Transcript Raheel Anwar Morel Lab CO2 Concentrations and Why They’re Important  The changing environment has led to higher dissolved cabron dioxide concentrations in the.

Raheel Anwar
Morel Lab
CO2 Concentrations and Why
They’re Important
 The changing environment has led to higher dissolved
cabron dioxide concentrations in the world’s oceans
due to global warming and the greenhouse gas effect
 Higher CO2 concentrations,which can easily be
converted to bicarobonate, cause changes in the pH or
acidity of the world’s oceans
http://switchboard.nrdc.org/blogs/lsuatoni/why_scie
ntists_agree_ocean_aci.html
The CDCA Enzyme
 Cadmium carbonic anhydrase
 Catalyzes the conversion of CO2 into bicarbonate and
viceversa
 Cadmium is a normally toxic metal, but here, it can act
as a coenzyme for the reaction
Why TW and CDCA?
 Knowing where and in what quanitities this enzyme is
located in the algae is important
 Tells us how this species and other algae like it can cope
with the changing conditions of the oceans
 Allow us to anticipate the effects of future changes
Overview of Work
 Studied the expression of the CDCA enzyme in TW
under different pH conditions
 Isolated chloroplasts to determine CDCA activity and
localization
CDCA Analysis
 Looked at TW cells growing under:
 High (8.6)
 Medium (8.2) and
 Low (7.8) pH conditions.
Western Blot
CDCA Expression under Different pH Conditions
Low
Medium
High
0
2000
4000
6000
8000
10000
12000
Intensity of CDCA band in units
14000
16000
18000
20000
Isolation of Chloroplasts
 Developed a procedure for the isolation of chloroplasts
 First, sonication was used to break open cells without
entirely destroying all the chloroplasts
 Cells are resuspended in a buffer designed to protect the
chloroplasts that are now floating free in the solution
 Solution is filtered on a 10 micron filter, allowing TW cells,
most of which are 10 micron or bigger to be caught on the
filter, while chloroplasts, which necessarily are smaller, pass
through.
 This reduces whole cell contaminants remarkably
 Chloroplasts are then isolated on a gradient for further
analysis
MIMS Assay of CDCA activity for
Chloroplast Extract
 Results showed that CDCA activity was not present in
the chloroplasts
 Reasons:




CDCA enzyme could be located outside of the chloroplasts
Whole cell activity is ruled out since the pellet of chloroplasts
is washed many times
Chloroplasts could get inactivated, but fluroescence
microscopy shows only slight decrease in output
Signals from the nucleus may be necessary for CA activity
Conclusions
 Overall, higher concentrations of CDCA are found
when the pH is higher, giving us clues on how algae in
the oceans are effected
 Isolation of chloroplasts showed no signs of CDCA
activity
Thanks to
 PEI/Grand Challenges
 Dr. Yan Xu
 Professor Morel