237_DE_S09__PS2_ANSWERS - Chem 237 DE June 18, 2009...

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Chem 237 DE June 18, 2009 Assignment #2 ANSWERS 1. A variant of HbA has been characterized in a published case report. The mutation is in the β chain. The analysis of the mutation showed that the C-terminal tryptic peptide had been altered. a. The amino acid composition of the C-terminal peptide 2 His residues. What is the mutation in the Hb β chain in this variant? ANSWER: 5 marks. Trypsin cleaves the peptide after Lys and Arg. When we look at the sequence of Hb (in the figure where we were talking about conservative vs non-conservative mutations). We see that amino acid #144 in the β chain is “K”, which according to the single letter amino acid code is Lys. Hence, trypsin will cleave the β normal chain after this Lys, to give a C-terminal peptide of Tyr-His. However, for this variant, the C-terminal peptide is His-His, NOT Tyr-His. So what is the mutation? It must be Tyr145 β His. Another notation would be Tyr H23(145) β→ His. Now (this is not part of the graded answer), if you were interested in the actual change in the DNA in this mutation, we note that on page 989 in the text, the following codons for Tyr and His: Tyr TAT His CAT TAC CAC A simple change of T into C could change a Tyr to a His. This is indeed what was looked into in this paper; they actually sequenced the DNA to see what the mutation is. And they find that a T has become a C, a TAT codon has become CAT, hence the Tyr His mutation.
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b. The variant Hb chains were “biosynthesized” with [3H]-Leu then separated by cation exchange chromatography (some urea was added to the buffer to ensure that the subunits did not form dimers and tetramers). [They do not say how the “biosynthesized” Hb was actually made, but I think it is safe to assume that the researchers did not feed the patient radioactive Leu, but that it was made in cultured cells from the patient.] Explain the column profile below from the cation exchange column. In particular, is the mutation you suggest above consistent with the column profile? (pay attention to the charges on the various subunits) ANSWER: 8 marks. The point of the [ 3 H]Leu is for at least 2 reasons that I can see. One, the label acts as a marker for the newly synthesized protein, but one could argue that A 280 is also a marker for the protein. Also, they wanted to be sure to get newly synthesized Hb tetramers, from the chromosomal DNA, not contaminating tetramers from the medium, and be adding the [
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This note was uploaded on 07/10/2010 for the course CHEM 237 taught by Professor Elisabethdaub during the Spring '10 term at Waterloo.

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237_DE_S09__PS2_ANSWERS - Chem 237 DE June 18, 2009...

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