Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials
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7.6 Molecular Chaperones: Biology's Effort to Overcome Improper Insolubility
315
FIGURE 7.44. Stereo view of a subunit of Apo-
GroEL with the black arrow pointing to the location
on the equatorial domain where the ATP triphos-
phate tail binds. Because of the large presence of
hydrophobic residues, ATP would be expected to
bind with a substantial apolar-polar repulsive free
energy of hydration,
AGap.
This
could drive clockwise
rotation of the apical domain that on combination
with ATP binding at other subunits would give rise
to positive cooperativity. (Prepared using the crys-
tallographic results of Xu et al.^^ as obtained from
the Protein Data Bank, Structure File lAON.)
consilient mechanism. The binding of seven
ATP molecules to a chaperonin surrounds the
hydrophobically misfolded protein with a cage
of charge. Why? The structural change on ATP
binding states simply that sufficient proximal
charge solubilizes hydrophobic groups, that is,
charge disrupts hydrophobic association. To the
best of our knowledge, only the consilient
mechanism, with a fundamental element being
the competition for hydration between charged
and hydrophobic groups, explains the phenom-
enon of what has been called the Anfinsen
cage. Only the experimentally calculable
apolar-polar repulsive free energy of hydra-
tion, AGap, embodies this concept. AG^p con-
tributes to the Gibbs free energy of
hydrophobic association,
AGHA-
Independent
experimental determinations show that under
the proper experimental variable, a change in
AGHA, determined from the change in heat of
an inverse temperature transition due to an
increase in hydrophobicity, and a change in
AGap, due to a change in pKa resulting from
the same change in hydrophobicity, yield the
316
7.
Biology Thrives Near a Movable Cusp of Insolubility
FIGURE 7.45. Stereo view of the inside of an asym-
metric cw-GroEL-GroES-(ADP)7. Residues D87
and D398 coordinate Mg^^ (along with a- and p-
phosphates of ADP), and residue R58 combines to
serve as a reference point; the black arrow points the
direction to the ADP binding site. Viewed from
within the large aqueous cavity, approximately at
right angles to the view in Figure 7.46. Figure pre-
pared using the crystallographic results of Xu et al.^^
as obtained from the Protein Data Bank, Structure
File lAON.
same change in kcal/mole (see Chapter 8,
section 8.1.8.1.9).
7.6.4.1
Explicit Mechanism for the
Function of an Anfinsen Cage
In
1973,
Anfinsen^^ stated the principle that the
sequence of a protein determined the resulting
full three-dimensional structure. The rationale
for calling the large aqueous cage that forms
upon binding of seven ATP molecules to the
GroEL/GroES molecular chaperone the Anfin-
sen cage was that this structure provided the
near ideal environment whereby the Anfinsen
principle would be unfailing. However, as
noted above, there is a physical reason why an
aqueous cavity surrounded by very polar walls
causes the unfolding of hydrophobically mis-
folded protein and why decreasing the polarity
by stepwise removal of the most polar group,
phosphate, would facilitate proper hydrophobic
folding. It is the physical process of competi-
tion for hydration between hydrophobic and
polar (especially charged) groups that causes
hydrophobic unfolding and the relaxation of
that competition by a decrease in charge
7.6 Molecular Chaperones: Biology's Effort to Overcome Improper Insolubility
317
that allows for proper refolding. That physical
process was demonstrated with care by a
series of experimental results in Chapter 5, and
it was called the apolar-polar repulsive free
energy of hydration for the reasons given
above.
Of course, a visualization of this GroEL/
GroES Anfinsen cage is incomplete without a
representation of the GroES cap of the cage.
The dome of the Anfinsen cage is shown in
Figure 7.47, and, to us, it seems to be every bit
as much of an architectural attraction as the
domes of the ancient cathedrals and the
modern sports arenas. The actual portion that
constitutes the inside enclosure contributed by
the dome represents a relatively small area of
the inside wall, but additional chains fold over
and into pores in the GroEL part of the
D87
FIGURE
7.46.
