Poster Text.docx

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.