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1. A biochemist discovers and purifies a new enzyme, generating the purification table below.
Procedure
Total protein (mg)
Activity (U)
Crude extraction
20000
4,000,000
Precipitation (salt)
5000
3,000,000
Precipitation (pH)
4000
1,000,000
Ion-exchange chromatography
200
800,000
Affinity chromatography
50
750,000
Size-exclusion chromatography
45
675,000
(1) From the information given in the table, calculate the specific activity of the enzyme solution after
each purification procedure.
(2) Which of the purification procedures used for this enzyme is most effective (i.e., gives the greatest
relative increase in purity)?
(3) Which of the purification procedures is least effective?
(4) Is there any indication based on the results shown in the table that the enzyme after step 6 is now
pure? What else could be done to estimate the purity of the enzyme preparation?
2. A 0.2-g sample of amylopectin was analyzed to determine the fraction of the total glucose residues
that are branch points in the structure. The sample was exhaustively methylated and then digested,
yielding 50 mol of 2,3-dimethylglucose and 0.4 mol of 1,2,3,6-tetramethylglucose.
(1) What fraction of the total residues are branch points?
(2) How many reducing ends does this sample of amylopectin have?
3. A new protein of unknown structure has been purified. Gel filtration chromatography reveals that
the native protein has a molecular weight of 240,000. Chromatography in the presence of 6 M
guanidine hydrochloride yields only a peak for a protein of Mr 60,000. Chromatography in the
presence of 6 M guanidine hydrochloride and 10 mM -mercaptoethanol yields peaks for proteins of
Mr 34,000 and 26,000. Explain what can be determined about the structure of this protein from these
data.
4. The artificial sweeteners Equal and Nutrasweet
contain aspartame, which has the structure:
(1) What are the two amino acids that are components of
aspartame?
(2) What kind of bond links these amino acids? What do
you suppose might happen if a solution of aspartame was heated for several hours at a pH near
neutrality? Suppose you wanted to make hot chocolate sweetened only with aspartame, and you stored
it in a thermos for several hours before drinking it. What might it taste like?
5. A previously unknown protein has been isolated in your laboratory. Others in your lab have
determined that the protein sequence contains 172 amino acids. They have also determined that this
protein has no tryptophan and no phenylalanine. You have been asked to determine the possible
tyrosine content of this protein. You know from your study of this chapter that there is a relatively
easy way to do this. You prepare a pure 50 M solution of the protein, and you place it in a sample cell
with a 1-cm path length, and you measure the absorbance of this sample at 280 nm in a UV-visible
spectrophotometer. The absorbance of the solution is 0.372. Are there tyrosines in this protein? How
many? (Hint: the molar absorptivity values of Phe, Trp and Tyr are approximately 2×102, 2×103 and
1.42×103 mol-1•L•cm-1, respectively)
6. A 10-kb DNA fragment digested with restriction endonuclease EcoRI yielded fragments 4 kb and 6
kb in size. When digested with BamHI, fragments 1, 3.5, and 5.5 kb were generated. Concomitant
digestion with both EcoRI and BamHI yielded fragments 0.5, 1, 3, and 5.5 kb in size. Give a possible
restriction map for the original fragment.
7. Assume that there is one nucleosome for every 200 bp of eukaryotic DNA. How many nucleosomes
are there in a diploid human cell? Nucleosomes can be approximated as disks 11 nm in diameter and 6
nm long. If all the DNA molecules in a diploid human cell are in the B-conformation, what is the sum
of their lengths? If this DNA is now arrayed on nucleosomes in the beads-on-a-string motif, what
would be the approximate total height of the nucleosome column if these disks were stacked atop one
another?
8. The plasmid pBR322 is a closed circular dsDNA containing 4363 base pairs. What is the length in
nm of this DNA (that is, what is its circumference if it were laid out as a perfect circle)? The E. coli
K12 chromosome is a closed circular dsDNA of about 4,639,000 base pairs. What would be the
circumference of a perfect circle formed from this chromosome? What is the diameter of a dsDNA
molecule? Calculate the ratio of the length of the circular plasmid pBR322 to the diameter of the DNA
of which it’s made. Do the same for the E. coli chromosome.
9. Erwin Chargaff did not have any DNA samples from thermoacidophilic bacteria such as those that
thrive in the geothermal springs of Yellowstone National Park. (Such bacteria had not been isolated by
1951 when Chargaff reported his results) If he had obtained such a sample, what do you think its
relative G C content might have been? Why?
10. Yeast (S. cerevisiae) has a genome size of 1.21×107 bp. If a genomic library of yeast DNA was
constructed in a vector capable of carrying 16 kb inserts, how many individual clones would have to
be screened to have a 99% probability of finding a particular fragment?
11. The South American lungfish has a genome size of 1.02×1011 bp. If a genomic library of lungfish
DNA was constructed in a vector capable of carrying inserts averaging 45 kb in size, how many
individual clones would have to be screened to have a 99% probability of finding a particular DNA
fragment?
12. Imagine that you are interested in a protein that interacts with proteins of the cytoskeleton in
human epithelial cells. Describe an experimental protocol based on the yeast two-hybrid system that
would allow you to identify proteins that might interact with your protein of interest.
13. Carbonic anhydrase catalyzes the hydration of CO2:
CO2 + H2O→H2CO3
The Km of carbonic anhydrase for CO2 is 12 mM. Carbonic anhydrase gave an initial velocity υo= 4.5
μmol H2CO3 formed/mL∙sec, when [CO2]= 36 mM.
a. What is Vmax for this enzyme?
b. Assuming 5 pmol/mL (5×10-12 moles/mL) of enzyme were used in this experiment, what is kcat
for this enzyme?
c. What is the catalytic efficiency of this enzyme?
d. Does carbonic anhydrase approach “catalytic perfection”?
