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Undergraduate Questions (50 Pts)

1. (4 pts) A chemist working in a pharmaceutical lab synthesized a new drug as a

racemic mixture. Why is it important that she separates the two enantiomers and test
each for its biological activity?

Biomolecules such as receptors for drugs are stereospecific, so each of the two enantiomers
of the drug may have very different effects on an organism. One may be beneficial, the other
toxic; or one enantiomer may be ineffective and its presence could reduce the efficacy of the
other enantiomer.

2. (6 pts) Phosphoric acid (H3PO4) has three dissociable protons, with the pKas
shown below. Which form of phosphoric acid predominates in a solution at pH 4?
Explain your answer.
Acid pKa
H3PO4 2.14

H2PO4 6.86

HPO42 12.4

At pH 4, the first dissociable proton (pKa = 2.14) has been titrated completely, and the second
(pKa = 6.86) has just started to be titrated. The dominant form at pH 4 is therefore H2PO4, the
form with one dissociated proton
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3. (4 pts) Mutations may arise during DNA replication when mispairing occurs as a
result of the transient formation of a rare tautomeric from of a base, such as the imino
tautomer of adenine shown below. Draw the structure of a base pair with proper
Watson-Crick geometry that contains a rare imino tautomer of adenine (see the
structure below). What base sequence change such a mispairing would cause?

This has the same geometry as C:G or T:A. In this case, the replicated strand has a C base pairing with A
rather than T. In the next round of replication, this mutatnt C di rects the incorporation of a G. The
mutation is A to G on the parent strand and T to C on the complement.

4. (4 pts) Methanol (wood alcohol) is highly toxic because it is converted to

formaldehyde in a reaction catalyzed by the enzyme alcohol dehydrogenase:

NAD+ + methanol NADH + H+ + formaldehyde

Part of the medical treatment for methanol poisoning is to administer ethanol (ethyl
alcohol) in amounts large enough to cause intoxication under normal circumstances.

Explain this treatment in terms of what you know about examples of enzymatic reactions.

Ethanol is a structural analog of methanol, and competes with methanol for the binding site of alcohol
dehydrogenase, slowing the conversion of methanol to formaldehyde, and allowing its clearance by the
kidneys. The effect of ethanol is that of a competitive inhibitor.
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5. (4 pts) The number of structurally different polysaccharides that can be made with 20
different monosaccharides is far greater than the number of different polypeptides that
can be made with 20 different amino acids even though both polymers contain an equal
number of 100 total residues. Is the statement true or false? Explain your reasoning.

True.
Because virtually all peptides are linear (i.e., are formed with peptide bonds between the -
carboxyl and -amino groups), the variability of peptides is limited by the number of different
subunits. Polysaccharides can be linear or branched, can be - or -linked, and can be joined 1
4, 1 3, 1 6, etc. The number of different ways to arrange 20 different sugars in a
branched oligosaccharide is therefore much larger than the number of different ways a peptide
could be made with an equal number of residues

This answer was based on a comparison of the primary structure of amino acids (peptide bond
formation) versus glycosidic bonds. However, if you discussed protein secondary structure and
all the various possibilities you will also receive credit as long as you justified your answer.

6. (4 pts) A plant breeder has developed a new frost-resistant variety of tomato that
contains higher levels of unsaturated fatty acids in membrane lipids than those found in
standard tomato varieties. However, when temperatures climb above 95 F, this frost-
resistant variety dies, whereas the standard variety continues to grow. Provide a likely
explanation of the biochemical basis of increased tolerance to cold and increased
susceptibility to heat of this new tomato variety.

More unsaturated fatty acids will cause an increase in membrane fluidity because unsaturated
fatty acids contain kinks and cannot pack as tightly as saturated fatty acids. At cold
temperatures, the fluidity increase from the extra unsaturated fatty acids counterbalances the
tendency of lipids to solidify at low temperature. At high temperatures, the fluidizing effects of
the extra unsaturated fatty acids add to the fluidizing effect of higher temperature, and the
membrane of the new plant loses its integrity
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7. (4 pts) Why is the mobility of H+ in ice only about an order of magnitude less than that
in liquid water, whereas, the mobility of Na+ in solid NaCl is zero?

