ABSTRACT
The heat shock response is a universal phenomenon in which an
environmental stressor causes an animal to produce large quantities of heat
shock proteins or HSPs. These proteins are thought to protect the cells from
the damaging effects of stress, allowing the organism to recover from the
shock and survive. In this study, the heat shock response was studied using
the western blotting technique in the benthic sea cucumber, Psolus
squamatus. An initial screening of the proteins from an unshocked specimen
revealed a total of five proteins that cross-react with the anti-heat shock
protein 60 antibody. P. squamatus generally lives in temperatures between
4°C and 7°C and in an ocean water salinity of 33.2 parts per thousand. Heat
shocking the animals along a temperature gradient revealed that heat shock
protein 60 production can be induced at 13°C, but protein synthesis appears to
shut down and protein degradation may occur at super-ambient temperatures
such as 20°C. Death occurs at 22°C. Salinity shock was at 10 g/L above and
below the natural salinity of 33.2 g/L (parts per thousand). Results of the
salinity shock appear a little more ambiguous and further study needs to be
done before any conclusions are drawn.
INTRODUCTION
Heat shock proteins are present in all organisms and serve basic
indispensable cellular functions (see 4, 6, 8 for review). They interact with
other proteins in important ways such as aiding in intracellular trafficking of
proteins and stabilizing proteins such as immunoglobulins. Heat shock
proteins are among the most highly conserved between different species and
therefore are very easy to detect. Their high degree of sequence homology
allows for probing with antibodies made in different organism. The proteins
occur in molecular weight families, of which there are four main types; the
large molecular weight HSPs, the HSP 70 family, the HSP 60 family, and the
small HSPs (15-30 KD). Proteins within each family share common amino
acid sequences and cross react with the same antibody. They can, however,
vary in size, but they generally fall into a narrow size range around which
they are named.
Heat shock proteins are present in the body at lower levels in the
absence of stress. The stress response is characterized by a large scale
induction of these proteins. The proteins are thought to protect the cells from
the damaging effects of stress, allowing the stressed organism to recover and
survive. Various stressors include heat shock, salinity shock, anoxia, pH
variations, and contamination from different chemicals or xenobiotics in the
environment. The stress response is universal.
For this study, the stress response was studied in Psolus squamatus., a
benthic sea cucumber that is found abundantly in the Monterey Canyon. It is
an interesting organism to study because it is found at a very large depth
distribution, ranging from 200 m to 3500 meters deep. The temperature
varies with the depth, with colder temperatures occurring at deeper depths.
P. squamatus is found to survive extreme environmental stresses with a great
amount of success. Its natural habitat is in temperatures ranging between 4°C
to 7°C. When exposed to a temperature gradient that ranged up to 25°C, the
organisms were seen to survive in temperatures up to 22°C.
Early studies of the heat-induced puffs in the fruit fly, Drosophila
busckii, demonstrated that heat shock protein induction tended to interfere
with oxidative phosphorylation or electron transport to protect cells from
respiratory stress (6). A recent study in P. squamatus demonstrated that
respiration increases with temperature between 2°C and 15°C (2). A heat
shock response may be induced in P. squamatus' to aid the organism in
surviving the temperature increase and the possible respiratory stress. The
main goals of this project were to identify the heat shock proteins present
constitutively in the animal and to study the effects of heat shock and salinity
shock on heat shock protein induction.
MATERIALS AND METHODS
COLLECTION AND MAINTENANCE OF SPECIMENS: Psolus squamatus used in
this study were collected with the MBARI ROV from various depths in the
Monterey Bay Canyon. They were kept in the Deep Sea Lab of Hopkins
Marine Station at an ambient sea water temperature of 5°C until needed.
INDUCTION OF STRESS RESPONSE:
• Heat Shock: Viable specimens were isolated in individual test tubes or jars
and acclimated in Deep Sea Lab sea water (5°C) for 12-18 hours. Heat shock
was induced by placing the jars containing the acclimated specimens into
water baths previously set to heat shock temperatures. The individuals were
shocked for a total of 2 hours. The control specimens were kept at 5°C for the
duration of the heat shock. Individuals were shocked over a gradient of
temperatures between 5.0 and 25.1°C. Individuals survived to an upper limit
temperature of 22.0°C.
