ABSTRACT
The presence of trimethylamine N-oxide (TMAO) was recently discovered in the
tissues of some Antarctic notothenioid fishes. This discovery raises the questions of
whether the effects of TMAÖ on protein function vary with temperature and whether
TMAO plays the same role in notothenioids as it does in elasmobranchs. Two
temperature-sensitive kinetic properties - apparent Kn of pyruvate and kea- of muscle A4-
LDH in Gillichthys mirabilis, a warm-temperature fish with negligible amounts of TMAC
in its cells, and Parachaenichthys charcoti, an Antarctic fish, are not significantly affected
by the presence of 150 mM TMAO at most temperatures from 0° to 30°C. The presence
of TMAO did not appear to significantly affect the loss of activity in the LDH of any of
the five teleost species studied over a period of 24 days. The Vmax of the LDH reaction in
these species was slightly inhibited by 150 mM TMAO at both 0° and 20°C. TMAO may
function by increasing the rigidity of the enzyme at hinge regions which play a role in
establishing the rates of conformational change.
TMAO is known to counteract urea’s perturbing effects on protein function in
elasmobranchs at an approximately 2:1 sureal:[TMAO] ratio. However, the effects of
temperature on this counteraction had not been studied. Below 20°C TMAO is able to
fully counteract the denaturing effect of urea, while TMAO’s ability to counteract urea at
those same concentrations appears diminished at 30°C or above. This varying
counteracting effect, as observed on the Michaelis-Menten constant, appears to be
consistent among species, including the subtropical cow-nosed ray (Rhinoptera bonasus)
the temperate catshark (Parmaturus xaniurus) and the Antarctic skate (Bathyraja easoni).
suggesting a lack of species-specific adaptation to solute effects.
INTRODUCTION
Trimethylamine N-oxide (TMAO), a small organic solute, was recently discovered in
the tissues of some members of the notothenioid suborder of teleost fish (Raymond and
DeVries, 1997). Notothenioids are primarily Antarctic species, with some members living
in warmer waters such as those off the tip of South America. The function TMAO serves
in these organisms is a matter of some speculation, as the molecule may not function in the
same context in which it has previously been studied in different organisms. TMAO has
been known to function as an osmolyte, but it also affects protein function and stability.
Its presence in notothenioids may indicate a common function in different marine
organisms, or it may reveal divergent roles for a single molecule.
The ability of TMAO to counteract urea’s denaturing effects on proteins has been
well studied in elasmobranchs, which use both molecules for osmotic regulation. While
urea is known to denature proteins, TMAÖ is thought to have a counteracting, stabilizing
effect. Past studies have shown that TMAO can counteract urea’s effects on protein
function when both solutes occur at an approximately 1:2 concentration ratio of TMAO to
urea (Yancey and Somero, 1980). Further studies, involving rabbit lactate dehydrogenase,
showed that the increased rate of loss of protein activity over time caused by urea can be
partially counteracted by the presence of TMAÖ, although TMAO alone causes a slightly
increased rate of loss of activity (Baskakov and Bolen, 1998).
Based on TMAO's effect on elasmobranch and rabbit proteins, TMAO may be
hypothesized to serve any of several functions in notothenioids, including:
1) Although elasmobranchs are osmoconformers, notothenioids are hypo-osmotic, so
they face the problem of freezing in Antarctic waters. In order to balance the high salt
concentrations in their blood, notothenioids may use TMAÖ as an osmolyte which could
function as a freezing point depressant.
2) Although urea is not present in significant amounts in notothenioids, TMAO may
serve as a stabilizer at low temperatures, reducing the natural rate of protein degradation.
If this were true, we would expect that the rate of loss of protein activity over extended
periods of time would be reduced in the presence of TMAO at physiological
concentrations.
3) TMAO may have adaptive value to notothenioids by altering the binding affinity
and/or efficiency of enzymes at low temperatures. If binding affinity were affected, we
would expect that in the absence of TMAO, K,, values (apparent Michaelis-Menten
constants) would be higher than the expected range for teleost fish, and that the presence
of TMAO would lower those values into the expected range. If per molecule efficiency of
enzymes were affected, we would expect that kea values (catalytic rate constants) would
rise in the presence of TMAO at low temperatures in order to counteract the reduced
protein flexibility caused by reduced thermal energy.
4) Finally, TMAO may serve no significant function at all in the context of protein
structure and function; outside of its role as an osmolyte, it may be a remnant of
evolutionary divergence, or a “Spandrel of San Marcos" (Gould and Lewontin, 1979). If