In NaCl, the sodium ion and chloride ion are rigidly held in a crystalline lattice and are therefore immobile
in the electric field. The same is true of entire water molecules in a crystal of ice. However, since the
water molecules in ice are orientated with their O-H bonds pointing at neighboring water molecules the
protons can easily jump from molecule to molecule. Consequently, the apparent mobility of protons in ice
is comparable to that in liquid water because the mechanism of proton migration is similar in both phases.

8. (6 pts) The amino acid histidine has a side chain for which the pKa is 6.0.
Calculate what fraction of the histidine side chains will carry a positive charge at pH
5.4. Be sure to show your work.

The amino acid histidine has a side chain for which the pKa is 6.0. Calculate what fraction of
the histidine side chains will carry a positive charge at pH 5.4. Be sure to show your work.

[conjugate base]
Ans: pH = pKa + log
[acid]

[acid]
pKa pH = log
[conjugate base]

[acid]
antilog (pKa pH) =
[conjugate base]

[acid]
antilog (6.0 5.4) =
[conjugate base]

4 = [acid]/[conjugate base], or
4[conjugate base] = [acid]

Therefore, at pH 5.4, 4/5 (80%) of the histidine will be in the protonated form.
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9. (4 pts) When a polypeptide is in its native conformation, there are weak interactions
between its R groups and when it is denatured there are similar interactions between the
protein groups and water. What then accounts for the greater stability of the native
conformation?

In the unfolded polypeptide, there are ordered solvation shells of water around the protein groups.
The number of water molecules involved in such ordered shells is reduced when the protein folds,
resulting in higher entropy. Hence, the lower free energy of the native conformation.

10. (2 pts) A polypeptide is subjected to the following degradative techniques resulting

in polypeptide fragments with the indicated amino acid sequences. What is the amino
acid sequence of the entire polypeptide?

I. Cyanogen Bromide treatment:

i. Asp-Ile-Lys-Gln-Met
ii. Lys
iii. Lys-Phe-Ala-Met
iv. Tyr-Arg-Gly-Met

II. Trypsin Hydrolysis:

i. Gln-Met-Lys
ii. Gly-Met-Asp-Ile-Lys
iii. Phe-Ala-Met-Lys
iv. Tyr-Arg

Try Arg Gly Met Asp Ile Lys- Gln Met Lys Phe Ala Met Lys
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11. ( 6 pts) A biochemist obtains the following set of data for an enzyme that is known
to follow Michaelis-Menten kinetics.

Substrate Initial
concentration velocity
(M) (mol/min)

1 49
2 96
8 349
50 621
100 676
1,000 698
5,000 699

A. Vmax for the enzyme is __________. Explain in one sentence how you
determined Vmax.

B. Km for the enzyme is _________. Explain in one sentence how you determined
Km.

A. Vmax is about 700. In a plot of V vs. [S], the asymptote is Vmax. Simple inspection of the data shows
the approach to Vmaxthe rate increases by only 1 unit when [S] increases fivefold.

B. Km is about 8 mM, the [S] at which the velocity is half-maximal. Because Vmax is about 700, 1/2 Vmax is
about 350. The [S] at that rate is about 8 mM.

12. (2 pts) Describe one biological advantage of storing glucose units in branched
polymers (glycogen, amylopectin) rather than in linear polymers.

Ans: The enzymes that act on these polymers to mobilize glucose for metabolism act only on
their nonreducing ends. With extensive branching, there are more such ends for enzymatic attack
than would be present in the same quantity of glucose stored in a linear polymer. In effect,
branched polymers increase the substrate concentration for these enzymes.
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Graduate Questions (50 Pts)

A patient has been admitted to the emergency room and is disoriented, having trouble
speaking and suffering from nausea and vomiting. The patient is also hyperventilating
and has admitted to taking an entire bottle of aspirin. She was admitted at 9 PM two
hours after taking the tablets. Blood work analysis results are shown in Table 1 and
suggest the patient is also experiencing mild respiratory alkylosis.

