1. What is the order of elution & explain. 2. Rank the pI values for the three h

Question

1. What is the order of elution & explain.

2. Rank the pI values for the three hemoglobins from most acidic to least acidic

Hb Kansas (Hb K) isa mutation of a asparagine to threonine at G4 of the beta domain, anamino aicd located at the a1b2interface. HbYoshizuka (HbY) is a mutation of an asparagine to aspartic acid at G10, anaminoacid locatedat the ablinterface. Consider howyouwouldseparate allthree hemoglobinsfrom one another. Ona DEAEcolumn The chromatogram is shownfor the situationwhere loadthe sample ontothe columnat pH8, and then apply a linear pHgradient. pH 8 A280 pH 7.5 volume, mL Youdetermine the Hill coefficient for each Hbisolated, andthe values are as follows: Hbfirst off the column, and represertedby lonthe chromatogram: 1.3 Hb represented by 2onthe chromatogram: 2.4 Hb last off the column, andrepresentedby 3onthe chromatogram: 23

Explanation / Answer

From the above shown picture,

The hill coefficient which represents the binding capacity relates to the elution order as follows,

1. Order of elution : As the binding ability goes down the elution increases,

So,

first to elute : 1 , then 3 and last would be 3.

2. pI of the three haemoglobins ranking from most acidic to least acidic would thus be in the following order,

2 (most acidic) > 3 (medium acidic) > 1 (least acidic)

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