Stereo view of the ADP binding site of
asymmetric (ADP)7-GroEL showing the a- and (3-
phosphates of ADP classically positioned at the base
of an aqueous cleft, that
is,
this
figure
provides a view
of the ADP binding site looking down at the interior
floor of the large aqueous cavity. The a- and (3-
phosphates within the circle and the D87 and D398,
all of which provide coordination for the Mg ion,
accurately define the nucleotide binding site. Note,
therefore, that the carboxylates of the D87 and D398
residues are not in position to contribute as directly
to the polar surface and are not included in the sum-
mation over AGHA for the surface. Note also that
inclusion of residue R58 provides easy reference to
the location of the nucleotide binding site for the
structures of Figures 7.43A,
7.44,7.45,
and 7.46. (Pre-
pared using the crystallographic results of Xu et al.^^
as obtained from the Protein Data Bank, Structure
File lAON.)
318
7.
Biology Thrives Near
a
Movable Cusp
of
Insolubility
FIGURE
7.47.
Stereo view
of the
aqueous chamber
side
of the
(ADP)7-GroEL-GroES cap, which
may
be called
the
dome
of the
Anfinsen cage
for
protein
folding.
ADP
molecules were included
to
provide
orientation.
(A)
Sevenfold symmetric structure
in
white
relief. (B)
Sevenfold symmetric structure with
black, dark gray, gray, light gray,
and
white repre-
senting aromatic, acidic, basic, other hydrophobic,
and neutral residues, respectively.
The
dome pro-
vides
a
very polar surface
as
demonstrated with
the
perspective
in
Figure 7.37. (Prepared using
the
crys-
tallographic results
of Xu et al.^^ as
obtained from
the Protein Data Bank, Structure File lAON.)
chamber,
as
shown
in
Figure 7.48. Interestingly,
a summation over AGHA
for a
comparison
of
the relative hydrophobicity,
as
done
for a
subunit
of the
walls indicated
in
Figures
7.44
and 7.45, indicates
a
very polar
cap
topping
the
aqueous chamber that
is
dominated
by
five
E,
three
D, and
three
K
residues
and
sums
to
approach -i-30kcal/mole-subunit
for a
relatively
small surface area.^^
7.6.4.2
Effect of ATP Binding
and
GroES
Capping
on the
Substrate's Movable Cusp
of Insolubility
AGHA
for the
apo-GroEL wall
of the
chamber
was approximated
to be
about -lOkcal/mole
subunit inner surface,
as
estimated
for the
surface shown
in
Figure 7.44.
ATP
binding
and
the resulting emergence mostly
by
rotation
of
7.6 Molecular Chaperones: Biology's Effort to Overcome Improper Insolubility
319
charge on the interior surface raises the tem-
perature of the movable cusp of insolubility to
hydrophobically unfold the substrate.
AGHA
for
the inner wall of the (ADP)7-GroEl/GroES
state approximates to be more than -i-50kcal/
mole subunit inner surface, as estimated by
the surface shown in Figure 7.45. With an
average radius to the inner wall of about 4nm^^
and with a mean diameter of the misfolded
protein of a 2 to
3
nm diameter, this means that
the misfolded protein would come within a
nanometer of the wall. This places the mis-
folded protein within the estimated reach,
for the apolar-polar repulsive free energy of
hydration, found for the elastic contractile
model proteins of Chapter 5. The apolar-polar
repulsive free energy of hydration,
AGap,
would
tend to center the hydrophobically misfolded
protein substrate, but Brownian motion and
transient openings would facilitate escape from
the metastable hydrophobically misfolded
state.
FIGURE 7.48. Stereo view of the aqueous chamber
side of the GroES cap at the GroEL-GroES junc-
tion for the asymmetric (ADP)7-GroEL structure.
The purpose is to estimate surface polarity by
obtaining a sum over AGHA for the face and to
compare the value to the inner faces of GroEL as
represented in Figures 7.44 and 7.45. Double-digit
numbers are for the GroES cap, and triple digit
numbers indicate the top of the apical domain of
GroEL. (Prepared using the crystallographic results
of Xu et al.^^ as obtained from the Protein Data
Bank, Structure File lAON.)
320
7.
Biology Thrives Near
a
Movable Cusp
of
Insolubility
7.6.4.3
Effect
of
Splitting
ATP to
Form
the Most Polar State,
ADP
Plus
Pi
Introduction
of the
potent charged species
ATP
prepares
the
Anfinsen cage. However,
the
most
potent collection
of
charge occurs when
ATP is
hydrolyzed, that
is,
splits,
to
give
ADP
plus
Pi.