14. The following graphs show the temperature and pH dependencies of four enzymes, A, B, X, and Y.
Problems 12 through 18 refer to these graphs.
(1) Enzymes X and Y in the figure are both protein-digesting enzymes found in humans. Where would
they most likely be at work?
a. X is found in the mouth, Y in the small intestine; b. X in the small intestine, Y in the mouth;
c. X in the stomach, Y in the small intestine; d. X in the small intestine, Y in the stomach.
(2) Which statement is true concerning enzymes X and Y?
a. They could not possibly be at work in the same part of the body at the same time.
b. They have different temperature ranges at which they work best.
c. At a pH of 4.5, enzyme X works slower than enzyme Y.
d. At their appropriate pH ranges, both enzymes work equally fast.
(3) What conclusion may be drawn concerning enzymes A and B?
a. Neither enzyme is likely to be a human enzyme;
b. Enzyme A is more likely to be a human enzyme;
c. Enzyme B is more likely to be a human enzyme;
d. Both enzymes are likely to be human enzymes.
(4) At which temperatures might enzymes A and B both work?
a. Above 40°C; b. Below 50°C; c. Above 50°C and below 40°C; d. Between 40° and 50°C
(5) An enzyme–substrate complex can form when the substrate(s) bind(s) to the active site of the
enzyme. Which environmental condition might alter the conformation of an enzyme to the extent that
its substrate is unable to bind?
a. Enzyme A at 40°C; b. Enzyme B at pH 2; c. Enzyme X at pH 4; d. Enzyme Y at 37°C
(6) At 35°C, the rate of the reaction catalyzed by enzyme A begins to level off. Which hypothesis best
explains this observation?
a. The temperature is too far below optimum; b. The enzyme has become saturated with substrate
c. Both A and B; d. Neither A nor B.
(7) In which of the following environmental conditions would digestive enzyme Y be unable to bring
its substrate(s) to the transition state?
a. At any temperature below optimum; b. At any pH where the rate of reaction is not maximum
c. At any pH lower than 5.5; d. At any temperature higher than 37°C
15. 18. Which statement is most likely to be true concerning obligate anaerobes?
a. These organisms can use oxygen if it is present in their environment.
b. These organisms cannot use oxygen as their final electron acceptor.
c. These organisms carry out fermentation for at least 50% of their ATP production.
d. Most of these organisms are vegetative fungi.
19. Foods rich in fiber are basically plant materials high in cellulose, a cell wall polysaccharide that
we cannot digest. The nutritional benefits provided by such foods result from
a. other nutrients present that can be digested and absorbed.
b. macromolecules (like cellulose) that are absorbed without digestion
and then catabolized inside the cells.
c. microbes that are the normal symbionts of plant tissues.
d. All of the above.
20. Regarding phosphofructokinase, which of the following statements is true:
a. Low ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits.
b. High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits.
c. High ATP stimulates the enzyme, but fructose-2,6-bisphosphate inhibits.
d. The enzyme is more active at low ATP than at high, and fructose- 2,6-bisphosphate activates the
enzyme.
e. ATP and fructose-2,6-bisphosphate both inhibit the enzyme.
21. Predict from the mode of lac operon whether the interaction of lac repressor with inducer might be
cooperative. Would it be advantageous for inducer to show cooperative binding to lac repressor?
Why?
22. RNA polymerase has two binding sites for ribonucleoside triphosphates: the initiation site and the
elongation site. The initiation site has a greater Km for NTPs than the elongation site. Suggest what
possible significance this fact might have for the control of transcription in cells.
23. What might be the advantages of capping, methylation, and polyadenylylation of eukaryotic
mRNAs?
24. Based on the information provided by following experiment, try to give the amino acid sequence
of the peptide.
26. In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both
catabolic and anabolic processes. What’s your understanding on this viewpoint?
25. Define the following terms:
1.
Structure and metabolic roles of cAMP
2.
PCR
3.
Western blotting
4.
Southern blotting
5.
DNA library
6.
Yeast two-hybridization
7.
Affinity chromatography
8.
G protein
9.
Allosteric effect
10. Domain
11. Motif
12. DNA melting temperature (Tm)
13. Chargaff’s rules
14. Isoenzyme
15. Irreversible inhibition
16. Reversible inhibition
17. Structure of -D-Glucopyranose
18. Structure of -D-Fructofuranose
19. Structure of sucrose/Lactose/Maltose
20. Lectin
21. Molecular chaperon
22. G protein
23. Nucleic Acid Hybridization
24. Gluconeogenesis
1.
胰岛素分子中包含 A 链和 B 链，是否代表有两个亚基？为什么？
2.
球状蛋白质在 PH7 时的水溶液中折叠成一定空间构象。这时通常非极性氨基酸残基侧链位
于分子内部形成疏水核，极性氨基酸残基位于分子表面形成亲水面。请问 Val、Pro 、Phe、
Asp、Lys、lle、和 His 中哪些氨基酸侧链位于分子内部？哪些氨基酸侧链位于分子外部？
3.
氨基酸残基的平均分子量是 120Da。有一个多肽链的分子量是 15120Da，如果此多肽完全
以 α-螺旋的形式存在，试计算该 α-螺旋的长度和圈数？
4.
简述 Watson-Crick 提出的 B-DNA 双螺旋结构的要点。
5.
简述 tRNA 二级结构的组成特点及其每一部分的功能。
6.
简述青霉素抑菌的作用机制。