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Unexpected spin-offs
Ralph W.G. Wyckoff

 

In 1935 we of the recently established Sub-
division of Biophysics at the Rockefeller
Institute for Medical Research had de-
cided that the time had come to move from
the X-ray analysis of simple organic com-
pounds towards an attempt to get data
from substances of more immediate bi-
ological interest. We had chosen for di-
verse reasons collagen, hemoglobin and
globulins in general. Alexis Carrel, with
whom I was closely associated, remem-
bered that some years earlier Nageotte and
Guyon [{] had shown that immature rat-
tail tendons, in contrast to mature connec-
tive tissues, could be solubilized by very
dilute acetic acid and then re-constituted.
We began studying the X-ray patterns of
such collagens [2], of hemoglobin [3] and
of other crystalline proteins (such as pep-
sin, insulin and the tryptic enzymes) which
were just becoming available. Our interests
centered around especially large globulins
present in the antipneumococcal sera then
in clinical use.

The recently perfected oil turbine ultra-
centrifuge of Svedberg had already proved
invaluable for the characterization of a
wide range of macromolecular substances
(see this issue p. 117). It clearly could pro-
vide the information we needed but was
quite beyond our financial reach. Beams
[4] in Virginia had, however, been experi-
menting for several years with the air-
driven spinning tops of the Belgian engi-
neers Henriot and Huguenard [5] and had
shown they could successfully rotate large
objects suspended by a thin shaft and en-
closed in a vacuum; he had constructed
various types of ultracentrifuge.

Believing that ultracentrifugal control
of the proteins we were investigating was
essential, I decided to build a simplified
analytical centrifuge based on this work of
Beams. I had come to the Rockefeller In-
stitute from the Geophysical Laboratory
where a first class instrument shop was at
the core of all research. The Rockefeller
Institute had no shop but I had been able
to include facilities in my department to
make the various pieces of apparatus
needed for our crystal structure investiga-
tions. This enabled us to undertake the de-
sign and development of an air-driven
analytical ultracentrifuge able to operate
up to the maximum speeds permitted by
the strength of its rotors. Pickels, who had
worked with Beams, had come to the
laboratories of the Rockefeller Founda-

tion with the object of constructing a cen-
trifuge for work with yellow fever. We
collaborated in the building of our first
ultracentrifuge and in the investigation of
light metals to replace the steel in its parts
[6]. This substitution was desirable because
much less energy would be required to spin
the lighter metal and the instrument would
thus be far safer. We obtained specially
treated aluminum-magnesium billets from
the Dow Chemical Company and devel-
oped rotors which could operate at nearly
as high a speed as the strongest steel.

The first ultracentrifuge built in my shop
had a steel rotor and was employed rou-
tinely for the measurement of sedimenta-
tion rates until it was wrecked one day
when either a vacuum leak developed or
the rotor failed. The vacuum chamber, of
two-inch armor plate, was knocked into an
egg shape, the turbine was driven through
the ceiling of the room and the rotor was
shattered. Fragments bounced several
times around the room and one piece flew
through a thick, wire-reinforced glass win-
dow without shattering it. The damage it
caused left no doubt of the desirability of
light metal rotors.

TIBS - May 1978

With our analytical ultracentrifuges we
measured the sedimentation constants of a
number of proteins including the pneumo-
coccal antibody globulins [7]. I noticed
during one of our runs that excellent
crystals of hemoglobin formed in the bot-
tom of the analytical cell. This immedi-
ately suggested that ultracentrifugation
might not only concentrate our globulins
but give an especially desirable way to ob-
tain the protein crystals we were seeking
for X-ray study. Quantity heads holding at
least 100 ml were constructed of different
alloys and used to purify, by differential
centrifugation, the proteins with which we
were then working. When the heads began
to show evidence of corrosion or metal
fatigue they were accelerated to bursting
in an outdoor pit, providing in this way
firm evidence of the relative merits of the
alloys and the safe speeds at which they
could be run.

