BIOL/CHEM 3361 Biochemistry I

(and BIOL 6352)

Fall 2015

Due: Mon., Sept. 14 at 5:00 pm in FO 3.617

(No late Problem Sets will be accepted.)
(To void last minute difficulties, you may turn
them in early -- at lecture or at FO 3.617 during
departmental office hours.)
PROBLEM SET 1

There are 30 lettered subparts to the questions below. Each will be graded on a 5 point scale, and then
the summed score will be converted to a percentage of the total points.
For full credit, all steps to the solutions of the following problems must be shown. You may work
together on the problems, but you may not copy or plagiarize. Your answers must be written, must
show your own math steps, and must be in your own words.
Round your answers as appropriate, but in no case to no more than three significant figures.
For problems 1 and 2, assume an activity coefficient of 1 for all substances and no effect of ionic
strength. Eliminate terms in quadratic solutions for [H+] only if the weak acid is dissociated < 5%.
Reported pKa values can vary depending on the conditions under which they were measured; therefore,
in solving the following problems use the pKa values given with the problems.
1. a. What mM concentration of HCl will have a pH of 2.0?
b. Acetic acid has one carboxylic group. What is the pH of 10 mM acetic acid?
c. Citric acid has three carboxylic groups. What is the pH of 10 mM citric acid?

(For citric acid, pKa1 = 3.13, pKa2 = 4.76, pKa3 = 6.40)

d. What mM concentration of citric acid will have a pH of 2?
e. If 3 volumes of 8 mM NaOH are mixed with 1 volume of 20 mM citric acid, what will be the pH
of the mixture?
2. a. If 3.0 moles of ammonia (pKa = 9.2) are generated in a 1.0 ml enzymatic reaction buffered by
50 mM Na-citrate, pH 7.0, what will be the final pH of the reaction mixture?
(For citric acid pKas, use the values given in problem 1c.)
b. A drop in the pH of blood by a few tenths of a unit can be life threatening. This acidosis can be
the result of either respiratory or metabolic disorders. Assume a patient is suffering from acute
loss of HCO3- ions in the blood plasma due to the inability of diseased kidneys to excrete enough
acid. The patients blood is pH 7.0. Normally blood pH is 7.4, the [HCO3- ] is 24 mM, and
[CO2(d)] is 1.2 mM. Assume a constant [CO2(d)] level in answering the following questions:
i) What is the percent increase in blood [H+] in the patient versus normal?
ii) What percent of the HCO3- ion has been lost compared to normal blood?
iii) Assuming 5.5 liters of blood in the body, what amount in grams of bicarbonate must be
given to the patient intravenously to raise blood pH to normal 7.4?

3. Triose phosphate isomerase catalyzes a middle step in glycolysis (aka Embden-Meyerhof pathway)
to interconvert the two halves of glucose after it is split:
dihydroxyacetone-phosphate2- (DHAP) glyceraldehyde-3-phosphate2- (G3P)
Only glyceraldehyde-3-phosphate can enter the next step of the pathway.
G at 25C for this reaction is +7.56 kJ/mol. Assume 25C in answering the following questions:
a. Does the G favor glyceraldehyde-3-phosphate2- formation? Explain.
b. What is Keq for this reaction?
c. What ratios of [glyceraldehyde-3-phosphate2-]/[dihydroxyacetone-phosphate2-] would favor the
forward reaction?
d. In vivo concentrations in human erythrocytes are 0.14 mM dihydroxyacetone-phosphate2- and
0.019 mM glyceraldehyde-3-phosphate2-. Do these concentrations drive the isomerization in a
forward direction? Explain. If the answer is no, how can glycolysis proceed?
e. Use Excel to plot G as a function of the [G3P]/[DHAP] ratio, changing the ratio in 10-fold
increments over the range Keq x 10-3 to Keq x 103. Starting at equilibrium, how much does G
change for each 10-fold decrease in the ratio [G3P]/[DHAP]? Again starting at equilibrium, how
much does it change for a 10-fold increase in the ratio?
4. Assume you are characterizing an enzyme that catalyzes the following reaction,
AB+C
By measuring equilibrium concentrations, you determine that Keq values for the reaction are
346 M, 378 M and 417 M at 25C, 30C, and 37C, respectively.
a. Using Excel or similar program, tabulate the data, make a vant Hoff plot with trendline and its
equation, and use it to determine H and S for the reaction.
b. What is G for this reaction at 25 C?
c. Is this reaction driven by entropy, enthalpy, or both? Explain.
d. I s the answer to part c in agreement with the type of chemical reaction you are studying?
5. The pentapeptide Lys-Gln-Met-Arg-Cys has N-terminal and C-terminal pKas of 9.2 and 4.7,
respectively. The side chain pKas are Arg 12.2, Cys 8.2, and Lys 10.5.
a. Write this peptide sequence using single letter abbreviations.
b. Draw its predominant chemical structure at pH 6.0, including full and partial charges. Assume
any group that is 99% charged to be fully charged and any that are 1% charged to be
uncharged.
c. What fraction of the peptide will exist in the predominant form at pH 6.0?
d. Calculate the pI of the peptide.
e. If the N-terminus of this pentapeptide is acetylated and the side chain of the lysine residue is
methylated, what will be the approximate pI of the product? Why cant you calculate an exact
pI?
f. To separate a mixture of the methylated and unmethylated peptide by ion exchange
chromatography at pH 8.2, would you choose a CM- or DEAE-matrix? Explain why.
g. Which peptide would elute first? Explain why.
6. Assume you are given a mixture of native proteins that you analyze by standard 2-D
electrophoresis, with the following results for isoelectric points and apparent molecular weights:
protein A (Mr 35,410; pI 7.25), protein B (Mr 45,650; pI 6.21) , and protein C (Mr 103,246; pI
8.26).

a. In the isoelectric focusing step, in what order will the proteins band, starting with the anode and
going to the cathode? Explain why.
b. Which protein would be considered the most acidic? Explain why.
c. If the mixture is subjected to salting in with KCl at pH 6.5, in what order would you expect the
proteins to be solubilized? Explain why.
d. If the mixture is subjected to phospho-matrix (P, pKa1 = 3, pka2 = 6) chromatography, what pH
range and optimal pH should you use for the buffer, and in what order would you expect the
proteins to emerge from the column? Explain why.
e. After gel filtration of the native protein mixture on Sephadex G-200, only two peaks with
absorbance at 280 nm were observed. In comparison to a set of protein Mr standards, these
peaks corresponded to apparent molecular masses of 162 kD and 103 kD. What can you deduce
from these results? Explain why.