this were the case, we would expect TMAÖ to have no effect on kinetic measurements
such as Kn or keat, or on rates of loss of residual activity over time.
I have addressed these questions using muscle lactate dehydrogenase (Az-LDH) from
various notothenioid and elasmobranch species. LDH is a useful enzyme to study because
it is a fairly universal element in the metabolic pathways of complex organisms.
Furthermore, the enzyme’s sequence is extremely well conserved; orthologous
homologues tend to differ at only a few positions. (Holland et al., 1997) LDH is easy to
work with in the laboratory because of its stability across a wide range of conditions. The
extensive research already completed on LDH provides a foundation for the
characterization of temperature and solute effects. Specifically, the effects of TMAO and
urea on protein stability and function have been studied using elasmobranch LDH, making
this study relevant to past data (Yancey et al., 1982).
The kinetic effects of TMAÖ were studied in a range of teleosts and elasmobranchs in
order to compare the notothenioids, which use TMAÖ alone, with elasmobranchs which
use the TMAO-urea counteraction system. Both warm and cold-water organisms were
studied in order to elucidate possible temperature adaptations. Two Antarctic
stenothermal notothenioids were studied: Parachaenichthys charcoti and Notothenia
coriiceps. These fish live from -1.86°C to -1°C year-round. Two South American
notothenioids also were studied: Patagonothen tessellata and Eleginops maclovinus, both
of which live at approximately 2-10°C. P. tessellata is probably phylogenetically closer to
the Antarctic species than E. maclovinus is, potentially revealing different solute
adaptations (Balushkin, 1992). A fifth teleost, Gillichthys mirabilis, was studied in order
to compare the four relative stenotherms to a temperate eurytherm. G. mirabilis, which
lives at approximately 5-30°C, might be expected to show less adaptation to the presence
of TMAO, because its tissue likely contains negligible amounts of the solute.
Three elasmobranchs were studied: Parmaturus xaniurus, a cold temperate catshark
species from Monterey Canyon which lives at approximately 2-10°C; Bathyraja easoni, an
Antarctic stenothermal skate which lives at -1.86° to -1°C; and Rhinoptera bonasus, a
cow-nosed ray species from Tampa Bay, Florida which lives at approximately 20-32°C.
The catshark is known to use the TMAO-urea counteraction system, but the solute
systems in the rajai remain to be characterized.
1 expected cold-water teleosts and elasmobranchs to have higher apparent Ky, curves
than warmer acclimated species, since closely related species should have similar apparent
Kn values at their respective physiological temperatures. Stenotherms would be expected
to have steeper sloped apparent K,, curves than eurytherms due to their adaptations to
narrow temperature ranges. Apparent Kn curves were generated for LDH in the presence
of TMAO for the teleosts and in the presence of TMAÖ and urea for the elasmobranchs to
determine whether shifting or flattening of the curves occurs, possibly indicating
temperature-related adaptations of protein function in the presence of these solutes.
Apparent Kn of NADH and Kn of pyruvate curves were generated in order to examine
possible differences.
In order to examine solute effects on the catalytic rate constants of LDII, I examined
the relative maximal velocities of LDH in the absence and presence of TMAO. If TMAO
slows enzyme activity by preventing substrate binding in a competitive manner, then
relative maximal velocities should be unaffected. However, if TMAÖ works by changing
the conformation or overall rigidity of LDH, then TMAO should lower the maximal
velocity.
MATERIALS & METHODS
Purification of LDII
White muscle was added to a 50 mM potassium phosphate (pHI 6.8) buffer in an
approximately 1:1 volume ratio, and homogenized. The mixture was centrifuged at
11,951 g for 40 min at 4°C. The supernatant was filtered through glass wool, and 0.5 M
potassium chloride, 0.2 mM dithiothreitol (DTT), and 200 uM NADH were added.
A gravity column with oxamate agarose was rinsed with 100 ml 50 mM KzPO4 buffer
(pH 6.8) containing 0.5 M KCl, and then with 100 ml of the same buffer containing 200
uM NADH. 100 ml of the homogenate supernatant solution were passed through the
column, followed by 100 ml of the buffer with NADH.
The LDH was eluted with 100 ml KzPO4 buffer containing 0.5 M KCl and 10 mM
pyruvate. Fractions of elutant were collected and assayed for LDH activity
spectrophotometrically at 340 nm. The active fractions were combined and placed in a
dialysis bag. The dialysis bag was placed in 2 L of dialysis buffer (potassium phosphate,
pH 6.80020°C) for 4 hours, and another 2 L for approximately 14 hours.
Kn of NADII
Kn of NADH values were measured for Az-lactate dehydrogenase at 0, 10, 20, and
30°C with and without 150 mM trimethylamine-N-oxide (TMAÖ Dihydrate, Sigma
