Methods:

 Take powdered proteins and dissolve in DDH2O

 Prepare TRIS and NaPO4 buffers and dialyze proteins in buffer
 Analyze dialyzed, diluted proteins using spectrophotometer
 Fit extinction curves into Ultrascan and calculate molar extinction coefficients
 Use extinction coefficients to prepare dilations and add varying concentrations of
NaCl
 Prepare titanium analytical ultracentrifuge (AUC) cells and run samples at 45000
RPM
 Upload and analyze data using Ultrascan fitting methods on supercomputing
clusters
 Graph finished values on Excel plot

Purpose:
The purpose of this experiment was, firstly, to validate using analytical
ultracentrifugation as a means of measuring the density and volume of a sedimenting
particle, and secondly, to determine the effects of electrolyte ions on the density and
volume of proteins in solution.

Materials Used:
Calibrated pipettes, Analytical Ultracentrifuge, UltraScan software and analysis
computer, Supercomputer, pH meter, 45mg powdered carbonic anhydrase, 270mg
powdered cytochrome c, 65mg powdered ribonuclease a, 250mg powdered lysozyme,
224mg powdered hemoglobin, 250mg powdered chymotrypsinogen a, 125µL 22mg/1mL
carboxypeptidase solution, milli-q water (double dialyzed), 5M NaCl solution, 60.55g
powdered TRIS, 34.4975g powdered NaH2PO4, 35.49g powdered Na2HPO4, 100mL
0.5M EDTA solution, Electronic pipette (various tips), Electronic scale, 8L fluid
containers, Erlenmeyer Flasks, Falcon test tubes, Eppendorf tubes, Spectrophotometer,
Sonicator, AUC cell parts, Vortex Genie, 1L beaker, 300µL spectrophotometer cuvettes,
pressurized purified nitrogen, lens paper, forceps, screwdriver, Ethyl Alcohol, Contrad
cleaning solution, Hot plate, Magnetic Stir Bars, Dialysis Tubing, Dialysis Clips, and
Sharpie fine-tipped pens.

Application: Recent biochemical developments in nanotechnology have led to the

creation of a new field, nanomedicine, in which nanoparticles carrying drugs are used to
treat patients. With the radius and mass of the nanoparticle alone determined by other
methods (e.g. electroscopy and mass spectrometry, respectively), the volume and mass
of cancer drug bound to the nanoparticle can be calculated using AUC. Because AUC is
able to determine the density and frictional coefficient of the nanomaterial-cancer drug
complex in solution, the volume of each individual complex can be obtained by either
using V = M/n*d or the Stokes-Einstein equation, f = 6 * pi * viscosity * r (using V = 4/3 *
pi * r^3 to find volume). The volume of drug can then be found by subtracting the
original nanoparticle volume from the volume of the complex, while the mass of drug
can be found by multiplying the density of the complex by its volume and then
subtracting the volume of the original nanoparticle.

Temporary Script
Good morning! How are you? Good; my name is Ryan Cao, and I did an experiment to
help determine the density of proteins in solution. To give you all a bit of background,
proteins are large, complex macromolecules that, even if overall are neutral, will have
functional groups resulting in local partial charges. So if you can imagine, these partial
charges will then cause dipole-dipole interactions and hydrogen bonding with the
surrounding water molecules, (explain those two, possibly?) resulting in a sort of water
“envelope” which is less dense and smooths out the external surface of the protein,
concealing the true shape of the protein. And so what we would really like to figure out
is, well, how does this shell of water affect the density of the protein in solution and
how does the said water affect the volume of the protein in solution. In order to
determine the effects of water on the protein, we said, hey! If we add salt ions to
neutralize the charges on the protein, that’ll get rid of the otherwise attached water!
Now to do this we used a novel technique in analyzing those properties, that is, the
density and volume. So we took these dried protein samples and dissolved them in
buffer solutions and purified them using diffusive dialysis. And so we spun them down in
the analytical ultracentrifuge (which I will call the AUC from now on; it’s quite a
mouthful), and we analyzed them. Now the analysis breaks down like this: (write this on
a whiteboard/paper/something) Essentially, in centrifugation, as the thing you are
centrifuging is being spun down, there are two main forces at play here: sedimentation
and diffusion. Sedimentation is the tendency of the molecule to move towards the
outside of the cell as a result of its inertia, whereas diffusion if the collective tendency of
all the molecules dissolved in solution to spread out evenly. Now as you can imagine,
these two clearly can’t work together, and so researchers have discovered separate
relationships for each one. S equals M times quantity 1 – v-bar rho over N f, while D
equals R T over N f. What do these mean? Well, because the AUC... (TBC)
So then as you can see, we are able to solve for v-bar using this equation and figure out
the density of the protein. Because v-bar is simply the inverse of density, we can take
1/v-bar and there’s your density for the sedimenting particle. Additionally, v-bar is
volume over mass, meaning that if we multiply by the molecular mass of the
sedimenting object, we can also obtain the volume of the individual object.