• Salinity Shock: Specimens were isolated in individual jars and acclimated
in a solution of Instant Ocean Synthetic Sea Salt, provided by Aquarium
Systems of Mentor, ÖH, at a concentration of 33.2 parts per thousand (33.2 g
Instant Ocean/1 L de ionized H2O) at 5°C for 12-18 hours. Individuals were
shocked by decanting the 33.2 g/L solution and adding hypotonic (23.2 g/L) or
hypertonic (43.2 g/L) solutions of Instant Ocean to the acclimated organism at
5°C for 3 hours. There were two salinity controls. The Instant Ocean control
organism was acclimated at 5°C in a 33.2 g/L solution of Instant Ocean which
was replaced with a new 33.2 g/L solution for the duration of the salinity
shock. The other control was acclimated in normal Deep Sea Lab sea water at
5°C which was replaced with fresh Deep Sea Lab sea water at 5°C for the shock.
The second control was to see whether the Instant Ocean Synthetic Sea Salt
itself elicits a heat shock response.
HOMOGENIZATION: Äfter shocking the organisms, the tentacle tissues were
dissected and placed in 200 ul of homogenization buffer, consisting of PBS,
EDTA, PMSF, pepstatin, leupeptin, chymostatin, and NP-40, and ground
using a Teflon homogenizer. The sample was transferred to a 1.5 ml
Eppendorf tube and centrifuged at 18,000 x g on a refrigerated microcentrifuge
MTX-150 for 15 min. The supernatant was transferred to a new Eppendorf
tube. Protein content was determined by adding 10 ul of the supernatant to 1
ml of de ionized H20 and 1 ml of Coomassie stain reagent and reading the
sample at 595 nm on a Beckman DU-7 Spectrophotometer. A standard curve
was used to determine the protein content from the spectrophotometer
reading. An equal volume of 2X solubilizing buffer (7) containing DL¬
Dithiothreitol was added to the supernatant and boiled for 3 minutes to
solubilize the protein. Samples were frozen at -20°C.
SDS-PAGE: SDS-PAGE (10 % polyacrylamide) was carried out by Laemmli's
method (5) to separate proteins according to size in reducing conditions.
WESTERN BLOTTING: Western blots were prepared by Burnette's standard
techniques (1). Two different transfer protocols were used, Hybond-ECL and
Immobilon-P Transfer PVDF. The primary antibodies used included rabbit
anti-60kD Heat Shock Protein (SPA-804), mouse anti-72kD HSP (SPA-801),
mouse anti-72/73KD HSP (SPA-802), and mouse anti-90KD HSP (SPA-803),
which were stored at -70°C and were provided by StressGen Biotechnologies
Corporation in Victoria BC, Canada (product numbers in parentheses) and
mouse anti-HSP 70 monoclonal antibody 3A3 that was stored at -20°C and was
provided by Sean Murphy of Northwestern University. Dilutions were as
follows: 1:1000 for anti-HSP 60, 1:10,000 for anti-HSP 70 3A3, 1:500 for anti
D 70
HSr 72, 1:500 for anti-HSP 72/73, 1:300 for anti-HSP 90. Secondary antibodies
included anti-mouse IgG peroxidase conjugate (Product No. A-4416) and anti¬
rabbit IgG peroxidase conjugate (Prod. No. A-4914) provided by Sigma
Immuno Chemicals in St. Louis, MO and stored at -20°C. Both secondary
antibodies were used at a 1:10,000 dilution. All antibodies were diluted in a
total volume of 2.5 ml. Visualization of antibody binding was done using
the Amersham Life Science ECL Western Blotting detection reagents
RPM2109 and Kodak X-ÖMAT Scientific Imaging Film. Film was developed
in a Konica X-ray film processor QX-60A.
