"SCOOP" -- Old Exams 1
BL401/CH401 Exam 1 Fall 1993 September 29, 1993 Page 1
1. 20 points - Cell structure (five points for each answer -- be brief!)
A. The prokaryotic and eukaryotic cell differ in what important features:
B. Mitochondria differ from other organelles of a liver cell in what features:
C. Double stranded DNA is found in what types of organisms:
D. Ribosomes of both prokaryotes and eukaryotes are composed of what:
2. 20 points - Protein Purification (five points for each answer -- be brief!)
A. Describe the purification method that takes advantage of protein net charge.
B. Describe the purification method that takes advantage of biological activity.
C. Describe how you determine enzyme purity.
D. Describe how you determine subunit composition of a pure protein.
3. 10 points - Special Topic Question -- Requires some thought!
An enzyme was chemically synthesized with all D-amino acids (R-amino acids) so it was the "mirror image" of the same enzyme normally made with all L-amino acids (S-amino acids).
How would it work (choose one answer and briefly defend it with some logic):
A. Catalyze the same reaction as the normal enzyme
B. Catalyze the "mirror-image" of the reaction catalyze by the normal enzyme
C. It would not fold and catalyzed no reaction.
BE SURE TO DO THE FOURTH PROBLEM
ON THE NEXT PAGE!
BL401/CH401 Exam 1 Fall 1993 September 29, 1993 Page 2
4. 50 points - Amino Acid Sequence Problem
The original peptide has the following amino acid composition after acid hydrolysis: Arg,Ser,Met, 2 Glu,Pro,Phe,Leu,Lys,Tyr
A. It had no N- or C-terminal found by danyslation and carboxypeptidase
B. After trypsin cleavage it have one tryptic peptide with same amino acid composition as the original peptide.
Edman of the tryptic peptide gave : 1st cycle Gln, 2nd Tyr, 3rd Ser
C. Chymotryptic cleavage gave two peptides:
Ch-1 had amino acid composition from acid hydrolysis: Arg,Phe,Ser,Pro
Edman on Ch-1 gave: 1st cycle Ser, 2nd Arg
Ch-2 had amino acid composition from acid hydrolysis: Leu,Lys,Tyr,Met, 2 Glu
Edman on Ch-2 gave: 1st cycle Leu, 2nd Met, 3rd Glu
D. CNBr Cleavage of the original peptide gave one peptide with the following amino acid composition: 2 Glu,Tyr,Pro,Phe,Leu,HSL,Lys,Ser,Arg
Edman on CNBr-1 gave: 1st cycle Glu, 2nd Lys, 3rd Gln
What is the amino acid sequence of the original peptide?
Draw the complete chemical structure of the Tryptic Peptide at pH 8.
What is the net charge on the Tryptic Peptide at pH 4?
What is the pI of the Tryptic Peptide?
pK information:
Glu 2.2, 4.1, 9.5
Asp 2.0, 3.9, 9.9
His 1.8, 6.0, 9.3
Lys 2.2, 9.2, 10.8
Arg 1.8, 9.0, 12.5
BL401/CH401 EXAM I - ANSWERS Fall 1993
1. a. Many differences between Eu- and Prokaryotes; see text book or my notes
b. Mitochondria have: 2 membranes, own DNA and synthesize some of their own proteins.
c. All organisms have double stranded DNA. d. Ribosomes are composed of RNA and proteins.
2. You needed to provide a brief description of each of these to get full credit.
a. Ion Exchange chromatography
b. Affinity Chromatography
c. Native PAGE (polyacrylamide gel electrophoresis)
d. subunit composition = native MW (gel filtration) / subunit MW (SDS-PAGE) = dimer etc.
3. B. Catalyzes the "mirror-image" reaction
4. AA sequence problem:
Circular peptide:
Glu-Lys-Gln-Tyr-Ser-Arg-Pro-Phe-Leu-Met
|____________________________________|
Trypsin cuts at Lys
Chymotrypsin cuts at Tyr and Phe
Draw: (+)NH3-Gln-Tyr-Ser-Arg(+)-Pro-Phe-Leu-Met-Glu(-)-Lys(+)-COO(-)
Charges are shown for sidechains at pH 7.
Charge at pH 4 = +2. pI = 10.2
BL401/CH401 Biochemistry I EXAM I Sept. 25, 1992
1. 25 Pts. Describe differences between cells of prokaryotes (bacteria) and eukaryotes (liver or plant) in how each system deals with the following:
A. Membrane permeability (import of metabolites and export of proteins)
B. Metabolic compartmentation (does it have organelles?)
C. DNA directed protein synthesis
D. Storage of DNA
E. Types of DNA
2. 40 Pts. Protein Sequence Problem
A peptide was found to have the following amino acid composition by acid hydrolysis:
Ala, Arg, Met, Tyr, Gly, 2 Lys, Ser, Pro, Glu
A. N-terminal analysis yielded Dansyl-Gly
B. Tyrptic digestion yielded 3 peptides: T-1, T-2, & T-3
T-1 was a dipeptide and Edman degradation showed it had N-terminal Gln
T-2 was a tripeptide with amino acid composition: Lys, Gly, Ser
T-3 was a pentapeptide and Edman degradation showed it had N-terminal Lys
C. Chymotyrptic digestion yielded 2 peptides: Ch-1 & Ch-2
Ch-1 had amino acid composition: 2 Lys, Pro, Gly, Ser, Tyr
Ch-2 yielded Met when degraded by Edman method
D. CNBr cleavage gave 2 peptides: CB-1 and CB-2
CB-1 contained homoSerlactone and had Gly as N-terminal by Edman method
CB-2 released PTH-Arg in first step of Edman and PTH-Gln in second step.
What is the sequence of the peptide??? Draw full chemical structure of the peptide and show its proper charges on all ionizable groups at pH 7.
3. 25 Pts. Protein Purification -- Provide short answers for the following:
A. A method for estimating the molecular weight of protein subunits
B. A method for separation of proteins based on their size
C. A method for separation of enzymes based on their biological activity
D. Method for determination of the purity of a protein
E. Method for separation of proteins by charge
4. 10 Pts. Why does Pro, when present in the position after a target amino acid for proteinases (i.e. trypsin or chymotrypsin), block the action of these enzymes?
(Hint - draw Lys-Pro in complete chemical form and identify the bond potentially to be hydrolyzed in these enzyme reactions, then explain why they cannot catalyze the hydrolysis of this bond)
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu