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Unformatted text preview: Chem 237 Midterm #2 March 10, 2004 ANSWERS regrade requests are due on March 31 (dont forget your note of explanation!) Isolated -subunits of HbA (normal hemoglobin) can be reassembled into 4 tetramers using a specific set of conditions (you do not need to know these conditions to do the problem). These 4 tetramers have a P 50 value of 6 torr and show no cooperativity. The isolated -subunits of HbA have a pI of 7.4, and the -subunits have a pI of 6.9. a. Assuming that you have purified HbA and have a method to selectively break the quaternary interactions between the and subunits, how might you separate the -subunits from the - subunits, using one of the separation techniques we have talked about in class? Be sure to include any pertinent details about the method you use (resin type, pH, salt concentration, etc). b. Draw an O 2 binding curve for the 4 tetramers; be sure to label the axes properly, and include an Mb and Hb curve for reference. Why did you draw the curve the way you did? c. Suppose that there is a person in the world with no -genes, and whose blood had only 4 tetramers. What physiological consequences would this condition have for that individual? ANSWER: a. (6 marks) There are lots of ways to do this. Ion exchange is a good method, either cation or anion exchange, will work. You would have to choose the pH and the type carefully. I can not go into all the details here, because there are so many possibilities. You had to mention the type of resin (cation or anion) and the pH. Also you had to say the expected result, i.e., which subunit would elute first, etc. Another technique that would work is isoelectric focusing because of the difference in pI values. Here you needed to give a bit of information about the technique (pH gradient, electric current, electrolytes, acid and base at the electrodes, use of a narrow gradient, etc). The techniques that wont work are SDS-PAGE, size exclusion chromatography or affinity chromatography. SDS- PAGE denatures the protein irreversibly, and therefore is not useful as a separation technique. The molecular weights of the subunits are too close for separation by this technique. Affinity chromatography would require a ligand, and there isnt one that is specific for one subunit over the other one. b. (4 marks) The axes are the usual, y-axis is degree of saturation, and the x-axis is pO 2 . the Mb and HbA curves are hyperbolic and sigmoidal, respectively. The curve for the 4 tetramers is in between (because P 50 = 6 torr, while Mb is 2 torr and HbA is about 25 torr), and is hyperbolic because it shows no cooperativity . c. (2 marks) I do not think that this person would be alive, but assuming they were, the very high affinity of the 4 tetramers means that O 2 binds fine in the lungs, but very little O 2 is dropped off in the body, resulting in hypoxia, etc. Not good. There are some naturally occurring mutants that have P 50 values as low as 12 torr, with no cooperativity. as 12 torr, with no cooperativity....
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