If
the ADP
plus
Pi
should stay
in
position
for a
few moments,
it
would present
an
extra jolt
of
charge, possibly amplified
as in the
original
ATP binding,
to
result
in the
pulse
of an
even
greater AGHA
in the
form
of
AGap.
It is, of
course,
the
AGap contribution
to
AGHA that
drives hydrophobic unfolding.
7.6.4.4
Stepwise Release
of
Inorganic
Phosphate Steadily Lowers
the
Temperature
of the
Substrate's Movable
Cusp
of
Insolubility
to
Promote
Systematic Hydrophobic Refolding
AGap peaks
at the
separation
of ATP to
form
the most charged state
of ADP
plus
Pi,
which
raises
the
movable cusp
of
insolubility
to its
highest temperature
(see
Figure
7.1) and in an
equivalent representation raises
the
Tfdivide
(also called
the
coexistence
or
bimodal line,
as
shown
in
Figure
5.3) to its
highest value.
The
subsequent stepwise release
of Pi
steadily
lowers
the
temperature
of the
movable cusp
of
insolubiUty
to
refold
the
substrate systemati-
cally
and
hydrophobically. This allows
the
sub-
strate surfaces
to
begin sorting
out the
most
favorable hydrophobic associations
as the in-
organic phosphates, HP04^, peel
off
stepwise
to
lower systematically
the
Tt-divides
for
different
cooperating hydrophobic domains toward
and
finally below
the
operating temperature.
7.7 Consequences
of
Excursions
into Excess Solubility
Due to
Oxidative Processes
7.7.1
The
Wide-ranging Impact
of
Oxidative Stress
From
all
that
we
have discussed
in the
forego-
ing process
of
developing
the
consilient mech-
anism
for
hydrophobic association,
the
proper
function
of
biology's great macromolecules,
proteins
and
nucleic acids, demands
a
graceful
balance between hydrophobic association
and
dissociation. Disruption
of
that fine ballet
of
"the movable cusp
of
insolubility" brings
disease
and
death.
In
general terms, unchecked
oxidative processes,
due to
oxygen directly
and
molecular species derived from oxygen,
are
among
the
most troublesome means
of
shifting
toward excess solubility. Hence, current
medical research interests emphasize disentan-
gling
the
role
of
numerous antioxidants
in
attenuating oxidative stress implicated
in the
full range
of
disease processes.
Oxidative stress
has
been implicated
in
disease states from cancers, heart disease,
Lou
Gherig's disease (amyotrophic lateral sclero-
sis),
infections, environmental toxicities,
to
primary
and
secondary aging neurovascular
disease processes. Oxidative stress occurs
in
general during critical illness
in the
intensive
care setting, where
the
conclusion
has
been that
"the oxidative damage
to
cells
and
tissues even-
tually contributes
to
organ failure."^^ Being
such
a
fundamental problem, nature
has
devised
a
direct attack
on the
most offensive
errant oxidants
by
means
of
superoxide dismu-
tase,
the
enzyme that disarms superoxide,
O2",
and hydrogen peroxide,
H2O2.
These
and
other
oxidants
are
given
off by
cells that attack
foreign bodies from coal dust particles
to
infec-
tious agents
Our focus here continues along
the
lines
of
studies
on the
elastic-contractile model
proteins. Examples will show that hydroxyla-
tion
of the
basic model protein, poly(GVGVP),
indeed moves
the
cusp
of
insolubiUty
to
higher
temperatures resulting
in
solubiUty that
becomes relevant during wound repair. Fur-
thermore,
the
argument will
be
presented that
the oxidative bursts
of
macrophages
in
their
attack
on
foreign agents
in the
lung lead
to the
development
of
pulmonary emphysema,
because
the
oxidative bursts
are not
well tar-
geted
to the
foreign agent alone. Defensive
oxidative bursts also lead
to the
destruction
of
natural elastic tissue
so
essential
to
lung
function.
7.7 Consequences
of
Excursions into Excess Solubility
Due to
Oxidative Processes
321
7.7.2 Solubility: The Consequence
of
Oxidative Hydroxylation
of
Proline
7.7,2.1
Demonstration with
the
Parent
Model Protein, (GVGVP)^
Early
in our
studies
it was
expected that
the
post-translational modification
of
proline
hydroxylation,
so
important
to
proper collagen
structure
and
function, would raise
the
value
of
the temperature, Tt,
for the
onset
of the
inverse
temperature transition
for
models
of
elastin.
