, thereaction accesses regions of the PES far re-moved from the minimum energy path. Fur-thermore, the use of DI significantly reducesthe previously noted (7, 8) space-charge ef-fects associated with the DBr precursor.

The broad range and high total energiesassociated with these experiments make thecorresponding theoretical calculations ex-tremely difficult. Noniterative, time-inde-pendent basis set expansion methods (10-14, 17-20, 22) have been the only meanspreviously available for carrying out sophis-ticated fully converged, three-dimensionalreactive QM scattering calculations. Al-though applicable to reactions at lower totalenergies, such methods become highly com-putationally intensive under the present ex-perimental conditions. New approaches areneeded to compare theory and experiment atthese high energies in which a large numberof reaction channels are open.

Among the theoretical techniques used,the newly developed time-dependent wave-packet approach (21, 23, 24) has four attrac-tive features. First, calculations are performedonly for the initial state of interest, whereasnoniterative time-independent basis set meth-ods necessarily include all initial states acces-sible at a given energy. Second, a singlepropagation of the wavepacket yields results at

Rotational level 11

many energies, thereby significantly reducingthe number of calculations required. Third, agrid representation is used so that the poten-tial energy is calculated by simple multiplica-tion. Fourth, the wavepacket can be propa-gated by using the Jacobi scattering coordi-nates of a single arrangement. Perhaps mostimportantly, the associated numerical com-plexity grows roughly linearly with the num-ber of grid points. This is a marked advantageover the noniterative basis set methods, inwhich the Hamiltonian matrix must be in-verted and the computational effort scales asN3, where N is the size of the basis set used.

Recent developments in the time-depen-dent approach include accurate methods forevaluating the kinetic energy terms and amixed grid and close-coupling approach forrepresenting the wave function. When thesemethods are coupled with an accurate algo-rithm for propagating the wavepacket, theresulting approach is decisively the mostefficient available for simulating inelasticmolecular collisions at high energies. Forreactive collisions, the major difficulty is thepresence of multiple asymptotic arrange-ments. Large grids are then ostensibly nec-essary for representing the wave functionbefore and after the scattering event. Thisdifficulty is circumvented (23-25) in threeways: (i) by using projection operators tocouple the one-channel wave function inthe reactant arrangement to the many-chan-

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0.15

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.0.05 --

0.000 2 4 6 8

Rotational level I'

Fig. 3. Comparison of experimentalretical relative rotational populationtions for the reaction D + H2(v = 1,HD(v' = 1, j') + H for a fixed-wphotolysis source (Ere, = 0.8 eV). Ttmental data (9) are represented byconnected by solid lines; the error baistandard deviation. The theoretical ca(26) are represented by circles conrdashed lines.

nel wave function in the interactionthat a many-channel representationonly in a small region; (ii) by c;accurate state-to-state probabilitiesoverlap-integral expression for thematrix that enables the asymptoticprobabilities to be calculated from tpacket within and just outside of thetion region; and (iii) by introducingthat absorb the wavepacket as it scathe interaction region, thereby redwavepacket propagation time.

The computational efficiency ofdependent wavepacket approach lcomplete simulation of the D + Iments (26). Specifically, the theoryculations include: (i) the variaticrelative collision energy as a functi(rovibrational state detected; (ii) thmental relative collision energ(-0.07 eV full-width at half-m,and (iii) the fast and slow D atomtions corresponding to the concurduction of I(2P312) and I(2Pl) intolysis of DI. In order to performtative comparison between theoryperiment, we must have a well-chaexperimental procedure. Numerouscarried out to check for possible sexperimental error. Among the mctant of these checks are: (i) verifithe validity of the procedure used lthe D + H2(v = 1, j = 1) distribu(ii) use of two photolytic D atom soand DBr) (8, 9); (iii) determinatiorproduct fly-out from the detectionnot perturbing the rotational disi(9); and (iv) use of an independewavelength photolysis source geonConsequently, we are confident th;experimental error nor incompleteof the experimental initial conditilsource of the remaining discrepatween theory and experiment.

