Hb Kansas (Hb K) is a mutation of an asparagine to threonine at G4 of the beta d
ID: 168444 • Letter: H
Question
Hb Kansas (Hb K) is a mutation of an asparagine to threonine at G4 of the beta domain, an amino acid located at the alpha1-beta2 interface. Hb Yoshizuka (Hb Y) is a mutation of an asparagine to aspartic acid at G10, an amino acid located at the alpha 1-beta 1 interface.
How would you separate the three hemoglobins from one another using a CM column
I need help with the last 3 questions, thank you!
The third hemoglobin is normal (without mutations).
Ona CM column The chromatogram is shownforthe situationwhere you our sample onto the columnat pH 7.3and then apply alinear pHgradient. pH 8 A280 pH 7.3 volume, mL Where do you expect each of these hemoglobirsto come off? Canyourationalize the pl for Hb Y? Does this hemoglobin have a pl that can be explained by this amino acidsubstitution? Discuss! Canyourationalize the pl for Hb K? Does this hemoglobin have a pl that can be explained by this amino on Discuss!Explanation / Answer
The CM (Carboxy methyl) column being used for the separation of the three types of Hemoglobin, is a cation exchanger. Among the three Hb, the ones which retain a positive charge when pH is altered from 7.3 to 8, will remain bound to the solid phase longer, and the one which is negatively charged will be eluted first.
Let us assume, the normal Hb contain the amino acid Asparagine at G4 and G10 position. Hb Kansas contain Threonine at G4 and Hb Yoshizuka contain Aspartic acid at G10.
These three amino acids differ by their charge state at different pH, because of the side chain groups. The order of elution of the respective Hb types are:
1. Hb Y
2. Hb Normal
3. Hb K
The Hb Y will have a lower pI as compared to normal Hb, because aspartic acid has a pI of 2.84 and asparagine has a pI of 5.41.
The Hb K have a slightly higher or almost equal pI as compared to Hb Normal, because threonine has a pI of 5.60.
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