Accordingly, hydroxyproline
(Hyp) was
incor-
porated
by
chemical synthesis into
the
basic
repeating sequence
to
give
the
protein-based
polymers poly[fvai(Val-Pro-Gly-Val-Gly),
fHyp(Val-Hyp-Gly-Val-Gly)], where
fva, + W
=
1 and
values
of
fnyp were
0, 0.01, and 0.1.
The effect
of
prolyl hydroxylation
is
shown
in
Figure 7.49.^^ Replacement
of
proline
by
hydroxyproline markedly raises
the
tempera-
ture
for
hydrophobic association. Prolyl
hydroxylation moves
the
"movable cusp
of
100
^
80
T3
^E60
#40
1^
1
20
0
a.
0%
Hyp
b. 1%Hyp
c. 10% Hyp
d.
100%
Hyp
e. enzymatically hydroxylated
'>•''•'
"^ "^
/''-
/
a//^b
-"c&e
'i
/
//
//
,1
II
10
30 40 50
y
/
/d
/
/
/
/
/
/
1
\
1
1
(
1
/
.
J 1 1
60
70 80
Temperature
°c
90
FIGURE
7.49.
Effect
of
prolyl hydroxylation
on
hydrophobic association (insolubility). Temperature
profiles show turbidity formation
due to the
aggre-
gation attending
the
onset
of the
inverse tempera-
ture transition. The value
for the
onset temperature
is taken
at
50%
of
the
maximal turbidity and
is
called
Tt. Hydroxylation markedly shifts
the
polymer
to
solubility. (Reproduced with permission from Urry
et al.^1)
insolubility"
to
higher temperatures
and
shifts
the molecular system toward solubiUty.
Poly(VPGVG)
is a
substrate
for the
natural
enzyme prolyl hydroxylase,^^ which uses mole-
cular oxygen
and the
cofactor vitamin
C
(ascor-
bic acid)
for the
reaction. Figure
7.49
also
contains
the
temperature profile
for
fiber
formation that results from hydroxylation
by
prolyl hydroxylase. When
100%
hydroxylated
as
in
poly(Val-Hyp-Gly-Val-Gly),
Tt is
about
65°C.
An
estimated
1%
hydroxylation
by
prolyl
hydroxylase results
in a
value
of Tt of
about
40°
C.
As
shown
in
Figure 5.1C,
Tt is
just
at the
very onset
of the
aggregation, such that rais-
ing
the
value
of Tt
from about
30° to
about
40°
C
would prevent assembly into fibers
at
body temperature. Prolyl hydroxylation
of the
polypentapeptide model
of
elastin impairs fiber
formation
at
physiological temperature.
7.7.2.2
Prolyl Hydroxylation Blocks
Elastic Fiber Formation
In
Vitro
and
In Vivo
7.7.2.2.1
In
Cell Culture
of
Vascular Smooth
Muscle Cells
The prediction
in 1979
that hydroxylation
of
proline (Pro,
P)
would impair fiber formation^^
was borne
out 6
years later
in
cell culture.^^
Using aortic smooth muscle cells
in
culture with
substantial amount
of the
cofactor ascorbate,
Barone
et al.^^
found
an
overhydroxylation
of
the
Pro
residues
in
elastin. Furthermore, over-
hydroxylated
the
elastin remained
in
solution
at 37°
C at a
higher than normal concentration
with hmited formation
of
insoluble elastin.
Thus,
as had
been predicted from what
is now
called
the
consilient mechanism
for
hydropho-
bic association, making
the
elastin more polar
by hydroxylation impairs fiber formation.
7.7.2.2.2
In
Vivo Scar Tissue Formation
of
Wound Repair
In wound repair
the
activity
of
prolyl hydroxy-
lase increases markedly
to
produce
the
neces-
sary quantity
and
quality
of
collagen. Although
this
is the
primary objective
of
hydroxylating
the proline
of
collagen,
a
side effect
of
hydrox-
322
7.
Biology Thrives Near
a
Movable Cusp
of
Insolubility
ylation
of
elastin
has the
effect
of
impairing
formation
of
elastic fibers.