Chemical Company) present. Each K„, was calculated using a buffer containing 80mM
imidazole-Cl (pH 7.0 ( 20°C), 2 mM pyruvate (Pyruvic acid, Sigma Chemical Company),
and eight different concentrations of NADH (Boehringer Mannheim). Reaction velocities
were measured by tracking NADH oxidation spectrophotometrically at 340 nm on a
Perkin-Elmer Lambda 3B UV/ViS Spectrophotometer. The pyruvate reductase reaction
was started by adding 25 ul of LDH at various dilutions depending on the reaction
temperature.
Apparent Kn, of pyruvate values were measured in the manner described above, except
that NADH concentration was maintained at 150 uM and pyruvate was varied among
eight concentrations. Kn of pyruvate values were measured for Az-LDH with no solute
present, or in the presence of 150 mM TMAO for teleost LDHI reactions and 200 mM
TMAO for elasmobranch LDH reactions. Elasmobranch enzymes were also measured in
the presence of 400 mM urea (Bio-Rad Laboratories) and in the presence of both 400 mM
urea and 200 mM TMAO. Teleost LDHs were incubated with 150 mM TMAO for the
appropriate reaction velocity measurements. In order to prevent rapid enzyme
degradation by urea, elasmobranch LDHs were not incubated with solutes present.
Cold Denaturation of teleost Aj-LDIIs
The cold denaturation experiments were designed to determine whether the rate of
loss of LDH activity at 0°C over time changes in the presence of TMAO. Enzyme activity
was measured at 20°C using reaction velocities measured spectrophotometrically at 340
nm with constant 150 uM NADH and 2 mM pyruvate concentrations. Three mixtures
were tested for denaturation over time: LDH with no solute (control); LDH with 150 mM
TMAO; and LDH with 600 mM TMAO.
Bovine Serum Albumin (Sigma Chemical Company) at a concentration of I mg/ml was
added to the enzyme to reduce LDH absorbance onto the walls of the container and to
control for the effects of protein concentration on loss of enzyme activity. DTT at a
concentration of 0.2 mM was added to the enzyme to prevent oxidation of cysteine side
chains. An 80 mM imidazole buffer (pH 7.0 (20°C) was used, and the reaction mixtures
were incubated on ice until the time point at which they were assayed. Activities were
determined spectrophotometrically as described above at 20°C
For each time point, duplicates were measured for each of the three reaction mixtures.
Time points were measured at 0, 6, 24, 48, 96, 192, 408, and 576 hours.
Relative Maximum Velocities
The activity of the Aj-LDH of each of the five teleost species was measured in a buffer
containing 80mM imidazole-Cl (pH 7.0 ( 20°C), 150 pM NADH, and 2mM pyruvate.
Five replicates were run for each species with and without 200 mM TMAO. Velocities
were measured spectrophotometrically at 340 nm at 20°C, and each reaction was started
by adding 25 ul of LDH at various dilutions depending on the species.
RESULTS
The apparent K» of NADH values for A4 LDH of G. mirabilis were not significantly
affected by 150 mM TMAO from 0°C to 30°C (Fig. 1). The apparent Kn of pyruvate
values were not significantly affected by 150 mM TMAO from 0°C to 30°C in G.
mirabilis, while the same values were not significantly affected in P. charcoti except at
30°C, where TMAÖ significantly decreased the apparent Kn of pyruvate value (Fig. 2).
The apparent K,, values rose with temperature for both species, with a steeper curve for
LDH of P. charcoti.
Whereas the apparent K„ of pyruvate for LDH of P. charcoti increased by 12% at
10°C in the presence of 150 mM TMAO, the apparent K» dropped by 19% in the
presence of 200 mM TMAO (from 0.2776 + 0.0108 without TMAO to 0.2248 + 0.0054
with TMAO)
The relative maximum velocities of the five teleost LDHs in the presence of 150 mM
TMAO were approximately 88-94% of the velocities without solute present at 0°C, and
approximately 93-99% of the velocities without solute present at 20°C. (Fig. 3). A chi¬
squared test showed that the (Vmag, 150 mM TMAO)/(Vmax, no TMAO) ratio was higher at 20°C
than at 0°C (p=1.54x10**), but an analysis of variance showed that the ratios are not
significantly different when variances are considered.
During cold denaturation, the relative rates of loss of activity of the teleost LDHs were
not significantly affected by the presence of 150 mM or 600 mM TMAO over 576 hours
(data not shown).
In all three elasmobranch species, the apparent K», values rose significantly in the
presence of 400 mM urea at 5°C or above and dropped significantly in the presence of
200 mM TMAO at 20°C or below, relative to the values in the absence of any solute. (Fig
4-9) For orthologs of the catshark and the cow-nosed ray, the values in the presence of
the TMAO-urea combination did not significantly differ from the values with only urea
present at 30°C or above. At 20°C or below in all three species, the TMAO-urea values
did not differ significantly from the values in the absence of any solute.
DISCUSSION
The lack of a significant effect of TMAO at a concentration of 150 mM on the binding