Table 1 Arterial blood gas concentration in patient

2 hours after 10 hours after Normal values
aspirin ingestion aspirin ingestion
pCO2 26 mm Hg 19 mm Hg 35 45 mm Hg
HCO3- 18 mM 21 mM 22 26 mM
pO2 113 mm Hg 143 mm Hg 75-100 mm Hg
pH 7.44 7.55 7.35 7.45
Blood saliclylate 57 117
concentration, mg/dL

The patient is given a stomach lavage with saline and two doses of activated charcoal to
absorb the aspirin. Eight hour later, nausea and vomiting become severe and the
respiratory rate has increase. The patient is now in severe respiratory alkylosis. Further
treatment involves a gastric lavage at ph 8.5 as well as further activated charcoal
treatments every 30 minutes. A bicarbonate drip is also administered to prevent blood
bicarbonate levels from dropping below 15mM. Over the next four hours, blood
salicylate concentrations decrease and the patients blood pH begins to drop finally
returning to normal after 60 hours.

13. (2 pts) Aspirin, or acetylsalicylic acid (structure shown in Figure 1), is hydrolyzed in
the presence of aqueous acid and stomach esterases to salicylic acid (the
pharmacologically active form of the drug) and acetic acid. Write the balanced chemical
reaction for this transformation.
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14. (8 pts) Since the patient was brought into the emergency room only two hours after
the overdose, you suspect that her stomach might contain undissolved aspirin that is
continuing to be absorbed. You decide to use a gastric lavage at pH 8.5 to effectively
remove any undissolved aspirin. This treatment solubilizes the aspirin so that it can easily
be removed from the stomach.

A. Calculate the percentage of protonated and unprotonated forms of salicylic acid at the
pH of the stomach, which is usually around 2.0.

These questions can be answered by using the Henderson-Hasselbalch equation:

At pH = 2, the percentage of salicylate (unprotonated) is 9% (0.1/1.1) and the percentage of salicylic acid
(protonated) is 91% (1/1.1).

B. Calculate the percentage of protonated and unprotonated forms of salicylic acid at the
pH of the gastric lavage. (Note: Assume that the pK values for the carboxylate group in
salicylic acid and acetylsalicylic acid are the same.)

At pH = 8.5, virtually 100% of the salicylate is in the unprotonated form.

C. Why does the gastric lavage result in increased solubility of the drug?

The gastric lavage increases the solubility of the drug because at this pH, the salicylate is unprotonated and
negatively charged. At the pH of the stomach, pH = 2, about 90% of the salicylic acid is in the protonated,
or uncharged form. Negatively charged species are generally more polar (and water soluble) than neutral
species, so the gastric lavage increased the solubility of the drug. This facilitates the removal of the aspirin
from the stomach.
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15. (8 pts) It has been shown that salicylates act directly on the nervous system to
stimulate respiration. Thus, our patient is hyperventilating due to her salicylate overdose.

A. Explain how the salicylate-induced hyperventilation leads to the values of pO2 and
pCO2 symptoms seen in the patient.

The patients pCO2 pressure increases between two hours and ten hours, and the pO2 pressure decreases in
the same time period, indicating the excessive oxygen intake and excessive CO2 exhalation that would be
expected in a hyperventilating patient.

B. Explain how the salicylate-induced hyperventilation causes the pH of the patients

blood to increase. Illustrate your answer with the appropriate equations.
The carbonic acid/bicarbonate buffering system relies on these three equilibria:

