Download Hemoglobin, or haemoglobin, is an iron

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