Hemoglobin is formed by the tertiary structure of 2 alpha chains and 2 beta chains. The alpha is normal in sickle cell anemia, but the beta chains have a mutation and form Hb-S.
But, in a sickle cell trait, there is a possibility of both Hb-S and Hb-A. This is because the cell can have 2 beta polypeptides. Lets call the normal one 'b' and the mutated one 'b*'. (referring to the polypeptides not the alleles)
So i'm assuming Hb-A is aabb while Hb-S is aab*b*. What stops the cell from making aab*b - i.e., a hybrid between the normal beta polypeptide and the mutated one in the same hemoglobin?
Hemoglobin electrophoresis works primarily on charge (rather than mol wt as SDS PAGE does). Since, the polypeptides are not separated, and whole Hb is loaded, in sickle cell trait, shouldn't we be expecting three bands?
The sickle cell mutation, glutamate is replaced by valine so there's a charge difference. So if in a Hb, there is a difference of one/two mutant polypeptides, the charge will also differ. So,
(the aa-bb notation here depicting phenotype of polypeptides not genes)
So if there are three electrophoretic possibilities (theoretically?), why only two bands? And since the 3rd possibility is midway of the two, I suppose, it can't be ignored as minor.
Does hemoglobin in sickle cell trait, have hybrids of normal and diseased beta polypeptide? -
I would think that in sickle cell trait there would be a roughly equal mixture of normal and mutant beta, randomly associating in hemoglobin. This would result in individual hemoglobin molecules containing either two normal betas (25%), two mutant betas (25%), or one of each (50%). In sickle cell electrophoresis, one beta band would be observed for a person with normal hemoglobin (homozygous), one band (at a different position) would be observed for someone with sickle cell anemia (homozygous), and two bands would be observed for someone with sickle cell trait (heterozygous).
In this explanation of sickle cell anemia/trait, the beta-globin gene can be in one of two forms in the hemoglobin alpha2beta2 tetramer, the wild-type (HbA) and the mutant (HbS). The alpha-globin gene is unaffected. I was referring only to the beta-globin gene. As far as I can tell, the hemoglobin subunits are not dissociated in the electrophoresis, so you would see only one band for unaffected people (HbA) or sickle-cell anemia patients (HbS), and 2 bands for people with sickle cell trait (HbA and HbS). This is an oversimplification because of the presence of minor hemoglobins and the existence of various other hemoglobinopathies.
The haemoglobin tetramer comprises 2 alpha and 2 non-alpha globin chains. In the case of HbA --> 2 alpha and 2 beta chains. The alpha1-beta1 interaction is very stable whereas the alpha1-beta2 interaction is much less so. In the inact red cell the Hb tetramer remains intact but outside the red cell, e.g. haemolysis, the tetramer dimerises into alpha1-beta1 and alpha2-beta2 dimers. This is the reason why upon Hb electrophoresis we do not observe the hybrid tetramers in globin chain variants.
To conclude, hybrid haemoglobins in above scenario exist in the in-vivo state but not the in-vitro state as they dimerise once haemolysed
@Nazeer Ali. Thanks a lot for the answer. For sake of completion, I would like to add a couple of references - modifications in methods to isolate the third band also known as the assymetric hybrid
http://pubs.acs.org/doi/abs/10.1021/bi00702a024?journalCode=bichaw
http://www.tandfonline.com/doi/abs/10.3109/03630268108997549?journalCode=ihem20
Thanks for the references. This provides further clarification on hybrid haemoglobin and also confirms its presence in vivo.
Can a single RBC of a sickle cell patient (trait or diseased) contain both HbS and HbA?
Since the hemoglobin in a red blood cell is produced in the nucleated cell that is a precursor to it by transcription of the hemoglobin genes in that cell, each red blood cell of a person with sickle cell trait would have an equal mixture of HbS and HbA, assuming that both of the genes are transcribed and translated at equal rates and both of the hemoglobin proteins have equal stability. If these assumptions are not met, then there might be more of one of the two types of Hb than the other, but both should be present in the cell.
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