The patient experiences salicylate-induced hyperventilation, which means that carbon dioxide is being
rapidly removed from the lungs. The patients laboratory values show a low pCO2 value (26 mm Hg two
hours after aspiring ingestion and 19 mm Hg after 10 hours of aspiring ingestion) and a high pO2 value (113
mm Hg two hours after aspiring ingestion and 143 mm Hg after 10 hours of aspirin ingestion), indicating
that oxygen is being taken in and carbon dioxide is being exhaled at a greater rate than normal. The
removal of CO2 shifts the equilibrium of the third equation above to the right, which means that carbon
dioxide moves out of the bloodstream and into the lungs. This causes CO2(aq) to be depleted, so the
equilibrium of the second equation also shifts to the right. This depletes the carbonic acid, so the
equilibrium of the first equation also shifts right to produce more carbonic acid. The result is that hydrogen
ions are depleted and the blood becomes more basic. This is verified by looking at the laboratory values,
which show that the patients blood pH after 10 hours of aspirin ingestion is 7.55 (normal is 7.35-7.45).
The shift of the first equation to the right causes bicarbonate ions to be depleted. This is why the
bicarbonate concentration in the patient is lower than normal.

C. Why was the bicarbonate drip necessary?

The shift of the first equation to the right depletes both hydrogen ions and bicarbonate ions. If the shift is
too dramatic, bicarbonate ions will be depleted and the ability of the carbonic acid/bicarbonate buffering
system will be compromised. A bicarbonate drip replaced the bicarbonate ions that were being depleted
until the salicylates could be removed from the patients system and thus remove the stimulus for
hyperventilation.

D. Sixty hours after aspirin ingestion, the patients blood pH has returned to normal (pH
= 7.4). Describe how the carbonic/bicarbonate buffering system responded to bring the
patients blood
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Diabetics lack the protein insulin, which is produced by the pancreatic -cells of the islets
of Langerhans. Insulin stimulates uptake of glucose from the blood into the tissues.
Diabetes is treated by replacing the missing insulin. Human insulin is produced
industrially by recombinant bacteria, but before this method was available, animal insulin
was used instead.
Insulin consists of two polypeptide chains, an A chain and a B chain, joined together by
disulfide bonds. The smaller of the two chains is referred to as the A chain and is 21
amino acids long in humans. The second chain is referred to as the B chain and is 30
amino acids long in humans. Insulin from various animals is similar to, but not identical
to human insulin, as illustrated in Table 2. A schematic diagram of the structure of insulin
is shown in Figure 2.
Table 2: Variation in positions A8, A9, A10, B1, B2, B27 and B30 of insulin. (All other
amino acids are the same.)

Species A8 A9 A10 B1 B2 B27 B30

Human Thr Ser Ile Phe Val Thr Thr
Cow Ala Ser Val Phe Val Thr Ala
Pig Thr Ser Ile Phe Val Thr Ala
Horse Thr Gly Ile Phe Val Thr Ala
Rabbit Thr Ser Ile Phe Val Thr Ser
Dog Thr Ser Ile Phe Val Thr Ala
Chicken His Asn Thr Ala Ala Ser Ala
Duck Glu Asn Pro Ala Ala Ser Thr

Figure 2. Structure of proinsulin. (From Voet and Voet, 1995, p.193)

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16. (2 pts) What animals would serve as the best sources of insulin to be used for treating
diabetics? Explain your answer.

Pig insulin is a good choice to replace human insulin. There is only one amino acid variation in B30. Cow
insulin has two differences in A8 and B30, and one difference in A10 (although these are conservative
changes). Pig and cow are the best sources because these animal products are readily available from
slaughterhouses, whereas horse, dog and rabbit are not. Chicken and duck have seven and six amino acid
differences as compared to human insulin and wouldnt be good choices. The primary sequence of amino
acids influences the tertiary structure of proteins. Thus it is possible that a change in the protein sequence
could alter the tertiary structure, thus giving the molecule a different shape, which means it might have
difficulty interacting with receptors.

17. (3 pts) Would the pI values of the animal insulins be the same as, greater than, or less
than human insulin?

The pI values for all of the animal insulins would be similar to human insulin (with the exception of
chicken and duck) because the amino acid changes all involved neutral amino acids. Chicken insulin would
be more basic (because of the histidine substitution) whereas duck would be more acidic (because of the
glutamate substitution.)

18. (2 pts) An SDS-PAGE gel is run of proinsulin and insulin. Samples were treated with
B-mercaptoethanol prior to electrophoresis. Draw a picture of the predicted results.