While this was going on, Stanley came to
New York to give a colloquium describing
his chemical isolation of the tobacco mo-
saic virus. There was evidence that the
virus was somewhat altered by the salting-
out he used and it seemed to me that
quantity ultracentrifugation offered a far
less damaging way to concentrate viruses.
Stanley gave me infectious plant juice
which we then differentially sedimented to
yield an especially pure product [8]. The re-
sult was so good that several other plant
viruses, latent mosaic, tobacco ring spot,
cucumber viruses, aucuba mosaic and to-

 

Ral. wie ky

Fig. 1. One of the first electron micrographs of tobacco necrosis virus crystals taken by K-M.-Smvith ( x ooo ).
N 106

bacce necrosis, were similarly purified [9].
In view of these successes it seemed ob-
vious that the same thing should be at-
tempted with animal viruses. Beard. work-
ing with the very stable Shope papillomas,
supplied material from which I obtained
the virus in pure form [10]. Next I was able
to concentrate the far less stable Western
encephalomyelitis virus [11] which Ten
Broek was growing in chicken embryonic
tissue. Beard and | utilized this observa-
tion to prepare a highly effective formal-
inized vaccine against the disease [12].

Simon Flexner had retired as Director
of the Rockefeller Institute at the time my
ultracentrifugal experiments were starting.
The full support he had always given was
not continued by the new director and |
was compelled to leave the Institute and
take employment at the Lederle Laborato-
ries. A serious epidemic of encephalo-
myelitis among horses in the United States
was arrested by the several million doses
of our vaccine [13] manufactured in my
new laboratory. While there I had the op-
portunity to encourage the sale to Ameri-
can Cyanamid, owner of the Lederle Lab-
oratories, of the first electron microscope
manufactured by RCA. Then during a
short stay in Ann Arbor in the midst of the
Second World War, I found unused the
# 2 microscope; with it and a sister instru-
ment Robley Williams and I developed
metal evaporation [14] as a way to measure
the heights of particles. The three-dimen-
sional effects thus obtained led to prelimi-
nary shadowed micrographs of the to-
bacco mosaic virus.

At the National Institutes of Health in
Bethesda when the war was over I was able
to resume, with the electron microscope,
the study of purified viruses that had been
arrested a decade earlier. While examining
preparations of tobacco mosaic virus {15]
concentrated by ultracentrifugation, it was
noted that their macromolecular rod-like
particles were in strikingly regular quasi-
crystalline arrangements. It happened that
Bernal visited my laboratory as I was
making the first of such electron micro-
graphs. At his urging I took them to a
meeting being organized to bring together
in London electron microscopists trom re-
cently liberated Western Europe. At the
time I made contact with Kenneth Smith,
then Director of the Molteno Institute in
Cambridge, who together with Roy
Markham had been preparing single crys-
tals of the tobacco necrosis virus protein.
Replicas of these crystals showed for the
first time the ordered arrangement of their
particles on various faces of a single crystal
[16]. Collaboration with Kenneth Smith
continued for many years and extended to
the insect viruses that were for him a major
interest.

During the years that followed im-
proved techniques of specimen prepara-
tion were used to visualize the particle
arrangement on the faces of many other
macromolecular crystals [17]. Crystalline
arrays are now being seen in all sorts of
biological material but at the outset it was
very exciting, and I should add esthetically
most satisfying, to see and not merely to
deduce the way in which the elementary

 

Fig. 2. A recent electron micrograph of tobacco mosaic virus rods. The rods are arranged in para-crystalline arrays
with the 300nM long axis of the virus particles vertical to the specimen plane. The central axial hole can he
seen to advantage in particles photographed as end-on arrays. Several TMV rods can be seen in horizontal positions
between the crvstalline forms ( x 110000). ( Electromicrograph supplied by R.W. Horne, J.W. Harnden and

R. Markham, John Innes institute, Norwich, U. Kui

TIBS - May 1978

particles of a substance arrange themselves
when crystallizing.