The
result
is
stated
v^ell
in the
treatise
on
w^ound repair
by
Peacock,^'*
"In a
scar, where there
are fev^
elastic fibers
and
where collagen fibers become
oriented primarily along
the
lines
of
tension,
there
is
little 'give'
and
stretching
and
relax-
ation
are not
possible." Furthermore, "Failure
to include
new
elastic fibers
in
repair tissue
until long after collagen fibers
are
formed
is
another example
of
the inferiority
of
scar tissue
to normal tissue... ."^'^
One
presumes that evo-
lution toward rapid closure
of a cut or
tear, pos-
sibly
to
limit infection,
has
proven
to be
more
important than ensuring quality
of
repair. Once
again, this time
in
vivo,
the
effect
of an
oxida-
tive process, that
of
hydroxylation, results
in
preventing
the
hydrophobic association
required
for
fiber formation.
7.7.3 Loss
of
Elastic Recoil
in
the Elastic Fiber
on
Oxidation
by
a Superoxide
and
Hydrogen
Peroxide Generating Natural
Enzyme
Applying what is now the consilient mechanism
for hydrophobic association
to the
elastic fiber,
the prediction became that oxidation
of the
elastic fiber
by a
natural enzyme with
the
biological role
of
producing superoxide
and
hydrogen peroxide would cause hydrophobic
dissociation evidenced
by a
swelling
and a
loss
of elastic recoil.
A
xanthine oxidase superoxide
generating system prepared
by
Bruce Freeman
was used.
The
stress-strain curve
was
deter-
mined
at
time zero,
and
then
the
superoxide
generating system
was
added
and
followed
for
2 hours, when
a new
stress-strain curve
was
again determined.
The
superoxide generating
system
was
replenished
and run for
another
2
hours,
and the
stress-strain curve
was
deter-
mined
for a
third time.
The
process
was
repeated
for
12
hours.
As shown in Figure 7.50,^^
each exposure
to
superoxide
and
hydrogen per-
oxide from
the
enzymatic generating system
caused
the
elastic modulus
to
decrease
and the
extension, required before force development
began,
to
increase.
The
interpretation
is
that
the formation
of
polar species
on the
elastin
chains by the processes
of
these highly oxidative
molecular species moves
the
"movable cusp
of
insolubility"
to
higher temperatures resulting
in
hydrophobic dissociation. Thus, the elastic fiber
is susceptible
to the
natural oxidative bursts
emitted
by
macrophages,
as
they encounter
a
foreign agent.
7.7.4 Relevance
to
Environmentally
Induced Lung Disease
7.7.4.1
Oxidative Attack
on
Foreign
Substances
by the
Body's Macrophages
The body's natural defense mechanism involves
macrophages (called monocytes when
in the
bloodstream) that give
off
oxidative bursts
on
encountering foreign agents
and
pathogens.
As
Young's Modulus
x10^dynes/cm2
3.93
1
2
A Length
(mm)
FIGURE
7.50. Effect of enzymatic
superoxide generating system
on
the elastic properties
of
ligamen-
tum nuchae elastin. With time
of
exposure
of
purified fibrous elastin
to
O2" and
H2O2,
the
elastic
modulus decreases,
and
larger
and
larger extensions
are
required
before development
of an
elastic
force occurs. This
is the
result
of swelling
due to
hydrophobic
dissociation. Once
the
fiber
is
hydrophobically dissociated,
it
becomes susceptible
to
proteolytic
degradation. (Reproduced with
permission from Urry
et
al.^^)
References
323
would be expected from the consilient mecha-
nism for hydrophobic association within macro-
molecular species, a most effective approach to
render the agent harmless would be to oxidize
it, thereby, causing it to become susceptible to
biodegradation, such as proteolysis, and even
to direct dissolution by oxidation. The problem
with such an oxidative attack is that it lacks the
capacity to be directed solely at the eliciting
agent. Because of this, natural constituents of
the extracellular matrix inadvertently become
targets of the oxidative bursts. Herein lies the
introduction to problems of environmentally
induced lung diseases such as pulmonary
emphysema and pulmonary fibrosis.