Comparisons of the experimental andtheoretical results for the reaction D +H2(v= 1,j = 1) --HD(v', j') + H arerepresented in Figs. 1 to 3. Because relativepopulations are measured, the experimentaland theoretical distributions in each of thecomparisons are normalized to the sum ofthe populations of their common levels.The distributions presented in Figs. 1 and 2

10 12 are measured with a variable-wavelengthphotolysis source, as described above. How-

and theo- ever, the D + H2(v = 1, j = 1) -- HD(v'i distribu- = 1, j') + H at Erel = 0.8 eV rotationalj = 1) -> distribution displayed in Fig. 3 was recorded,avelength with a fixed-wavelength photolysis arrange-ie experi- ment. For this geometry, an independent/ squares laser pulse was added to generate the fast Drsalrae onnes atoms through photolysis of DI. This pro-iected by cedure allows the DI photolysis and HD

ionization steps to be decoupled.Very good agreement is observed be-

tween experiment and theory for the vibra-region so tional distribution (see Fig. 1); they agreeis needed within the experimental error bars for eachalculating vibrational level. For the rotational distribu-with an tions, however, the extent of agreement

scattering varies from moderately good (Fig. 2, A andscattering B) to quantitative (Figs. 2C and 3). Thethe wave- very good agreement observed for the HD(v'ieinterac- = 2, j') distribution at Erel 1.3 eV (Fig.functions 2C) and HD(v' = 1, j') distribution at Ereitters from = 0.8 eV (Fig. 3) shows that the discrepan-ucing the cies in Fig. 2, A and B, are not caused by

the vibrational state of the HD product. Asthe time- the fraction of energy grows in the HDpermits a product translational degree of freedom, the12 experi- theoretical distributions become increasinglyetical cal- hotter than the corresponding experimentaln in the ones (Figs. 2 and 3). Thus, both the overallon ofHD shape and the peak of the distributions are inie experi- good agreement for the HD(v' = 2, j')y spread distribution at E, = 1.3 eV and the HD(v'aximum); = 1, j') distributions at EM1 = 0.8 eV (Figs.contribu- 2C and 3). In contrast, neither the shaperrent pro- nor the peak of the distributions is wellthe pho- matched by theory for the HD(v' = 0, j')a quanti- distribution at Erei 1.5 eV and the HD(v'f and ex- = 1, j') distribution at Erel 1.4 eV (Fig. 2,racterized A and B). Specifically, the calculated distri-s tests are butions peak one to two quanta higher thansources of the measured ones in Fig. 2B and two to)st impor- three quanta in Fig. 2A.ication of The consistency of the experimental andto extract theoretical results for the vibrational distri-tions (8); bution (Fig. 1) and for the rotational dis-urces (DI tributions HD(v' = 1, j') at Erel = 0.8 eVi that HD (Fig. 3) and HD(v' = 2, j') at Erei 1.3 eVvolume is (Fig. 2C) suggests that the observed dis-tributions crepancies do not arise from an error in thent, fixed- QM scattering calculations. Instead, wenetry (9). believe the differences are most likely at-at neither tributable to inaccuracies in the PES used,modeling which was the double many-body expan-ons is the sion (29). This hypothesis is consistentncies be- with the good agreement observed for the

lower energy reactions, because the high

SCIENCE * VOL. 257 * 24 JULY 1992

energies accessed in these experiments al-low the reactants to explore regions of thePES far removed from the minimum energypath of the reaction. The PES is not as wellcharacterized in these regions because abinitio points that are used to generate thesurface are concentrated around the mini-mum energy path. Buntin et al. (4, 5) andContinetti et al. (6) measured differentialcross sections for the D + H2 reaction andsimulated the experiments with the QMcalculations of Zhao et al. (18) and ofZhang and Miller (10, 14). These workersalso found discrepancies that they attribut-ed to errors in the H3 PES.

As the above comparisons demonstrate,the new wavepacket approach is able to pro-vide a full simulation of the dynamics of the D+ H2(v = 1, j = 1) -- HD(v' = 0, 1, 2; j')+ H reaction at high total energies. Theaccuracy of these calculations is apparentlylimited only by the accuracy of the PES forthe reaction and the validity of the assump-tion that this reaction is electronically adia-batic (30). The wavepacket approach hasmatured to the stage where it affordably yieldscomplete dynamical information. Further-more, the reduced computational effort, rela-tive to the noniterative time-independent ba-sis set expansion methods, establishes thewavepack approach as a powerful method forpredicting the dynamics of systems with highenergy or complexity or both.

REFERENCES AND NOTES

1. D. P. Gerrity and J. J. Valentini, J. Chem. Phys. 81,1298 (1984).

2. H. B. Levene et al., Chem. Phys. Lett. 143, 317(1988).

3. K.-D. Rinnen, D. A. V. Kliner, R. N. Zare, J. Chem.Phys. 91, 7514 (1989).

4. S. A. Buntin, C. F. Giese, W. R. Gentry, ibid. 87,1443 (1987).

5. ._ , Chem. Phys. Lett. 168, 513 (1990).6. R. E. Continetti, B. A. Balko, Y. T. Lee, J. Chem.

