biochem problem set

  1. (10 points) From a kinetics experiment, kcat was determined to be 95sec-1. For the kinetic assay, 0.3mL of a 0.25mg/mL solution of enzyme was used, and the enzyme has a molecular weight of575,000 g/mole. Assume a reaction volume of 3mL. Calculate Vmax (µM∙min-1) for the enzyme and catalytic efficiency ( in M-1sec-1) for the enzyme. The Km for the enzyme was determined to be 1.5 x 10-2M.
  1. (8 points) Consider the following peptide: Lys-Tyr-Glu-His-Cys-Glu-Ala-Cys-Arg-Asp-His-Arg
  • What is the net charge of this peptide at pH = 1 (you must show work for full credit!)?
  • What is the net charge of this peptide at pH = 14 (you must show work for full credit!)?


  1. Some characteristics of three proteins are listed in the table below:
Protein Molecular Weight (Da) Isoelectric point (pI) Does the Protein Contain a heme moiety?
1 65,000 5.0 No
2 54,000 9.8 No
3 15,500 4.8 Yes

a.(1 point) What type of chromatography separates proteins based their charge?


b. (1 point) What type of chromatography separates proteins based on their size?

c.(6 points) Could gel filtration chromatography be used to separate a mixture containing Protein 1 and 3? Clearly explain why or why not. If gel filtration chromatography can be used to separate Protein 1 from Protein 3, which protein would elute first (clearly explain why)? After collecting the fractions from the column, the absorbance of each fraction will be measured using a spectrophotometer. Can both proteins (Protein 1 and Protein 3) be monitored at 280nm and 400nm (clearly explain)?

d. (5 points) Which 2 proteins listed in the table above could be separated by ion exchange chromatography but NOT by gel filtration chromatography (clearly explain why)?

e.(5 points) Which 2 proteins listed in the table above could be separated by gel filtration chromatography but NOT by ion exchange chromatography (clearly explain why)?

4. The ionization of p-nitrophenol is shown below (pKa = 7.0):

a.(4 points) Identify the weak acid and conjugate base.

b.(4 points)At pH 7, what are the relative concentrations of ionized and un-ionized p-nitrophenol?

c.(4 points)If enough concentrated hydrochloric acid is added to a solution of p-nitrophenol to lower the pH from 7 to 5, what will happen to the relative concentrations of the ionized and un-ionized forms?

d.(4 points) Ionized p-nitrophenol has a yellow color, while the un-ionized form is colorless. The yellow color can be measured using a spectrophotometer at 400nm. In order to determine the total amount of p-nitrophenol in a solution, would you perform the spectrophotometer reading at an acidic or basic pH? Clearly explain why?

  • (8 points)A solution of p-nitrophenol at pH 8.05 was found to have an A400 of 0.375 . What is the total concentration (in µM) of p-nitrophenol (ionized plus un-ionized) in the solution? The molar extinction coefficient of p-nitrophenol is 17,500 M-1cm-1 and the pKa is 7.
  • (18 points) Use the data in the table below to plot a standard curve (this must be submitted with the problem set) and determine the concentration of the unknowns if possible (use graph paper from lab manual, DO NOT PLOT USING EXCEL- if you plot using Excel, 10pts will be deducted!). If it is not possible to determine the concentration of the unknown(s), state why and what you would need to do (explain in detail) in order to determine the concentration of the unknown(s). If the concentration of the unknown(s) can be determined, clearly illustrate this on the graph and clearly explain how you determined the concentration of the unknown(s).


610 nm

Standard 1 1 mg/ml 0.064
Standard 2 2.5 mg/ml 0.154
Standard 3 5 mg/ml 0.304
Standard 4 7.5 mg/ml 0.449
Standard 5 10 mg/ml 0.613
Unknown A 0.870
Unknown B 0.375

6. (10 points) A protein was purified and then analyzed on a size exclusion column. Its MW was determined to be 90,000 Dalton. The SDS-PAGE gel shows two bands, corresponding to molecular weights of 10,000 and 35,000 Dalton. Describe the native conformation of this protein in terms of the number of subunits present and their molecular weight.

(12 points) You have a 250mM stock of glucose, a 25mM stock of asparagine, and a 0.050M stock of NaH2PO4, and magnesium chloride powder (solid form) , MgCl2 (Formula Weight; 95.21g/mole). How would you prepare (be specific) 150mL of a solution that contains 0.05M glucose, 5mM asparagine, 5mM NaH2PO4 and 150mM MgCl2?v

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