It is instructive to note that all the more
interesting results of this work (the ultra-
centrifugal purification of viruses, the
three-dimensional consequences of metal
shadowing, the direct visualization of the
molecular arrangement in crystals) were
unforeseen products of the research rather
than the fulfillment of its original intent.

Ralph W.G. Wyckoff. F_R.S. is Professor of Physics
at the University of Arizona, Tucson, Arizona, U.S.A.

Now in his 81st vear Wyckoff was a pioneer in the
use of X-rays to determine atomic positions in crystals,
the development of the ultracentrifuge and the use of
ihe electron microscope to study and visualise viruses
and large molecules. He has been Professor of Physics
at the University of Arizona since 1959, spending from
1959-1962 as Directeur de Recherche, at the Centre
National de la Recherche Scientifique in France. From
1952-1954 he was Science Attache at the US Embassy
in London and this was the first Embassy to he fitted
with an electron microscope.

He is also the author of several books on crystallo-
8raphy, the electron microscope and most recently on
the biochemistry of animal fossils.

References

The following is a partial, condensed bibliography
dealing with the work described above.
Nageotte, J. and Guyon, L. (1934) C. R. Assoc.
Anat. ( Bruxelles}, 25 mars
2 Corey, R.B. and Wyckoff, R.W.G. (1935)
Science 82, 175; (1936) J. Biol. Chem. 114, 407:
116, 51: (1936) Proc. Soc. Exp. Biol. Med. 34,285
Corey, R.B. and Wyckoff, R.W.G. (1935)
Science 81, 365: (1936) J. Biol. Chem. 116, 51
4 Beams, J. and Pickels, E.G. (1935) Rev. Sci. Instr.
6, 299; (1937) J. Appl. Phys. 8. 795
Henriot. E. and Huguenard, E. (1925) Compt.
rend Acad. Sci. ( Paris) 180, 1389: (1927) J. Phys.
et le Radium 8, 443
6 Biscoe, J., Lagsdin, J.B.. Pickels, E.G. and
Wyckoff. R.W.G. (1936) J. Exp. Med. 64, 39:
(1936) Rev. Sci. Instr. 7,246; (1937) ibid. 8,74 and
427
7 Biscoe. J.. Hercik, F. and Wyckoff, R.W.G.
(1936) Science 83, 602: 84, 291
8 Corey, R.B. and Wyckoff, R.W.G. (1936)
Science 84, 513
9 Loring. H.S., Price, N.C.. Stanley, W.M. and
Wyckoff, R. W.G. (1937) J. Biol. Chem. 121, 225:
(1938) 124, 585; (1939) 128, 729: (1938) Nature
( London) 141, 685: (1939) Phytopathology 29, 83
10 Beard, J.W. and Wyckoff, R.W.G. (1937)
Science 85, 201: (1937) Proc. Soc. Exp. Biol. Med.
36, 562: (1938) J. Biol. Chem. 123, 46]
If Wyckoff, R.W.G. (1937) Proc. Soc. Exp. Biol.
Med. 36, 771
12 Beard, J.W., Finkelstein, H., Sealy, W.C. and
"Wyckoff, R.W. G. (1938) Science 87, 89 and 490
13 Eichhorn. A., Lyon, B.M. and Wyckoif. R. W.G.
(1938) J. Am. Vet. Med. Assoc. 93NS, 285: (1938)
Vet. Med. 33, No.9
[4 Williams, R.C. and Wyckoff, R.W.G. (1944) J.
Appl. Physiol. 15,712: (1945) Science 10L, 594
15 Wyckoff, R. W.G. (1947) Biochim. Biophys. Acta
1, 139
16 Markham. R., Smith. K.M. and Wyckoff,
R.W.G. (1947) Nature ( London } 159,574: (1948)
161, 760
17 Labaw, L.W. and Wyckoff, R.W.G. (1948) Acta
Crystallogr. 1, 292: (1955) Exp. Cell Res., Suppl.
3, 395; (1957) Arch. Biochem. Biophys. 67, 225

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