Phys. 93, 5719 (1990).7. D. A. V. Kliner and R. N. Zare, ibid. 92, 2107

(1 990).8. D. A. V. Kliner, D. E. Adelman, R. N. Zare, ibid. 95,

1648 (1991).9. D. E. Adelman, N. E. Shafer, D. A. V. Kliner, R. N.

Zare, unpublished work.10. J. Z. H. Zhang and W. H. Miller, Chem. Phys. Lett.

153, 465 (1988).11. M. Mladenovic et al., J. Phys. Chem. 92, 7035

(1988).12. D. E. Manolopoulos and R. E. Wyatt, Chem. Phys.

Lett. 159, 123 (1989).13. J. M. Launay and M. L. Dourneuf, ibid. 163, 178

(1989).14. J. Z. H. Zhang and W. H. Miller, J. Chem. Phys.

91, 1528 (1989).15. W. H. Miller, Annu. Rev. Phys. Chem. 41, 245 (1990).16. H. Buchenau, J. P. Toennies, J. Arnold, J. Wolfrum,

Ber. Bunsenges. Phys. Chem. 94,1231 (1990).17. N. C. Blais, M. Zhao, D. G. Truhlar, D. W. Schwenke,

D. J. Kouri, Chem. Phys. Lett. 166,11 (1990).18. M. Zhao, D. G. Truhlar, D. W. Schwenke, D. J.

Kouri, J. Phys. Chem. 94, 7074 (1990).19. J. V. Michael, J. R. Fisher, J. M. Bowman, 0. Sun,

Science 249, 269 (1990).20. M. D'Mello, D. E. Manolopoulos, R. E. Wyatt, J.

Chem. Phys. 94, 5985 (1991).21. D. Neuhauser, ibid. 95, 4927 (1991).22. S. L. Mielke, R. S. Friedman, D. G. Truhlar, D. W.

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29. A. J. C. Varandas et al., J. Chem. Phys. 86, 6258(1987).

30. B. Lepetit and A. Kuppermann, Chem. Phys. Lett.166, 581 (1990).

31. D.N. is a James Franck postdoctoral fellow, D.E.A. isan NSERC postgraduate fellow, and D.A.V.K. is anNSF postdoctoral fellow. This work was supportedby several sources: R.S.J. acknowledges supportfrom the DOE under the contract DE-AC04-76DP00789; D.J.K. from the NSF under grant NSFCHE-89-07429, and through a grant of supercom-puter resources from the NSF DMsion of AdvancedScientific Computing at NASA-Ames; and R.N.Z.from the NSF under grant NSF CHE-89-21 198.

2 April 1992; accepted 16 June 1992

Magnetoferritin: In Vitro Synthesis of aNovel Magnetic Protein

Fiona C. Meldrum, Brigid R. Heywood, Stephen Mann*The iron storage protein ferritin consists of a spherical polypeptide shell (apoferritin)surrounding a 6-nanometer inorganic core of the hydrated iron oxide ferrihydrite(5Fe203-9H20). Previous studies have shown that the in vitro reconstitution of apoferritinyields mineral cores essentially identical to those of the native proteins. A magnetic mineralwas synthesized within the nanodimensional cavity of horse spleen ferritin by the use ofcontrolled reconstitution conditions. Transmission electron microscopy and electron dif-fraction analysis indicate that the entrapped mineral particles are discrete 6-nanometerspherical single crystals of the ferrimagnetic iron oxide magnetite (Fe304). The resultingmagnetic protein, "magnetoferritin," could have uses in biomedical imaging, cell labeling,and separation procedures.

The ability of ferritin to sequester andstore iron in a bioavailable form arises froma quaternary structure of 24 polypeptidesubunits assembled into a spherical hollowshell that is penetrated by two types ofintersubunit channels (1). The mechanismsby which iron is accumulated within the8-nm internal cavity have been elucidatedthrough reconstitution experiments of re-

combinant apoferritins modified by site-directed mutagenesis (2). In brief, two keysites have been implicated: a ferroxidasecenter residing in the intrahelical domainof H-chain subunits (3) and a nucleationsite comprising three Glu residues on thecavity surface. These sites appear to actcooperatively in affecting the kinetics ofiron oxide deposition such that mineral-ization occurs specifically within the pro-

tein cavity and not in bulk solution. Suchprecise molecular control of inorganic pre-

cipitation within a confined volume couldbe generally relevant to the synthesis ofinorganic clusters and nanoparticles. Wehave recently shown that Mn(III) oxidecores can be reconstituted in ferritin byincubation of apoferritin molecules withaqueous Mn(II) under controlled reactionconditions (4).

