Hemoglobin isoelectric point

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

Table 9.2 Migration Time and Isoelectric Point Reproducibility for Human Hemoglobins A and S Analyzed by CIEF...

Figure 8.8. Resolution by isoelectric focusing in an immobilized pH gradient (pH range 7.35-7.55 over 20 cm path) of two human fetal hemoglobins, one with glycine (lower) instead of alanine (upper) in position 136 of the gamma chains. The isoelectric points of the hemoglobins differ by only 0.003 pH units. (Courtesy of P. G. Righetti, University of Milano.)...

Hemoglobin A (the major, normal form in humans) has an isoelectric point of pH 6.9. The variant hemoglobin M has a glutamate residue in place of the normal valine at position 67 of the a chain. What effect will this substitution have on the electrophoretic behavior of the protein at pH 7.5 ... 

Since pH 7.5 is above the isoelectric point of hemoglobin A, the protein carries a negative charge and will migrate to the anode. At pH 7.5 the glutamate side chain has a negative charge, while valine... 

Hempe JM, Graver RD. Separation of hemoglobin variants with similar change by capillary isoelectric focussing value of isoelectric point for identification of common and uncommon hemoglobin variants. Electrophoresis 2000 21 738-43. 

Effective comparison of patterns of proteins on lEF/PAGE gels requires some method of normalizing the protein patterns to compensate for distortions in the patterns due to gel-to-gel variation in the analytical conditions in either the first- and/or second-dimension separation. Internal standards are valuable in monitoring variation in analytical conditions Isoelectric point standards for lEF based on carbamylated hemoglobin, fluorescein-labeled-hemoglobin, and creatine phospho-kinase (All, M5) can be prepared, as can molecular weight standards based on heart muscle proteins (G8). 

To test the quantitative predictions of the model, we measured the disaggregation of the hemoglobin tetramer by the increase in osmotic pressure with pH (5). When the pressure is compared to that observed in solutions of bovine serum albumin (BSA a protein of comparable molecular weight that does not disaggregate), we can ascribe the difference in pressure to disaggregation. In the titration of both protein solutions, the minimum pressure occurs at the isoelectric point (IEP), which is 6.82 0.06 for hemoglobin at 25.0 °C. 

In a schematic elution pattern of some standard proteins, peroxidase was eluted first with saline, BSA came next with glycine buffer at pH 6.6 and hemoglobin and catalase were eluted at a pH of nearly 8.0. Aldolase, lysozyme, chymotrypsinogen A, malate dehydrogenase, and cytochrome c were not eluted under these conditions, but were eluted with 0.1% SDS. The adsorption order does not depend on the isoelectric point, the molecular mass, or the content of basic amino acids. However, adsorption may depend on the o -helix content, and the secondary structure of those proteins may be important. We have also reported on protein adsorption and separation on siliconized glass surfaces (30), and on the adsorption and separation of nucleic acids on those same surfaces (31-35). 

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