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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.

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 have found?

B.  (2 points) Explain your answer.
 

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 as follows.
 

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.)
 

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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 Dintzis’ experimental design take into account the possibility of multiple leucines in a single peptide? (Note: Dintzis did not analyze every leucine-containing peptide.)
 

MVLSPADKTNIKTAWEKIGSHGGEYGAEAVERMFLGFPTTKTYFPHF

DFTHGSEQIKAHGKKVSEALTKAVGHLDDLPGALSTLSDLHAHKLRV

DPVNFKLLSHCLLVTLANHHPSEFTPAVHASLDKFLANVSTVLTSKYR
 

Rabbit Hemoglobin beta chain

VHLSSEEKSAVTALWGKVNVEEVGGEALGRLLVVYPWTQRFFESFGDL

SSANAVMNNPKVKAHGKKVLAAFSEGLSHLDNLKGTFAKLSELHCDKL

HVDPENFRLLGNVLVIVLSHHFGKEFTPQVQAAYQKVVAGVANALAHKYH
 

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