Although we have synthesized iron sul-fide cores in ferritin by in situ reaction ofnative iron oxide cores with gaseous H2S(4), the formation of iron oxide cores otherthan ferrihydrite has not been achieved (5).Indeed, it has generally been consideredthat the ferrihydrite structure of the bio-mineral is functionally important in provid-ing a source of relatively labile iron formetabolic use. To determine the extent ofstructural specificity of iron oxide mineral-ization in ferritin, we carried out reconsti-tution experiments under conditions tai-lored to the synthesis of Fe304 (Fig. 1) (6).Iron was removed from native horse spleenferritin by dialysis against thioglycolic acidat pH 4.5 (7). The resulting apoferritinsolution was buffered at pH 8.5 and main-tained at a temperature of 550 to 60°Cunder argon in a water bath. Ten incre-ments of Fe(II) solution were added over

200 min (8). Slow oxidation was achieved

Fig. 1. Schematic showingthe synthetic route to mag-netoferritin. Step involvesthe removal of native ferri-hydrite cores from horsespleen by dialysis againstthioglycolic acid, under N2,at pH 4.5. Step 11 involvesthe reconstitution of apo-

Native ferritin

during this procedure by the introduction ofair into the solution with a Pasteur pipette.On completion of the Fe(II) additions,samples of the reconstituted ferritin were

taken immediately for transmission electronmicroscopy (TEM) analysis (9). Severalsamples were left open to the atmospherefor 15 min before TEM investigation toascertain their stability to possible oxida-tion in air. Some of these grids were neg-

atively stained with 1% uranyl acetate so-

lution to determine whether any thermaldegradation of the protein had occurred andto verify that the crystals had indeed beenproduced within the protein shell. Protein-free control reactions, involving an analo-gous procedure in which 0.15 M saline was

substituted for apoferritin, were also under-taken (10).

Addition of Fe (II) to the apoferritinsolutions resulted in an initial increase inturbidity followed by a blackish discolora-tion after - 1 hour. The final solutionremained black, and no bulk precipitationwas observed. Exposure to air resulted in a

red-brown coloration within a few min-utes. Neither the black nor the red-brownprotein solutions responded to a bar mag-

net placed against the side of the samplecontainer. In contrast, a bulk white pre-

cipitate [Fe(OH) 2] was initially formed inthe control reaction, transforming into a

green gelatinous deposit and subsequentlyinto a black and magnetic precipitate after1 hour. The sample remained black underargon and after exposure to the atmo-sphere for 2 weeks.

Examination of the ferritin and controlsamples by TEM showed particles of re-

markably different size and morphology(Fig. 2). The majority of particles synthe-sized in the presence of apoferritin were

discrete, spherical nanometer-sized crystals(Fig. 2A) of uniform size (mean diameter =6 nm, a = 1.2 nm). Negative stainingshowed that the individual particles were

surrounded by a protein shell (Fig. 2B),indicating that the discrete crystals were

formed specifically within the apoferritincavity. In contrast, particles formed in thecontrol preparations were aggregated andheterogeneous in size (range = 4 to 70 nm)and morphology (irregular spheroidal, cu-

bic, and cubo-octahedral) (Fig. 2C). Elec-tron diffraction patterns identified the crys-

Apoferritin Magnetoferritin

ferritin with Fe(ll) solution under slow oxidative conditions at 600C and pH 8.5.

SCIENCE * VOL. 257 * 24 JULY 1992

Schwenke, Chem. Phys. Lett. 188, 359 (1992).23. D. Neuhauser, M. Baer, R. S. Judson, D. J. Kouri,

ibid. 169, 372 (1990).24. D. Neuhauser, J. Chem. Phys. 93, 7836 (1990).25. _, R. S. Judson, M. Baer, D. J. Kouri, in

Advances in Molecular Vibrations and CollisionDynamics, J. M. Bowman, Ed. (JAI Press, Green-wich, CT, in press), vol. 2.

26. D. Neuhauser, R. S. Judson, D. J. Kouri, unpub-lished work.

27. K.-D. Rinnen, D. A. V. Kliner, R. N. Zare, W. M.Huo, Isr. J. Chem. 29, 369 (1989); K.-D. Rinnen,M. A. Buntine, D. A. V. Kliner, R. N. Zare, W. M.Huo, J. Chem. Phys. 95, 214 (1991).

28. K.-D. Rinnen, D. A. V. Kliner, R. S. Blake, R. N.Zare, Rev. Sci. Instrum. 60, 717 (1989).

School of Chemistry, University of Bath, Bath BA27AY, United Kingdom.*To whom correspondence should be addressed.

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