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C L A S S I C E X P E R I M E N T
BRINGING AN ENZYME BACK TO LIFE
C. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry 236:1362
By the 1950s, scientists realized that
DNA held the code that allowed pro-
teins to be synthesized. Nevertheless,
how a chain of amino acids folds into
a fully functional protein, with the
proper three-dimensional structure, re-
mained a mystery. A mechanism must
exist to assure the proper folding of the
protein. But where did that informa-
tion come from? In 1957, Christian
Anfinsen published the first evidence
that the information for proper folding
was held within the protein itself.
Background
Proteins are made from combinations
of 20 amino acids that then fold into
complex structures. The unfolded
amino acid chain is called the primary
structure. To have biological activity,
the protein must fold into proper sec-
ondary and tertiary structures. These
structures are held together by interac-
tions between the side chains and
backbone atoms of the amino acids,
including hydrogen bonds, hydropho-
bic interactions, and, at times, covalent
bonds. How these higher structures
form had long been a mystery. Does
the protein fold correctly as it is syn-
thesized or does it require the action
of other proteins to correctly fold it?
Can it correctly fold on its own spon-
taneously?
In the 1950s, Anfinsen was a bio-
chemist interested in the proper fold-
ing of proteins. Specifically, he was
investigating the formation of disulfide
bridges, which are covalent bonds be-
tween cysteine side chains that serve
as one of the major anchors holding
together the structure of secreted pro-
teins. He believed that the protein itself
contained all the information neces-
sary for proper protein folding. He
proposed the “thermodynamic hypoth-
esis,” which stated that the biologically
active structure of a protein was also
3 . 1
the most thermodynamically stable un-
der in vivo conditions. In other words,
if the intracellular conditions could be
mimicked in a test tube (in vitro), then
a protein would naturally fold into its
active conformation. He began his work
on a secreted enzyme, bovine pancreatic
ribonuclease, and studied its ability to
properly fold outside of the cell.
The Experiment
Proteins perform a wide variety of func-
tions in the cell. Regardless of its func-
tion, a protein must be properly folded
to carry out its biological role. For pro-
tein folding studies it is best to study an
enzyme whose biological activity can be
easily monitored by performing a test,
or “assay” of its activity in vitro. Anfin-
sen chose a small, secreted protein, the
enzyme ribonuclease, in which he could
monitor proper folding by assaying its
ability to catalyze the cleavage of RNA.
Ribonuclease, a secreted protein, is
active under oxidizing conditions in
vitro. The tertiary structure of active ri-
bonuclease is held together by four
disulfide bonds or bridges. Adding a re-
ducing agent reduces the disulfide bond
between two cysteine side chains to two
free sulfhydryl groups, and can disrupt
this covalent interaction. Complete
denaturation of ribonuclease requires
treatment with a reducing agent. An-
finsen monitored the reduction of
ribonuclease by measuring the number
of free sulfhydryl groups present in the
protein. In the oxidized state, there are
no free sulfhydryl groups in ribonucle-
ase because each cysteine residue is
involved in a disulfide bond. In the
completely reduced state, on the other
hand, ribonuclease contains eight free
sulfhydryl groups. Anfinsen exploited
this difference to assess the extent of re-
duction by using a spectrophotometric
assay to titrate, or count, the number of
free sulfhydryl groups.
To study protein folding outside
the cell, one must first denature the
protein. Proteins are easily denatured
by heat, mechanical disruption such as
shaking, and chemical treatment. Pro-
teins with disulfide bridges require an
additional measure of treatment with a
reducing agent to break apart these
covalent bonds. To denature ribonu-
clease, Anfinsen first reduced the disul-
fide bridges with thioglycolic acid. He
then denatured the protein by using a
high concentration of urea and incubat-
ing the solution at room temperature.
He demonstrated that this treatment
rendered the enzyme inactive by show-
ing that ribonuclease was now unable
to catalyze the cleavage of RNA. Using
the spectrophotometric assay, he went
on to show that the inactive ribonucle-
ase contained eight sulfhydryl groups,
which corresponded to the four bro-
ken disulfide bridges. With a com-
pletely reduced, denatured protein in
hand, Anfinsen then could ask: Can a
denatured enzyme correctly fold in
vitro and become active again?
To find the answer, Anfinsen al-
lowed a solution of reduced, denatured
ribonuclease to oxidize. He removed
the urea from the denatured enzyme by
precipitation. Next, he resuspended
the urea-free denatured ribonuclease in
a buffered solution and incubated it
for two to three days. Exposure to
molecular oxygen in the atmosphere
oxidized the cysteine residues. He then
compared the activity of this renatured
ribonuclease to that of the native en-
zyme. In initial experiments, 12–19
percent of the previously inactive pro-
tein were able to catalyze the cleavage
of RNA once again. Proteins aggregate
at high concentrations, which makes it
difficult for them to fold properly. By
decreasing the overall concentration
of ribonuclease in solution, Anfinsen
showed that up to 94 percent of the
protein could be refolded (see Table 1).
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contained in its primary sequence. His

TABLE 1 Cell-free Refolding of Ribonuclease careful analysis of the chemistry of this
process answered a fundamental ques-
ACTIVITY AS A PERCENT OF EQUIVALENT tion in biology. He went on to demon-
CONCENTRATION OF PROTEIN (MG/ML) CONCENTRATION OF NATIVE RIBONUCLEASE strate the cell-free refolding of other
enzymes, including proteins lacking
7.0 31% disulfide bridges. While it is possible to
properly fold a number of proteins out-
4.8 70% side of the normal protein-processing
machinery in the cell, this process is
2.3 75% greatly accelerated in vivo by a number
of proteins. Anfinsen continued to
0.9 77%
study the protein-folding problem. Al-
though the “thermodynamic hypothe-
0.35 94%
sis” does not hold true for all proteins,
Anfinsen’s demonstration of the cell-
[Data adapted from C. B. Anfinsen and E. Haber, 1961, Journal of Biological Chemistry
236:1362.] free refolding of ribonuclease made a
mark on the field of biochemistry. In
1972, he received the Nobel Prize for
The enzyme had folded back to its ac- Discussion Chemistry for his work.
tive conformation outside of the cell,
demonstrating that the information for Through careful experiments, Anfin-
the protein folding is contained in the sen demonstrated that the information
protein itself. required to properly fold a protein is