affinities of either G. mirabilis or P. charcoti LDH across a wide range of temperatures
suggests that the solute does not affect teleost LDH by competitive binding. Since the
binding affinity is apparently unaffected but maximum velocities are slightly lowered by
TMAO, the solute may affect LDH by altering only the aspects of the enzyme which are
operational once the substrate is bound. The catalytic domain of LDH is comprised
primarily of alpha helical regions connected by regions known as hinges (Abad-Zapatero
et al., 1987); these hinges are where much of the inter-species sequence variation occurs
(Holland et al., 1997). Because the enzyme has a catalytic loop which closes around the
substrate and cofactor during the pyruvate reduction, the flexibility of the hinge regions
has been hypothesized to be a key determinant of the overall reaction speed. TMAO may
alter the rigidity of a specific hinge in the enzyme, slowing down the closing of the
catalytic loop prior to pyruvate reduction or the opening of the catalytic loop after the
conversion. The similarity of TMAO's effect on apparent Ky values and maximum
velocities between the Antarctic and South American stenotherms and the temperate
eurytherm suggests that no species-specific adaptation to the presence of TMAO has
taken place, despite the fact that TMAÖ is present in much larger quantities in the
notothenioids than in the goby
The larger effect on apparent Kn, of P. charcoti LDH seen in the presence of 200 mM
TMAO compared to 150 mM TMAO may indicate that teleosts contain the solute in
levels low enough to avoid any significant effect of enzyme function. If these fish were to
accumulate greater than 200 mM TMAO, their enzymes might be affected the way
elasmobranch enzymes are in those conditions.
TMAO’s effect on the maximum velocity of LDH’s pyruvate reduction is only slightly
affected by temperature, with a reduced inhibition at 20°C. The reduction of maximum
velocity seen in the presence of 150 mM TMAÖ is consistent with TMAO's hypothesized
protein-stabilizing role, since decreased flexibility would reduce the per-molecule turnover
rate of the enzyme. According to the mechanism proposed by Arakawa and Timasheff
(1985), TMAO’s unfavorable interaction with water would result in the exclusion of the
solute from the enzyme's contact area with the water around it, making a small surface
area more favorable for the protein and thereby causing stabilization. If this mechanism is
true, the change in the water’s surface free energy caused by the presence of TMAO does
not appear to vary significantly with higher thermal energy. A slightly lessened inhibition
of Vmax at 20° relative to 0° complements the data showing a decreased counteraction
effect of TMAÖ at higher temperatures; at higher thermal energies, TMAO's effect on
enzyme function may be decreased.
The cold denaturation portion of the study revealed long-term (at least 24 days)
resistance to denaturation of LDH of all five teleost species. The data (not shown)
illustrate the lack of any significant difference in the rate of loss of activity in the presence
of different TMAO concentrations. The percent of initial activity data generated by
Baskakov and Bolen (1998) for rabbit Az-LDH showed a large drop (approximately 25%)
in relative activity in the presence of 600 mM TMAO relative to no TMAO by about one
week. By comparison, none of the five teleost species I studied showed less than 90%
relative activity after more than three weeks. This discrepancy may be due to the different
buffer used by Baskakov and Bolen (0.2 M tris-Cl, versus 0.16 M imidazole-Cl in my
experiment) or inherent differences between mammalian and teleost orthologs of the
enzyme.
The effects of TMAO and urea on the apparent K» values of Aj-LDHs of the
elasmobranchs are consistent with TMAO’s role as a protein stabilizer and urea’s role as a
denaturant. TMAO increases LDH's binding affinity for pyruvate, while urea lowers this
affinity. In the catshark and the Antarctic skate, the 1:2 ratio of TMAO to urea appears to
give almost complete counteraction of these kinetic effects, as is consistent with past
knowledge of solute counteraction. This does not necessarily mean that these two species
have adapted to the presence of TMAO and urea; the ability of the two solutes to
counteract each other may depend on conditions other than LDH sequence variation, such
as other small molecules present in the organism, or the counteraction may be inherent to
aqueous protein systems (Lin and Timasheff, 1994). The lack of complete counteraction
at high temperatures in the warm water ray further supports the idea that the
elasmobranch LDH has not adapted to the presence of these two solutes, since we would
expect that TMAO and urea’s combined effect on LDH function would not differ
significantly over temperatures within the animal’s physiological range. This change from
almost complete counteraction at 20°C and below to only slight counteraction at 30°C and
