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Ryan Cao
Intel ISEF
Research Plan
accurately determine whether the salt concentration of a solution affects the partial spe-
cific volume and/or frictional ratio of the protein solute. Large, complex biological macro-
molecules have many amino acid side chains and residuals on the outer, interacting
portion of the molecule. These side chains often are locally charged, and thus attract
polar water molecules in solution, creating an envelope of water that changes the ap-
parent shape, volume, and mass (and therefore density) of the protein as it is recorded
sedimenting. Salt ions added into solution will be more strongly attracted to these local
charges and cancel them, causing the envelope of water to disperse and revealing the
true form of the protein in solution. Analyzing this type of species using an analytical ul-
tracentrifuge, highly accurate results for sedimentation and diffusion coefficients can be
determined and thus highly accurate frictional ratio and partial specific volumes can be
terms of applicability, this experiment actually serves a triple purpose. Firstly, it provides
modeling values. Secondly, it tests and proves the technique of using analytical ultra-
measured such as frictional coefficient and partial specific volume, and lastly, it proves
Cao 2
that for multiple well studied proteins, the addition of salt into solution does make a sig-
nificant, consistent impact on the partial specific volumes and frictional coefficients of all
the proteins.
The tested hypothesis for this project was that if proteins hemoglobin, cyto-
placed into TRIS and sodium phosphate solutions with various salt concentrations rang-
ing from 5mM to 155mM, then the partial specific volume will decrease with increasing
salt concentration and the frictional ratio will increase, because as the water envelope,
initially forming a smooth, low-density cover around the protein (artificially increasing
partial specific volume by making the protein overall much larger and less dense and
smoother, artificially decreasing frictional ratio) is removed, the true form of the protein,
which will be less smooth and more dense than its water-coated fourme, will give back
readings showing a higher density (lower partial specific volume) and more resistance
feel it is redundant to re-state that we’re trying to find the f/f0 and v-bar of proteins. Also,
I don’t believe that the engineering goal(s) portion is applicable for my research. If it is,
please tell me and I’ll try and fix this. Thanks! Additionally, I believe that the expected
outcomes are addressed sufficiently in the hypothesis, along with the reasoning. If the
ISEF people really really like redundancy though, I guess I can just say it again…)
Procedures-
45 mg of dry, powdered carbonic anhydrase was measured out and mixed with
200 mg of dry, powdered hemoglobin was measured out and mixed with 4mL of
200 mg of dry, powdered cytochrome c was measured out and mixed with 4mL
200 mg of dry, powdered lysozyme was measured out and mixed with 4mL of
200 mg of dry, powdered chymotrypsinogen A was measured out and mixed with
60 mg of dry, powdered ribonuclease A was measured out and mixed with 1.2mL
12.5 mg of dry, powdered carboxypeptidase was measured out and mixed with
Twenty approx. 30cm long sets of porous dialysis tubing were cut, rinsed with
ddH2O, boiled in a solution of 50% ethanol for 10 minutes, rinsed again thoroughly with
ddH2O to prepare for dialysis, boiled again in ddH2O for another 10 minutes, rinsed one
last time with ddH2O, and stored in a 50% ethanol solution in the fridge (insert temp.
here)
The 1M stock TRIS buffer was made by mixing 60.55g of TRIS with 400mL
ddH2O and 100mL of 0.5M EDTA solution, then adding HCl to lower the pH to 8.0.
The 1M sodium phosphate buffer was made by mixing 305mL of 1M di-basic so-
lution with 195 mL of 1M mono-basic solution, then adding NaOH to raise the pH to 7.0.
5L of 20mM TRIS buffer solution (i.e. the one that the proteins would actually be
in) was made by mixing 100mL of the 1M TRIS solution with 4.9L of ddH2O, while 5L of
5mM NaPO4 solution was made by mixing 25mL of the 1M NaPO4 solution with 4.975L
of ddH2O.
The total volume of each 50mg/mL protein solution was halved, and that amount
The dialysis tubes were dropped into 4- and 6-liter Erlenmeyer flasks containing
4 liters of phosphate and TRIS buffer, respectively, and left to dialyze for 16 hours.
The buffer was then changed (i.e. another 4 liters of 20mM TRIS and 5mM EDTA
solution was made, then replaced the old buffer) and the proteins dialyzed for another
22 hours.
The proteins were then extracted from the dialysis tubing and placed into 15mL
300µL of 1mg/mL protein solution was made from each dialyzed 50mg/1mL solu-
tion.
These samples were run through the spectrophotometer and repeatedly diluted
These spectra curves were then transferred into the Ultrascan II global extinction
fit program and fit with 25 Gaussians to determine a molar extinction curve, and fitted to
the given extinction coefficient produced for that specific protein sequence by Ultrascan.
After using the molar extinction curve to determine the factor of dilution needed
to make a 0.9OD solution, 800µL of 0.9OD samples in all salt concentrations were
The AUC cells were cleaned, built, and loaded once to twice a day. (PPE was
After each run, samples had to be extracted and cell windows and centerpieces
After each run, the data was transferred to a local database (run by UTHSCSA)
fined by their sequence from the Protein Data Bank) and buffers (TRIS and sodium
phosphate). Each run (datum) was associated with a solution, rotor, centerpiece, and
instrument.
The data (runs) were then edited, specifying a meniscus position, data range,
and plateau range, removing spikes, and deleting baseline/light-scattering scans as well
as end scans all showing complete sedimentation. The data was then analyzed.
A meniscus fit was then run to determine the true meniscus position, as well as
An iterative 2DSA was performed to optimize fitting for the experimental data
curve.
A 100 iteration Monte-Carlo analysis was performed using a reduced 2DSA grid
with 100 different random noise plots being generated on top of the iterative fit, taking
the fits from the Monte-Carlo and averaging them to produce a Monte-Carlo fit for the
Finally, if the Monte-Carlo fits were close (root mean square deviation of 5x10^-2
sis) was performed using the sedimentation and diffusion coefficients measured from
the Monte Carlo analysis as well as a molecular weight derived from mass spectropho-
tometry.
The resulting PCSA values for frictional coefficient and partial specific volume
were then recorded and graphed, revealing an extremely consistent trend among three
Potential risks involved the centrifuge rotor being unbalanced and thus exploding,
as well as the very real danger of loading needles causing unwanted harm.
The plots of the v-bar and f/f0s show a consistent decrease in the v-bar and a
consistent increase in the frictional ratio, the exact opposite of what had been hypothe-
sized. Conclusions based on biophysics cannot be drawn yet about why the salt may
have affected the properties of the proteins in such a way, although the trend is ex-
Bibliography:
Brooks, Emre, and Borries Demeler. "Parallel Computational Techniques for the
Brooks, Emre, Borries Demeler, Suresh Marru, Marlon Pierce, Mattia Rocco, and
Brooks, Emre, Weiming Cao, and Borries Demeler. "A Two-dimensional Spec-