Haptoglobin is a genetically polymorphic plasma protein which possesses the property of combining specifically with hemoglobin. Haptoglobin binds hemoglobin stoichiometrically in a stable and essentially irreversible complex. The present studies are concerned with the nature of the hemoglobin
combining site on haptoglobin in relation to its function.
Large quantities of homogeneous haptoglobin have been prepared from ascites fluid by a newly developed method involving first, ammonium sulfate precipitation, followed by DEAE-cellulose chromatography and finally purification by Sephadex G-200 gel filtration. A Sephadex G-200 assay which separates the free hemoglobin from the Hb-Hp complex and enables direct measurement of the amount of hemoglobin bound to haptoglobin has been developed.
In order to determine whether the site of polymerization of haptoglobin polymers is distinct from the hemoglobin binding
site, haptoglobin polymers partially resolved on Sephadex G-200, were examined for their ability to bind hemoglobin both by the peroxidase assay and the Sephadex assay. Localization
of the hemoglobin binding site on the component haptoglobin
chains was achieved by determination of the ability of isolated a¹, a² and β chains to bind hemoglobin by means of the Sephadex assay. The binding of globin, myoglobin and various vertebrate hemoglobins were also examined by the Sephadex assay. The effects of environmental factors on the combination of haptoglobin with hemoglobin has been studied by alteration of ionic strength and of pH and by addition of phenol as a tyrosine analogue. The involvement of amino groups of haptoglobin in binding with hemoglobin has been studied by selective chemical modification of haptoglobin amino groups with three reagents of increasing severity followed by measurement of the ability of the chemically modified
proteins to bind hemoglobin. An approximate determination
of the area of the binding site in the Hb-Hp complex has been made by comparison of the extent of guanidination of amino groups of the individual components with that of the complex and also by comparison with the extent of modification
in dissociated hemoglobin and haptoglobin from the guanidinated complex.
The results of these studies show that the hemoglobin combining site is distinct from the site of polymerization and that the β haptoglobin chain carries the hemoglobin binding site. Haptoglobin combines with globin but not with myoglobin and it binds stoichiometrically with all animal hemoglobins examined except those of the frog and fish, which may be the result of a conformational change in the Hb-Hp complex which causes expulsion of the heme group. It is shown that electrostatic forces cannot be the sole interrnolecular
forces involved in the binding. Chemical modification studies show that the area of contact between the hemoglobin and the haptoglobin in the complex involves only a small area of the haptoglobin molecule of else the area is particularly deficient in lysyl-side chains. It is also shown that amino groups are not directly involved in the binding site and that acylation of amino groups particularly with succinyl group cause a profound change in the haptoglobin molecule and its ability to bind hemoglobin is very much reduced. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/35840 |
| Date | January 1968 |
| Creators | Chan, Gwendolyn Faye Quen |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
Page generated in 0.0024 seconds