1. (30 points) A student working in Dr. Selbys research lab orders a new lab buffer system from Sigma chemical company. The chemical formula is proprietary and the manufacturer refuses to release the complete chemical formula. From the companys technical data sheet, the following information was provided, the buffer system is a triprotic buffer system with each proton being attached to a unique functional group that equilibrates as shown in the reaction below Sigma also includes that A represents the polyatomic chemical species that Sigma chemical refuses to release;
H3A + H2O H2A- + H3O+ HA-2 + H3O+ A-3 + H3O+
Its known that the A portion of the buffer has a molecular weight of 135.25 g/mol. The four components of the buffer system are packaged separately as dry powders with one in the fully protonated form and the others in the three sodium salt forms (H3A, NaH2A, Na2HA, and Na3A) where the sodium (atomic weight 22.99 g/mol) dissociates as a spectator ion when the buffer is prepared and does not participate in the acid-base equilibrium.
The student finds there are no pKa values on the packaging and gets upset. She cant Google the pKa values since she doesnt know the exact chemical formula, so she takes 182.23 grams of Na2HA and 64.58 grams of Na3A and measures the pH with her pH meter. She finds the pH is 8.5 after these components have mixed and equilibrated. This pH is too highugh!
She then takes 36.36 grams of Na2HA and 160.24 grams of NaH2A and finds the pH is 6.3. This pH is too lowugh!
She needs to prepare a buffer with a pH of 7.5 to show Dr. Selby her assay results in the morning. As she hurries around the lab due to this taking so long, she breaks the pH meter and cannot use it for the final buffer preparationugh!
She is left with only the values she has found from her previous measurements and her biochemistry class experience to guide her. She takes a deep breath, focuses, and asks herself;
How much, and which, dry compound(s) should I use to make 250 mL of my buffer at pH 7.5 that is 0.225 M?
Give her some assistance;
a. Show all relevant chemical equations involving the equilibrium expressions (5-points),
b. mathematical equations for your choice of which preliminary values to use (10-points), and
c. steps to find your final solution (15 points).
***Use the following page(s) for your answers. Add more pages as needed to the final document you submit.
2. (40 points) A student doing research at St. Petersburg College Cancer Center is working on a protein purification and kinetic assay for an enzyme found to be upregulated in patients with type-2 diabetes who have lung cancer. The protein is being prepared in a stabilized human cell line and the student has a significant amount of human cancer cells to prepare the enzyme.
When the enzyme is first isolated, the student uses gel filtration (GF, or size exclusion) chromatography and finds the molecular weight of the enzyme is 120,000 based on GF column standards. This form of the enzyme has 1,000 units of activity per mg of isolated protein. This result was repeated and verified.
The enzyme is purified a second time, but the student working with the GF column prior to the current purification has equilibrated the gel filtration column with a buffer containing a high concentration of urea. This was unknown at the time the enzyme was being purified, but was determined a few days later. This preparation showed a molecular weight of 40,000 based on GF column standards and had 2,000 units of activity per mg of protein. This result was repeated and verified.
The student was very confused and began considering changing her major. She then ran a SDS-PAGE gel with beta-mercaptoethanol added to both the sample that was found to be 120,000 MW on the GF column and the sample where urea was added where the molecular weight appeared to be 40,000 MW. These samples were run on the SDS-PAGE gel in separate lanes for analysis.
After staining her SDS-PAGE gel, she found the molecular weight of both her protein samples was 20,000 MW on the gel based on SDS-PAGE molecular weight standards. The student was even more confused and began perusing the course catalog for career options. She calls you to get career advice and explains her observations. You inform her you took biochemistry at St. Petersburg College and that the world needs devoted scientists right now more than ever–and she should not change her major. You tell her you can explain exactly whats occurring, and you do;
a. (10 points) How do you explain the different apparent molecular weights using the different methods based on your knowledge of protein structure? You must include both GF column purifications and the SDS-gel in your explanation.
b. (10 points) Is there a molecular difference in the enzyme activity for the 120,000 MW form and the 40,000 MW form of the enzyme? Which is more active and by how much?
c. (5 points) Is this an allosteric enzyme? What properties support it being allosteric from the data collected thus far? What experiments should be done to prove that it is? (2-3 sentences)
d. (5 points) Are there any likely post-translational modifications? What is the evidence for (or against) this?
e. (5 points) Show the structure(s) of the amino acids that might be modified based on the data collected (both prior and after modificationtwo total structures).
f. (5 points) What is the role of these post-translational modifications on the structure and/or function of the enzyme (one sentence)?
***Use the following page(s) for your answers. Add more pages as needed to the final document you submit.
3. (15-points) A student at St. Petersburg College isolates an enzyme with trypsin-like specificity from an organism that thrives in a volcanic deep sea vent where temperatures are typically above 100 Celsius. The student working with this enzyme finds there is no activity below 37 Celsius, only moderate activity around 50 Celsius, and optimal activity at 90 Celsius. He discusses these results with three other students who are attending the University of Florida, Florida State, and UCF and they exchange ideas on why this might be occurring. Student-A believes this is due to a completely different set of amino acids that exist in deep sea creatures which have special functional groups that only work at elevated temperatures. Student-B states the amino acids are the same as everywhere else on planet earth, but the temperature-activity observations are due to the pKa values that are only relevant at elevated temperatures and Student-C states this is due to the amount of Gibbs free energy that this organism needs for its chemical transitions due to its environment. As you listen to these ideas, you develop your own concepts related to the discussions in our biochemistry course, and decide which of these ideas are likely true are and which are not likely true;
a. Are Student-As ideas supported by any known biochemical information? Give 2-3 sentences why or why not these ideas are reasonable. (5 points)
b. Are Student-Bs ideas supported by any known biochemical information? Give 2-3 sentences why or why not these ideas are reasonable. (5 points)
c. Are Student-Cs ideas supported by any known biochemical information? Give 2-3 sentences why or why not these ideas are reasonable. (5 points)
***Use the remainder of this page and the following page for your answers
4. (25 points) The same enzyme from the question above is characterized using kinetic assays based on its specificity to develop a competitive inhibitor. Based on the specificity of trypsin substrates;
a. (5 points) Provide the complete chemical structure of an affinity-labeling molecule that might find use to determine which amino acids are near the active site.
b. (5- points) Provide the complete chemical structure of a chromogenic substrate molecule that might be used to determine the kinetics values of kcat and KM.
c. (5-points) Provide the complete chemical structure of a substrate analog that might find use as a reversible competitive inhibitor.
d. (5- points) Sketch the reaction scheme (E + S going to ES, and so on) for this inhibition and provide a double-reciprocal plot of the enzyme with substrate alone and with inhibitor added at three (3) increasing inhibitor concentrations.
e. (5- points) How do we determine if the inhibition is purely competitive? If the inhibitor works, but is not competitive, what might we observe on the graphs?
***Use the remainder of this page and the following page(s) for your answers. Add more pages as needed to the final document you submit.