QUANTITATIVE ANALYSIS: Protein bands on western blots were analyzed
using Jandel Scientific JAVA Version 3 Video Analysis Software. Eight
intensity readings were taken of background and four intensity readings were
taken of each band of interest. Background readings were averaged and
subtracted from band intensity averages to produce relative intensities of the
heat shock protein bands. It is assumed that there is a direct correlation
between the intensity of the protein band and the amount of protein that was
present in the specimen at the time of homogenization.
RESULTS
INITIAL SCREENING FOR HEAT SHOCK PROTEINS: Before the heat shock and
salinity shock experiments were performed, two untreated specimens were
homogenized in order to determine which heat shock proteins are present in
the non-stressed organism. Table 1 (4/8 - 4/20) lists the results of this initial
screening. There are at least two, and possibly three heat shock proteins that
are recognized by the anti-HSP 60 antibody. The molecular weights of the two
more prevalent HSP 60s are about 58 kD and 48 kD based upon the pre-stained
molecular weight marker. The third HSP 60 protein, which appears fainter
than the other two, appears at a molecular weight of about 55 kD. Figure 1
shows a western blot of untreated P. squamatus protein probed with anti-HSP
60. The lane on the left contains twice as much protein as the lane on the
right. The 58 kD and the 48 kD proteins are visible in both lanes. Small
quantities of the 55 kD protein can be seen in the lane on the right.
Screening for HSP 70 proteins was done with three different primary
antibodies, each of which has a small difference in binding affinity. Results
show that there were no HSP 70 proteins present in the untreated organism.
Screening with anti-HSP 90 showed that HSP 90 proteins were absent as well
(see Table 1).
P. SQUAMATUS PRODUCES AT LEAST FIVE HSP 60 PROTEINS: There appears
to be five proteins that vary widely in molecular weight and cross-react with
the anti-HSP 60 antibody. These five proteins can be seen in Figure 2. The
lane on the left shows the three proteins that cross-react with the anti-HSP 60
antibody seen in Figure 1. The second lane contains the proteins of a control
P. squamatus in a heat shock experiment that is producing an HSP 60 protein
with a molecular weight of about 116 kD in addition to the three HSP 60
proteins seen in the left lane. The lane on the far right shows the proteins of
a control P. squamatus in a salinity shock experiment that is producing a 40
kD protein that cross-reacts with the anti-HSP 60 antibody. Three of the
proteins that are recognized by anti-HSP 60 (116, 55, 40 KD) appear sporadically
throughout the experiments under different shock conditions and sometimes
not at all. However, the 58 kD and the 48 kD proteins appear in every protein
sample at different concentrations and intensities. The rest of this study
focuses on these two proteins and the relative intensities of these HSP 60
bands as they appear on the western blots.
EFFECT OF HEAT SHOCK ON THE 58 KD AND 48 KD PROTEINS RECOGNIZED BY
ANTI-HSP 60: Figure 3 demonstrates the induction of protein synthesis at
12°C and the shutdown of protein synthesis and possible protein degradation
at 19°C by western blot analysis of the HSP 60 proteins present in P. squamatus
under different temperature shock conditions. The first lane contains the
proteins of the control organism which was kept at 5°C during the heat shock.
The second lane did not run properly. It contained the proteins of the
organism shocked at 8°C. The third and fourth lanes contain the proteins of
organisms shocked at 12°C and 19°C respectively. Equal amounts of protein
were loaded into each of the lanes. Analysis of the darkness of each of the
bands illustrates trends in the relative amount of protein in each shock
condition compared to the control. Heat shock protein induction occurs at
milder heat shock conditions and protein degradation occurs in the more
drastic shocks. Figure 4 contains the quantitative analysis of the intensities of
the 58 kD and the 48 kD protein bands on the western blot in Figure 3 based
on data from the JAVA image processor. Specific values of the relative
intensities are given in Table 1 (Expt. 5/25/93) under the "HSP 60 (58 kD)" and
the "HSP 60 (48 kD)" columns. The numbers reported are the relative
intensity (intensity of band minus the intensity of the background) values per
ug of protein loaded per lane. In the higher molecular weight protein (58 kD),
there is a dramatic increase in the amount of protein at 12°C and a decrease at
19°C. Levels of the lower molecular weight protein (48 kD) stay relatively
constant in the organism between 5°C and 12°C but decrease at 19°C. It
appears that heat shock protein production is induced during heat shock to
aid the organism in dealing with the stress. However, there is a threshold
temperature beyond which protein synthesis is inhibited and the organism is
unable to manufacture any new proteins.
