Aggregation, Protein
The aggregates formed by either heat or
pH denaturation can be disrupted in 6 M
GdnHCl into monomeric unfolded species
and then renatured under optimal condi-
tions to yield an active enzyme. Only strong
denaturants such as high concentrations
of guanidine hydrochloride are efFcient in
this disruption process.
The presence of covalent cross-links
such as disulFde bridges in a protein
molecule can complicate the refolding of
the denatured and reduced protein re-
sulting in the formation of incorrect and
intramolecular disulFde bridges leading
to further aggregation. The Frst well-
documented studies were performed by
AnFnsen and his group on the refold-
ing of reduced ribonuclease. The authors
showed that the reoxidation of the enzyme
produces a great number of species with
incorrectly paired disulFde bonds. This
scrambled ribonuclease is capable of re-
gaining its native structure in a slow step,
a process that is accelerated by the addi-
tion of a small quantity of reducing reagent
such as
-mercaptoethanol yielding about
100% of active enzyme. The reshuffling
of a protein’s disulFde bonds takes place
through a series of redox equilibria ac-
cording to either an intramolecular or
an intermolecular exchange. To prevent
a wrong pairing of half-cystine and fur-
ther aggregation, the addition of small
amounts of reducing reagents or redox
mixture is frequently used as investigated
by Wetlaufer.
The detection and characterization of
aggregates represent an important aspect
of folding studies. The aggregation phe-
nomenon can occur without precipitation.
Indeed, the degree of association of pro-
tein intermediates during folding might
be small, depending on the intermolecular
interactions, and does not necessarily lead
to a visible insolubility. The association
state may be determined in several ways.
The most common methods, available
in any biochemistry laboratory, are gel
permeation and sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-
PAGE), used both with and without cross-
linking. The detection of aggregates can
also be monitored by other hydrodynamic
tion or classical light scattering. The latter
method also gives information on the size
of the aggregates. Quasi-elastic light scat-
tering is a dynamic technique that can be
used to determine macromolecule diffu-
sion coefFcients as a function of time, that
is, to follow the kinetics of aggregation.
Neutron scattering can also be used to
detect protein aggregates, and mass spec-
trometry has become a useful tool as well.
Mechanisms of Protein Aggregation
A substantial body of information sup-
ports the idea that protein aggregation
arises from partially folded intermediates
formation of aggregates has often been
considered as a trivial phenomenon, a
nonspeciFc association of partially folded
polypeptide chains to form a disordered
precipitate. However, several analyses in-
dicate that aggregation occurs by speciFc
intramolecular associations involving the
recognition of a sequence partner in an-
other molecule rather than in the same
molecule during the folding process. Anal-
yses of the aggregation mechanisms of
various proteins, such as bovine growth
hormone and phosphoglycerate kinase,
has permitted the identiFcation of speciFc
sites that are critical in the association.
An elegant demonstration of the speci-
Fcity of aggregation was provided by King
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