above is consistent between the Florida ray and the catshark, even though 30° is well
above the physiological range of the shark. This similarity suggests that TMAO’s ability
to counteract urea’s effects on protein function diminishes as temperature increases, and
furthermore that this diminishing effect is not the result of species-specific adaptation.
The implications of the solute effects studied in this paper depend on the assumption
that the elasmobranchs studied share common solute concentrations. Measurements of
the urea and TMAO levels in the tissues of these species could reveal different TMAO
levels among species which live in very different environments. If this is the case, I expect
that elasmobranchs which live in warm, shallow waters would contain more TMAO than
those which live in cold, deep water. Increased TMAÖ levels may compensate for
TMAO’s possibly diminished counteraction of urea at high temperatures.
LITERATURE CITED
Abad-Zapatero, C., et al. 1987. Resined crystal structure of dogsish M, apo-lactate dehydrogenase. Journal
of Molecular Biology. 198:445-467.
Arakawa, T., and S. N. Timasheff. 1985. The stabilization of proteins by osmolytes. Biophysical Journal.
47:411-415.
Balushkin, A. V. 1992. Classification, phylogenetic relationships, and origins of the families of the
suborder Notothenioidei (Perciformes). Voprosy ikhtiologii. 32(3):3-19.
Baskakov, I., and D. W. Bolen. 1998. Time-dependent effects of TMAO/urea on LDH activity: An
unexplored dimension of the adaptation paradigm. Biophysical Journal. In press.
Baskakov, I., et al.. 1998. TMAO counteracts urea essects on rabbit muscle LDH function: A test of the
counteraction hypothesis. Biophysical Journal. In press.
Gould, S. J., and R. C. Lewontin. 1979. The spandrels of San Marco and the Panglossian paradigm: a
critique of the adaptationist programme. Proceedings of the Royal Society of London. B 205:581-598.
Holland, L. Z., et al. 1997. Evolution of lactate dehydrogenase-A homologs of barracuda fishes (Genus
Sphyraena) from different thermal environments: Disserences in kinetic properties and thermal
stability are due to amino acid substitutions outside the active side. Biochemistry. 36:3207-3215.
Lin, T., and S. N. Timashesf. 1994. Why do some organisins use a urca-methylamine mixture as
osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with
protein. Biochemistry. 33:12695-12701.
Raymond, J. A., and A. L. DeVries. 1997. Elevated concentrations and synthetic pathways of
trimethylamine oxide and urea in some telcost sishes of McMurdo Sound, Antarctica. Fish Physiology
and Biochemistry. In press.
Yancey, P. H., et al. 1982. Living with water stress: Evolution of osmolyte systems. Science. 217:1214-
1222.
Yancey, P. H., and G. N. Somero. 1980. Methylamine osmoregulatory solutes of clasmobranch fishes
counteract urea inhibition of enzymes. Journal of Experimental Zoology. 212:205-213.
FIGURE LEGENDS
Fig. 1: Temperature vs. K» of NADII for A--LDII of G. mirabilis. LDH was incubated
on ice and added in 25ul aliquots to reaction mixture containing 80 mM imidazole-Cl
buffer (pH 7.00020°C), 2 mM pyruvate and varying concentrations of NADH.
Reaction velocities were determined spectrophotometrically at 340 nm in triplicate.
Error bars indicate one standard deviation as determined by Willman 4.0 software.
The presence of TMAO had no significant effect on apparent K,, values from 0° to
30°
Fig. 2: Temperature vs. Kn of pyruvate for A--LDII. Reaction mixtures contained 80
mM imidazole-Cl buffer (pH 7.0(020°C), 150 uM NADH, and varying concentrations
of pyruvate. Reaction velocities and error bars were determined as described above
Except for P. charcoti at 30°C, apparent Kn, values were not significantly affected by
150 mM TMAO.
Fig. 3: Relative Vmax for Ag-LDII (150 mM TMAO over no solute). Reaction mixtures
included 80 mM imidazole-Cl buffer (pH 7.00020°C), 150 uM NADH and 2 mM
pyruvate. Error bars represent one standard deviation based on five velocity
measurements made at each temperature for each species.
Fig. 4: Temperature vs. Kn of pyruvate for A--LDII of Parmaturus xaniurus. See
description of Fig. 2 for procedures.
Fig. 5: Temperature vs. K» of pyruvate for A--LDII of Bathyraja easoni. See
description of Fig. 2 for procedures.
Fig. 6: Temperature vs. K., of pyruvate for AJ-LDII of Rhinoptera bonasus. See
description of Fig. 2 for procedures.
Fig. 7: Relative Kn of pyruvate for A--LDII of Parmaturus xaniurus. Values are
relative to apparent Kn values with no TMAÖ or urea present.
Fig. 8: Relative Kn of pyruvate for A--LDII of Bathyraja easoni. Values are relative to
apparent Kn values with no TMAO or urea present.
Fig. 7: Relative K,, of pyruvate for A--LDII of Rhinoptera bonasus. Values are
relative to apparent K,, values with no TMAÖ or urea present.
Figure 1
15
..............
10
5
— No solute
-E- 150 mM TMAO
5
10
15
20
Temperature (?C)
Figure 2
0.8
—— G. mirabilis, no solute
0.7
E— G. mirabilis, 150 mM TMAO
-Q— P. charcoti, no solute
0.6
—+ P. charcoti, 150 mM TMAO
—

