1. Hemoglobin was one of the first proteins crystallized and one of the first proteins to have its three-dimensional structure determined by X-ray crystallography. Appropriately for Valentine’s Day, the cover of this year’s February 14th issue of Science displays a bright red crystal of Hemoglobin C, a human hemoglobin variant most common in West Africa. In maximum dimension it is about 0.6 mm, much smaller than Zinoffsky’s crystals of horse hemoglobin and produced in a much more controlled way using vapor diffusion. The procedure is as follows: a small drop (microliters) of a purified and concentrated, but not supersaturated, protein sample dissolved in some aqueous solution (e.g. polyethylene glycol in a buffer), is placed on see-through lid (“hanging drop”) of a sealed chamber containing a much larger volume of a more concentrated, the same solution without protein. The sample is kept in a refrigerator and examined periodically through the lid with a microscope. If the conditions are right, crystals will form in a few days as the system come to equilibrium. Based on the information provided. [Polyethylene glycol is a water-soluble, non-volatile, high molecular weight polymer used to precipitate proteins.]A. Draw a picture of the crystallization set up.
B. Using words and illustrations as needed, describe conceptually what happens in the hanging drop over time and why this is a gentle and controlled way to obtain protein crystals.
2a. How do the results of Bohr et al. explain Stokes’ observation that carbonic acid “behaved like a reducing agent” in the presence of oxyhemoglobin?
2b. Svedberg and Fåhraeus determined the molecular weight of hemoglobin to be four times that estimated by Zinoffsky. Given the known structure of hemoglobin, could Zinoffsky have obtained the actual molecular weight of hemoglobin with his methods? Explain.
2c. Why does the reduction of methemoglobin to deoxyhemoglobin require one electron per mole of hemoglobin while the reduction of oxyhemoglobin to deoxyhemoglobin that Stokes studied was shown by Conant to require four electrons?
2d. Diggs and coworkers did not know that red blood cell
sickling was caused by polymerization of the deoxy form of a variant hemoglobin.
In retrospect, what observations in the Diggs et al. article could have
led them or others to suspect hemoglobin was involved more than a decade
before Linus Pauling made the connection?
3. Table 4 of Zinoffsky’s article reports the data for
calculating the percent iron in the crystalline hemoglobin he purified.
Two lines from Prep III are provided below. The first set of data is from
the titrimetric determination and the second is from the gravimetric determination.
Set up the equations with numbers that yield the 0.334% Fe that Zinoffsky
calculates for each analysis. (You do not need a calculator to do this
for full credit.)
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___ Fe2+ + ___ MnO4- + ___ H+ ---> ___ Fe3+ + ___ Mn2+ + ___ H2O