to Biochemistry Group Members________________________
Final Examination - Group Part
Friday, 25 May 2001
5:15 - 6:30 PM
H. B. White - Instructor
Important - Please read this before you turn the page.
Each group member must sign his or her name on this page
to receive the group grade.
If you cannot come to consensus, you may submit separate
answers for a separate grade that would be substituted for the group grade.
In that case, do not sign the group exam.
You may refer to your notes, course reader, handouts, textbook,
or graded homework assignments. Reference books in the course library may
be consulted briefly and returned.
Please read the questions carefully and make sure that you
have thought them through with everyone=s
input before converging on a solution.
1. On the last page, there
is a representation of the amino acid sequences of the alpha and beta globin
chains of rabbit hemoglobin.
A. (3 points) If Zinoffsky had used rabbit
hemoglobin instead of horse hemoglobin for his determination of the iron
and sulfur content of hemoglobin, what stoichiometry of Fe to S would he
B. (2 points) Explain your answer.
2. (Someone in your group read this
aloud.) Around 1960, biochemists knew from the amino acid sequences of
a few small proteins that proteins were linear polymers. They also knew
that there was a linear correspondence between genetic maps (e.g. DNA)
and protein sequence and that ribosomes catalyzed the synthesis of proteins.
However, they had no idea of how the polymerization occurred. Messenger
RNA and the Genetic Code were not yet known.
Howard M. Dintzis [Proc.
Natl. Acad. Sci. USA, 47, 247-261 (1961)] designed an experiment to
determine whether proteins were polymerized from their amino terminus to
their carboxyl terminus, vice versa, or in some less orderly way.
He knew that animals like rabbits produce large numbers
of reticulocytes when exposed to phenylhydrazine. Unlike the mature erythrocytes,
these immature red blood cells are making hemoglobin and, at any given
moment, all stages of synthesis are present. When incubated with radioactive
amino acids, reticulocytes will incorporate the amino acids into hemoglobin.
In order to make the system experimentally manageable, Dintzis slowed down
the process of protein synthesis by lowering the incubation temperature
to 15ºC. He reasoned that, if synthesis proceeded from one end to
the other, successive peptides in the amino acid sequence should display
a gradient of labeling at short time periods. His experiment was designed
A. At the time Dintzis did
his experiment, he did not know the amino acid sequences of rabbit hemoglobin
alpha and beta chains. He inferred the order of the peptides by the gradient
of labeling. Later Naughton & Dintzis [Proc.
Natl. Acad. Sci. USA, 48, 1822-1830 (1962)] determined the order of
the peptides by comparison to the known human hemoglobin sequences and
plotted their data as shown below.
B. (12 points) Which direction of protein synthesis
do these data support?
(You may know the answer, but credit will be
given only for an answer that clearly and unambiguously explains
how the data support the conclusion. Diagrams are welcome.)
B. (8 points) Several leucine-containing
tryptic peptides have more than one leucine and would be expected to contain
correspondingly more radioactivity, yet there are no "spikes" in the data.
The numbers increase regularly. How did Dintzisí experimental design take
into account the possibility of multiple leucines in a single peptide?
(Note: Dintzis did not analyze every leucine-containing peptide.)
C. Bonus Question (5 points) The rate of protein synthesis
at 15ºC is about 1/6th of the rate at 37ºC. Based
on the data provided, make a reasonable, rough estimate of the time in
seconds that it takes for a ribosome to make a single peptide bond in hemoglobin.
Describe your reasoning.
Rabbit Hemoglobin alpha chain
Rabbit Hemoglobin beta chain
The single-letter abbreviations
for the 20 amino acids are:
A = Alanine
I = Isoleucine R = Arginine
C = Cysteine
K = Lysine
S = Serine
D = Aspartic Acid
L = Leucine T = Threonine
E = Glutamic Acid
M = Methionine V = Valine
F = Phenylalanine
N = Asparagine W = Tryptophan
G = Glycine
P = Proline Y = Tyrosine
H = Histidine
Q = Glutamine
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Posted 14 June 2001 by Hal
Copyright 2001, Harold B.
White, Department of Chemistry and Biochemistry, University of Delaware