Figures 5, 6, and 7 show the JAVA image processing analysis of the
other three heat shock experiments that were run. Again, specific values can
be found in Table 1. Figure 5 shows an increase in the amount of both the 58
kD and the 48 kD proteins after a heat shock of 2 hours at 13°C. Figure 6
shows a decrease in the amount of both the 58 kD and the 48 kD proteins after
a heat shock of 2 hours at a super-ambient temperature of 19.6°C. Figure 7
shows a small increase in the amount of the 58 kD protein and a more
significant decrease in the amount of the 48 kD protein present after a shock
at a super-ambient temperature of 20.4C for two hours. The results of these
three experiments serve to support the trend seen in Figures 3 and 4. The
only inconsistency is in the increase of the 58 kD protein after a super-shock,
but the amount of increase is very small and may be an anomaly.
EFFECT OF SALINITY SHOCK ON THE 58 KD AND 48 KD PROTEINS RECOGNIZED BY
ANTI-HSP 60: Figure 8 shows the western blot analysis of the heat shock
proteins present in different conditions of salinity shock. P. squamatus are
thought to naturally reside in sea water at a salinity of 33.2 parts per thousand
(ppt). Salinities of 10 ppt above and below this value were tested to see if a
heat shock response was induced. In the figure, the first and third lanes
contain the proteins from the shocked animals. Lanes two and four contain
the instant ocean and the normal control respectively. The fifth lane contains
the HSP 60 molecular weight standard. Analysis of the darkness of each
protein band reveals that the levels of protein that cross-react with the anti-
HSP 60 antibody increase in both of the shock conditions relative to the
controls. This trend is observed in both the higher molecular weight (58 KD)
and the lower molecular weight (48 kD) proteins, although much more
dramatically in the 58 kD protein. Figure 9 shows this trend more clearly in a
quantitative analysis of the relative intensities of each protein band based on
the JAVA image processing data. The intensity reading directly correlates to
the amount of protein present. Another point of interest is that there does
not appear to be a significant difference in the amount of protein present
between the instant ocean control and the normal sea water control.
Therefore, it may be reasonable to assume that the instant ocean does not
have any profound effects on heat shock protein activity in the organism.
Figures 10 and 11 show the JAVA image processing analysis of the two
other salinity shock experiments performed. Again, the specific relative
intensity values of each protein band can be found in Table 1 under the "HSP
60 (58 kD)“ and the "HSP 60 (48 kD)" columns. Figure 10 reports a decrease in
the amount of the 58 kD protein in the shock condition relative to the control
(33.2 ppt) and a steady amount in the 48 kD protein. Figure 11 shows the
control organism (33.2 ppt) to have a dramatically higher amount of both the
58 kD and the 48 kD proteins. However, this result may not be significant
because the water that was removed after the experiment appeared to be
contaminated with some sort of milky substance. This contamination may
have accounted for the increased levels of heat shock proteins in that
particular animal. If the 33.2 ppt control is disregarded, and the shock
conditions are compared to the normal sea water control, it appears as if the
salinity change induces a small increase in the levels of the heat shock
proteins. However, the change is not dramatic enough to be able to draw any
conclusions. This and the contradictory data seen in Figure 10 make it
difficult to predict any conclusive trends.
DISCUSSION
The initial screening to determine which heat shock proteins are
present in Psolus squamatus revealed that there are five proteins that cross¬
react with the anti-HSP 60 antibody. These five proteins are 116 kD, 58 KD, 55
KD, 48 kD, and 40 kD in size and span a molecular weight range of 76 kD. A
heat shock protein family is generally characterized by sequence homology
among its members and specific binding of its members to the same antibody.