7.7.4.2 Molecular Basis for Defective
Elastic Tissue in Environmentally Induced
Lung Disease: Pulmonary Emphysema
Pulmonary emphysema is a chronic progressive
disorder wherein the elastic fibers of the lung
become fragmented and dysfunctional with the
result of a loss of elastic recoil.^^'^^ One result is
that the patient must consciously and forcibly
exhale. A prominent proposal for the etiology
of the disease has been the lack of proteinase
inhibitors that would otherwise prevent prote-
olytic degradation of the fiber.^^"^^ To help
study the disease, a successful animal model for
pulmonary emphysema was developed in which
direct instillation of the proteolytic enzyme
elastase into the lung became the causative
agent.^^i'i^^
Another element of the story is the finding
that the antiprotease, al-proteinase inhibitor is
damaged by superoxide. We, of course, argue
that inactivation by oxidation of this inhibitor
protein would be due to hydrophobic dissocia-
tion, that is, due to moving the "movable cusp
of insolubility." Furthermore, from our studies
to develop elastic model proteins as materials
for medical applications (see Chapter 9), the
finding is that these materials are essentially
nonbiodegradable in situ as long as the value of
Tt remains about
15°
C below body tempera-
ture.
Only after the value of Tt increases above
body temperature, as when carboxamide
chemical clocks convert to carboxylates, does
degradation occur.
Thus our scenario for the pathogenesis of
pulmonary emphysema becomes a primary
event of macrophages encountering foreign
agents inspired into the lungs and emitting
oxidative bursts that inadvertently oxidize the
elastic fiber. As shown in Figure 7.50, this causes
the elastic fiber to hydrophobically dissociate,
that is, to swell and lose its elastic recoil, and to
become susceptible to proteolytic digestion.
ThuSy
underlying the pathogenesis of pulmonary
emphysema resides a "movable cusp of insolu-
bility" where excursion into the realm of
increased solubility means disease and death.
References
1.
D.W.
UrryT. Hugel,
M.
Seitz,
H.
Gaub,
L.
Sheiba,
J. Dea, J. Xu, and
T.
Parker, "Elastin: A Repre-
sentative Ideal Protein Elastomer." Philos.
Trans.
R.
Soc.
Lond.,
B
357,169-184.2002.
2.
D.W. Urry and T.M. Parker, "Mechanics of
Elastin: Molecular Mechanism of Biological
Elasticity and its Relevance to Contraction."
/. Muscle
Res.
Cell
Motil, Spec. Issue,
23,
2002.
3.
D.W. Urry, "Physical Chemistry of Biological
Free Energy Transduction as Demonstrated by
Elastic Protein-based
Polymers."
/.
Phys.
Chem.
B,
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5.
Two curiously complementary and impeding
truisms face any newly proposed concept or
outlook in science. They may be demonstrated
by two quotations. The first, attributed to
Maurice Maeterlinck, indicates the struggle of
those who would make new proposals, "At
every crossway on the road that leads to the
future, tradition, has placed against each of us,
10,000 men to guard the past." The second sug-
gests why it is so easy, even for those without a
direct personal stake, to hold with tradition,
"Keep in mind that new ideas are common-
place, and almost
always
wrong'' (E.O. Wilson,
Consilience:
The Unity
of
Knowledge,
Alfred E.
Knopf,
New York,
1998).
Accordingly, to stand
against a new idea is to be almost
always
right,
with neither intellectual effort nor actual
understanding
required.
Those
who might prac-
tice default to pretense could seldom find a
better opportunity.
324
7.
Biology Thrives Near a Movable Cusp of Insolubility
There is another aspect noted by Francis
Bacon in The New Organon, "The human
understanding is no dry light, but receives an
infusion from the will and affections; whence
proceed sciences which may be called 'sciences
as one would wish.' For what a man had rather
were true he readily believes." It is particularly
difficult for anyone to allow ready marginaliza-
tion of a hard-won and once very empowering
viewpoint.
Of course, the basic issue in science always
remains the extent to which any concept con-
tinues to bear fruit. The wonderful thing about
scientific pursuit is that there is a correct and
unchanging truth and that progress is best
achieved by hewing to that path as closely as
possible. This was attempted, as reported in
part in Chapter 5, by repeatedly designing new
protein-based machines and testing those
designs for intended function.
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1993,
p.
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7.
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bulk water gives a negative change in
AS,
which
makes the (-TAS) term positive for this process,
whereas the conversion of hydrophobic hydra-
tion to bulk water results in a positive change
in AS such that the (-TAS) term is negative for
this process.
8. Borland's Illustrated Medical Dictionary, 2T^
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of the English Language, Unabridged. Merriam-
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1993,
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