0.5
0.4-

0.3

0.2

0.1


0.0
5
10
20
Temperature (°C)

25 30 35



....................
25 30 35
Figure 3
1.4
G. mirabilis

P. charcoti
1.2
N. coniceps

E. maclovinus
1.0
IP. tessellata

0.8
0.6
8 0.4
0.2
0.0
20
Temperature (?C)
Figure 4
0.45
— No solute
0.40
—A- 200 mM TMAO
0.35
•- 400 mM Urea
-E— TMAO + Urea
0.30

0.25



0.20



0.15


0.10

005

....
......
0.00 -
-5 0
5
10
25 30 35
20
Temperature (°C)
Figure 5
0.15
—— No solute
E- 200 mM TMAO
—A- 400 mM Urea
•- TMAO + Urea
0.10






0.05
0.00
-5
15
20
Temperature (°C)
Figure 6
0.50
— No solute
.......................................
0.45
-A- 200 mM TMAO

0.40
- 400 mM Urea
-E— TMAO + Urea

0.35


**.................
.........

0.30

0.25


0.20



0.15

W
0.10
O5
0.00
30 35 40
10
15
20
25
Temperature (?C)
25
Figure 7
2.0

1.8
5 16 4
214
.
1.2 —

.


.......
1.0 —
9



06

•- 200 mM TMAO
04

- 400 mM Urea
02
• TMAO + Urea
0.0
5
10 15
20
25 30 35
Temperature (?C)
Figure 8
2.0 —
18

E
4—
1.2
1


0.8

- 200 mM TMAO

....
0.4
- 400 mM Urea

—- TMAO + Urea
E 0.2
0.0
10
20
Temperature (°C)
25
Figure
2.0 -
18-
1.6
.................................................


1.4

1.2
1.0


S
—
0.8



0.6

200 mM TMAO

0.4
400 mM Urea

-0.2
• TMAO + Urea
0.0
25
10
20
30
35
Temperature (°C)
40