The anti-HSP 60 antibody used to probe the blots was polyclonal. This may
have decreased the specificity of the cross-reaction between the antibody and
the proteins on the blots. Of particular interest is the 116 kD protein found in
P. squamatus that cross-reacts to the anti-HSP 60 antibody. This protein
occurs greatly out of the size range of the other anti-HSP 60 binding proteins
and has not been documented before. This binding may be a result of the
polyclonal nature of the primary antibody used and may not necessarily mean
that the protein belongs to the HSP 60 protein family. Nevertheless, the fact
that there are a total of five proteins that cross-react with the anti-HSP 60
antibody may give some indication as to why Psolus squamatus has been so
successful at surviving in such a wide variety of environments. The sum of
these proteins may better prepare the organism to survive environmental
changes and stresses, accounting for its abundance and success in the
Monterey Canyon.
In addition to the constitutive HSP 60 proteins that aid the organism in
its survival, this study has also documented a marked increase in the levels
of these same proteins under stress conditions. The heat shock experiments
showed that HSP 60 protein production is amplified under a temperature
shock of about 13°C. However, at super-shock conditions of about 20°C, the
amount of protein present in the animal drops significantly. It appears as if
the organism can respond to the heat shock with the HSP induction response
to a certain threshold temperature. Beyond this point, the organism may be
impaired in its ability to synthesize new proteins. Furthermore, the
constitutive heat shock proteins may also be breaking down at these super-
ambient temperatures. Again, protein degradation was seen at temperatures
of about 20°C and P. squamatus have been seen to survive in temperatures of
up to 22°C. There appears to be a direct correlation between the decrease in
heat shock protein activity and the deaths of the animals, as they occur within
the same general temperature range.
The results of the salinity shock experiments were not as clear cut.
Much more work can be done to continue this project. The salinity and heat
shock experiments can be rerun in gradients with smaller salinity and
temperature increments in order to better predict the trends occurring. Time
course experiments could be run in order to determine at which specific
points of the shock protein activity is occurring. The five different proteins
that cross-reacted with the anti-HSP 60 antibody could be isolated, sequenced
and studied in order to determine whether or not each of these proteins are
really HSP 60 proteins. This would be especially interesting to do to the 116
kD protein because it seems to be a previously unidentified heat shock
protein. The proteins could also be separated on two-dimensional gels on the
basis of both size and charge in order to get better resolution of the proteins.
The induction of HSP 60 proteins could be studied at the level of gene
expression by isolating and quantifying RNA during different stages of the
shock. All of these experiments would be useful in better characterizing the
heat shock response in this organism. Learning more about the heat shock
proteins and the biochemical activity occurring in Psolus squamatus may give
important insights into its heartiness, its successful survival in many
different environments, and the reasons for its dominant presence in the
benthic seas.
ACKNOWLEDGMENTS
First and foremost, I would like to thank the Levine lab. It got a little crazy at
times, but it was always fun. Thank you to my advisor, Dr. Paul Levine, for
his advice, support, and encouragement throughout my project. Thank you
to Julie Golden for her patience and kindness and to Jan Van Kampen for
letting us invade his lab space. Thanks to my lab mates Theresa, Dan, and
Greg for helping me out in lab, for our Big Mac lunches, our pizza and
Chinese food dinners, and for making this quarter a blast. Thank you to
Chuck Baxter for all of his wise advice and for sharing his enthusiasm.
Thanks to all of Hopkins Marine Station and the spring quarter class. It was
great getting to know everyone. I had a great time and learned a lot. Thanks
for making this the greatest quarter ever!!
LITERATURE CITED
1. Burnette, W.N. 1981. "Western blotting": electrophoretic transfer of
proteins from sodium dodecyl sulfate-polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with antibody
and radioiodinated protein A. Anal. Biochem. 112 : 195-203
2. Carlsten, Christopher. 1991. Respiration in Psolus squamatus.
Unpublished manuscript available at Hopkins Marine Station in
Pacific Grove, CA.
3. Harlow, Ed and David Lane (eds.). 1988. Antibodies, a Laboratory Manual.
Cold Spring Harbor Laboratory. Cold Spring Harbor Laboratory Press.
