Passage
Beta-thalassemia is a blood disorder caused by reduced production of the β-globin subunit of hemoglobin. The erythrocytes in patients with beta-thalassemia are abnormally small, lack sufficient functional hemoglobin, and are decreased in number. These individuals require lifelong blood transfusions to correct their erythrocyte shortage. Each unit of blood transfused contains many milligrams of iron and, consequently, blood iron levels increase due to repeated transfusions. The excess iron is then continuously deposited in the organs, leading to organ damage.Transferrin (Tf) is a protein that binds free iron in the blood and plays a key role in erythropoiesis, which requires uptake of iron from the blood in a process facilitated by Tf and its cell surface receptor (TfR). Iron-bound Tf, known as holotransferrin (holoTf), binds TfR to form a complex that is brought into the developing erythrocyte in an endosome. Acidification of the endosome triggers a conformational change in holoTf, which allows the release of free iron into the cell and the recycling of the remaining complex components to the cell surface. Non-iron-bound Tf is referred to as apotransferrin (apoTf), and due to its iron-binding capability, researchers believe that apoTf injections may help prevent deposition of excess iron in the organs of patients with beta-thalassemia.To investigate the effect of apoTf administration in a mouse model of beta-thalassemia, mice homozygous for an inactivating deletion in the murine β-globin gene (Hbb th−3 /Hbb th−3 ) were given blood transfusions followed by apoTf injection. Measurements of non-transferrin-bound iron in the blood (NTBI), plasma holoTf, and urinary holoTf were taken immediately and at 15 days post-injection. Results revealed that NTBI decreased over the tested period. Experimenters hypothesized that apoTf mediated certain regulatory changes that reduced NTBI in Hbb th−3 /Hbb th−3 mice by increasing urinary holoTf excretion.
-ApoTf and TfR were analyzed by SDS-PAGE under nonreducing and reducing conditions. Based on the gels shown, which conclusion is LEAST likely to be true?

Question

Passage
Beta-thalassemia is a blood disorder caused by reduced production of the β-globin subunit of hemoglobin.  The erythrocytes in patients with beta-thalassemia are abnormally small, lack sufficient functional hemoglobin, and are decreased in number.  These individuals require lifelong blood transfusions to correct their erythrocyte shortage.  Each unit of blood transfused contains many milligrams of iron and, consequently, blood iron levels increase due to repeated transfusions.  The excess iron is then continuously deposited in the organs, leading to organ damage.Transferrin (Tf) is a protein that binds free iron in the blood and plays a key role in erythropoiesis, which requires uptake of iron from the blood in a process facilitated by Tf and its cell surface receptor (TfR).  Iron-bound Tf, known as holotransferrin (holoTf), binds TfR to form a complex that is brought into the developing erythrocyte in an endosome.  Acidification of the endosome triggers a conformational change in holoTf, which allows the release of free iron into the cell and the recycling of the remaining complex components to the cell surface.  Non-iron-bound Tf is referred to as apotransferrin (apoTf), and due to its iron-binding capability, researchers believe that apoTf injections may help prevent deposition of excess iron in the organs of patients with beta-thalassemia.To investigate the effect of apoTf administration in a mouse model of beta-thalassemia, mice homozygous for an inactivating deletion in the murine β-globin gene (Hbb th−3 /Hbb th−3 ) were given blood transfusions followed by apoTf injection.  Measurements of non-transferrin-bound iron in the blood (NTBI), plasma holoTf, and urinary holoTf were taken immediately and at 15 days post-injection.  Results revealed that NTBI decreased over the tested period.  Experimenters hypothesized that apoTf mediated certain regulatory changes that reduced NTBI in Hbb th−3 /Hbb th−3 mice by increasing urinary holoTf excretion.
-ApoTf and TfR were analyzed by SDS-PAGE under nonreducing and reducing conditions.  Based on the gels shown, which conclusion is LEAST likely to be true?

Quizplus · Accepted Answer

11ecba2d_0ffc_9975_a3bf_3debd2b25a7f_MD0008_00 Protein electrophoresis is a method of separating proteins according to factors that (depending upon the specific technique used) can include shape, molecular weight, and charge. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) is an electrophoresis technique used to separate proteins based on molecular weight (size). SDS is a strong anionic detergent that denatures and confers a net negative charge on the protein, eliminating its shape and charge as separation factors. Using this technique, larger proteins will migrate a shorter distance toward the positive electrode than smaller proteins. However, SDS disrupts only noncovalent bonds. Therefore, a protein containing multiple covalently bound polypeptide subunits will present as a single band in SDS-PAGE electrophoresis. When SDS-PAGE is performed under reducing conditions, a reducing agent (eg, 2-mercaptoethanol) is added to disrupt disulfide bridges, allowing each subunit to separate as individual polypeptides. Because apoTf does not split into multiple bands in either gel and migrates to the same (40 kDa) position under both nonreducing and reducing conditions, it can be concluded that apoTf is a 40-kDa monomeric protein. Although TfR does not split into multiple bands, it does migrate to different positions within the gels depending on the experimental condition. In Gel 1, TfR appears as an 80-kDa band, and in Gel 2 it appears as a thicker 40-kDa band. Accordingly, it can be concluded that when subjected to reducing conditions, the disulfide bridges holding TfR together are disrupted, and the 80-kDa TfR dimer splits into two 40-kDa subunits. (Choices A, B, and C) Gel 1 represents the nonreducing condition (because it contains the whole 80-kDa TfR protein), and apoTf has a smaller total molecular weight (40 kDa) than TfR (80 kDa). Because of the nature of SDS application, both proteins were conferred a net negative charge before being electrophoresed. Educational objective: Comparing the results of reducing and nonreducing SDS-PAGE, both techniques that separate proteins based on molecular weight, can be useful in determining whether disulfide bonds contribute to the tertiary structure of a protein.

Passage Beta-Thalassemia Is a Blood Disorder Caused by Reduced Production of of the β-Globin