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Transcript
Binding of haptoglobin to hemoglobin Srikanth Dosapally Hemoglobin (Hb) is an iron-containing protein in the red blood cells of mammals and other vertebrates. Hb is released under certain pathological conditions, such as malarial infection and allergic drug reaction. This free Hb is toxic and causes damage to blood vessels and surrounding tissues. Haptoglobin (Hp), a plasma protein made mainly in the liver, stops these Hb-mediated reactions by forming tight complexes with Hb. It is not clear how Hp and Hb bind to each other. The aim of this work was to identify amino acids in Hp that are involved in its binding to Hb. The approach used was to first select amino acid residues that are likely to be involved in binding by comparing the sequence of human Hp with those of related proteins, and then to prepare recombinant Hp in which these amino acids were mutated and measure the effect of the mutations on binding to Hb. Two different Hps in which one amino acid was mutated were expressed in a mammalian cell line. However, the amounts of secreted Hp were too low for binding experiments. Scaling up of the procedure was unsuccessful probably because of a technical failure. In contrast, expression of wild type Hp under the same conditions yielded enough protein for quantification with ELISA and determination of binding to Hb with ELISA. Taken together, these results show that with the techniques described in this study, with some modifications, it should be possible to identify amino acid residues in Hp involved in binding to Hb. Examensarbete i biologi 20 p, 2005 Degree project in Biology Biology Education Centre and Department of Medical Biochemistry and Microbiology, Uppsala University Supervisor: Prof.Erik Fries