Cold Spring Harbor, New York.
4. Kaufmann, S.H.E. (ed.). 1991. Heat Shock Proteins and Immune Response.
Springer-Verlag. Berlin Heidelberg.
5. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature (London) 227 : 680-685.
6. Lindquist, Susan. 1986. The Heat-Shock Response. Ann. Rev. Biochem.
55: 1151-1185.
7. Lowry, O. H., N.J. Rosenbrough, A.L. Farr, and R.J. Randall. 1951. Protein
determination with the Folin phenol reagents. J. Biol. Chem.
193: 265-275.
8. Morimoto, Richard, Alfred Tissieres, and Costa Georgopoulos (eds). 1990.
Stress Proteins in Biology and Medicine. Cold Spring Harbor
Laboratory Press. Cold Spring Harbor, New York.
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FIGURE LEGEND
FIGURE 1: Western Blot: HSP 60 Proteins Present in Initial Screening of P.
squamatus: The two lanes contain protein from an unshocked P. squamatus.
The left lane contains twice as much protein as the right lane. The blot was
probed with anti-HSP 60 antibody.
FIGURE 2: Western Blot: Five Proteins Cross-React with Anti-HSP 60: The
three lanes contain protein from an unshocked specimen (same as in Figure
1), protein from a control organism in a heat shock experiment, and protein
from a control organism in a salinity shock experiment respectively. The
blots were probed with anti-HSP 60 antibody. The five different proteins that
cross-react with the antibody are marked.
FIGURE 3: Western Blot: HSP 60 Proteins Due to Heat Shock Conditions: The
first three lanes contain protein from organisms shocked for two hours at 5°C
(control), 12°C, and 19°C. The last lane contains the HSP 60 standard. The
blot was probed with anti-HSP 60 antibody.
FIGURE 4: Relative Intensities of HSP 60 Protein Bands Under Different
Heat Shock Conditions (5/25/93)
FIGURE 5: Relative Intensities of HSP 60 Protein Bands Under Different
Heat Shock Conditions (5/20/93)
FIGURE 6: Relative Intensities of HSP 60 Protein Bands Under Different
Heat Shock Conditions (5/18/93)
FIGURE 7: Relative Intensities of HSP 60 Protein Bands Under Different
Heat Shock Conditions (5/13/93)
FIGURE 8: Western Blot: HSP 60 Proteins Due to Salinity Shock Conditions:
The first four lanes contain protein from organisms shocked at 23.2 g/L, 33.2
g/L (instant ocean control), 43.2 g/L, and in normal sea water (normal
control). The HSP 60 standard is in the fifth lane. The blot was probed with
anti-HSP 60 antibody.
FIGURE 9: Relative Intensities of HSP 60 Protein Bands Under Different
Salinity Shock Conditions (5/11/93)
FIGURE 10: Relative Intensities of HSP 60 Protein Bands Under Different
Salinity Shock Conditions (5/13/93)
FIGURE 11: Relative Intensities of HSP 60 Protein Bands Under Different
Salinity Shock Conditions (5/18/93)
HSP 60 Proteins
present in Psolus
116-
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Figure 1
Five Proteins Cross-react
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HSP 60 Proteins due to
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Figure 3
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Heat Shock
5/25/93


HSP 48
HSP 58
Heat Shock Proteins
Figure 4
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Heat Shock
5/20/93


HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 5
5.0 C
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Heat Shock
5/18/93
HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 6
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2
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Heat Shock
5/13/93


HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 7
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E 20.4 C
HSP 60 Proteins due to
Salinity Shock

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Salinity Shock
5/11/93


HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 9
23.2 ppt
33.2 ppt
43.2 ppt
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15-
0 +
Salinity Shock
5/13/93


HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 10
23.2 ppt
E 33.2 ppt
0 +
Salinity Shock
5/18/93


HSP 60 (58 kD)
HSP 60 (48 kD)
Heat Shock Proteins
Figure 11
23.2 ppt
E 33.2 ppt
43.2 ppt
Normal SW*