Question

Protein molecules in solution can be separated from each other by taking advantage of their net charges. In the electric field between two electrodes, a positively charged particle moves toward the negative electrode and a negatively charged particle moves toward the positive electrode. This movement, known as electrophoresis, varies with the strength of the electric field, the charge of the particle, the size and shape of the particle, and the buffer/polymer gel combination through which the protein is moving.
The net charge on a protein is determined by how many of the acidic or basic side-chain functional groups in the protein are ionized, and this, like the charge of an amino acid, depends on the pH. Thus, the mobility of a protein during electrophoresis depends on the pH of the buffer. If the buffer is at a pH equal to the isoelectric point of the protein, the protein does not move.
By varying the pH of the buffer between the electrodes and other conditions, proteins can be separated in a variety of ways, including by their molecular weight. Once the separation is complete, the various proteins are made visible by the addition of a dye.

Electrophoresis is routinely used in the clinical laboratory for determining which proteins are present, and in what amounts, in a blood sample. One commonly used test is for the diagnosis of sickle-cell anemia (p. 568). Normal adult hemoglobin (HbA) and hemoglobin showing the inherited sickle-cell trait (HbS) differ in their net charges. Therefore, HbA and HbS move different distances during electrophoresis. The accompanying diagram compares the results of electrophoresis of the hemoglobin extracted from red blood cells for a normal individual, one with sickle-cell anemia (two inherited sickle-cell genes) and one with sickle-cell trait (one normal and one inherited sickle-cell gene). With sickle-cell trait, an individual is likely to suffer symptoms of the disease only under conditions of severe oxygen deprivation.

Question 1: The proteins collagen, bovine insulin, and human hemoglobin have isoelectric points of 6.6, 5.4, and 7.1, respectively. Suppose a sample containing these proteins is subjected to electrophoresis in a buffer at pH 6.6. Describe the motion of each with respect to the positive and negative electrodes in the electrophoresis apparatus.

Question 2: Three dipeptides are separated by electrophoresis at pH 5.8. If the dipeptides are Arg-Trp, Asp-Thr, and Val-Met, describe the motion of each with respect to the positive and negative electrodes in the electrophoresis apparatus.

Answer 1

Answer 1. Buffer at pH 6.6, the charge on the protein

Collagen = zero, So no movement in electrophoresis

Bovine insulin = - ve, so it will move towards positive electrode

Human hemoglobin = +ve, it will move towards negative electrode.

Answer 2.

Isoelectric point of dipeptides

Arg-Trp = (9.04+12.48)/2 = 10.76

Asp-Thr = (3.65+2.20)/2 = 2.925

Val- Met = (2.28+9.60)/2 = 5.94

At pH 5.8, charge on dipeptides and motion

Arg- Trp = +ve, towards negative electrode

Asp-Thr = - ve, towards positive electrode

Val-Met = negligence positive ( very less ionized ), no moment