Essential Cell Biology 5th edition

Macromolecules in Cells
61
ultracentrifuge. Using hemoglobin as his test protein,
Svedberg found that the centrifuged sample revealed a
single, sharp band with a molecular weight of 68,000
daltons. The finding strongly supported the theory that
proteins are true macromolecules (Figure 2–33).
Additional evidence continued to accumulate throughout
the 1930s, when other researchers were able
to obtain crystals of pure protein that could be studied
by x-ray diffraction. Only molecules with a uniform size
and shape can form highly ordered crystals and diffract
x-rays in such a way that their three-dimensional structure
can be determined, as we discuss in Chapter 4.
A heterogeneous suspension could not be studied in
this way.
We now take it for granted that large macromolecules
carry out many of the most important activities in living
cells. But chemists once viewed the existence of such
polymers with the same sort of skepticism that a zoologist
might show on being told that “In Africa, there are
elephants that are 100 meters long and 20 meters tall.”
It took decades for researchers to master the techniques
required to convince everyone that molecules ten times
larger than anything they had ever encountered were
a cornerstone of biology. As we shall see throughout
this book, such a labored pathway to discovery is not
unusual, and progress in science—as in the discovery
of macromolecules—is often driven by advances in
technology.
the sample is loaded as a
narrow band at the top of
the tube
sample
CENTRIFUGATION
tube
heterogeneous
aggregates would
sediment to
produce a
diffuse smear
stabilizing
sucrose
gradient
(A)
BOUNDARY SEDIMENTATION
BAND SEDIMENTATION
hemoglobin
protein
sediments as a
single band
CENTRIFUGATION
CENTRIFUGATION
(B)
Figure 2–33 The ultracentrifuge helped to settle the debate about the nature of macromolecules. In the ultracentrifuge,
centrifugal forces exceeding 500,000 times the force of gravity can be used to separate proteins or other large molecules. (A) In a
modern ultracentrifuge, samples are loaded in a thin layer on top of a gradient of sucrose solution formed in a tube. The tube is placed
in a metal rotor that is rotated at high speed in a vacuum. Molecules of different sizes sediment at different rates, and these molecules
will therefore move as distinct bands in the sample tube. If hemoglobin were a loose aggregate of heterogeneous peptides, it would
show a broad smear of sizes after centrifugation (top tube). Instead, it appears as a sharp band with a molecular weight of 68,000
daltons (bottom tube). Although the ultracentrifuge is now a standard, almost mundane, fixture in most biochemistry laboratories, its
construction was a huge technological challenge. The centrifuge rotor must be capable of spinning centrifuge tubes at high speeds for
many hours at constant temperature and with high stability to avoid disrupting the gradient and ruining the samples. In 1926, Svedberg
won the Nobel Prize in Chemistry for his ultracentrifuge design and its application to chemistry. (B) In his actual experiment, Svedberg
filled a special tube in the centrifuge with a homogeneous solution of hemoglobin; by shining light through the tube, he then carefully
ECB5 e2.31/2.33
monitored the moving boundary between the sedimenting protein molecules and the clear aqueous solution left behind (so-called
boundary sedimentation). The more recently developed method shown in (A) is a form of band sedimentation.