Haptoglobins are a group of glycoproteins found in normal human serum and are capable of binding hemoglobin both in vivo and in vitro. Haptoglobin 2-1, one of the common haptoglobin phenotypes was isolated in large quantity from the ascites fluid. Upon reduction and alkylation in the presence of 8 M urea, haptoglobin dissociates into two different classes of polypeptide chains, the α and the β chains.
The haptoglobin β chain, which is the larger and the only component that contains carbohydrate has been studied in the present investigation. The homogeneity of this polypeptide chain was examined using different criteria, such as DEAE-cellulose chromatography, gel filtration chromatography, urea starch gel and SDS disc gel electrophoresis. The β chain was found to be homogeneous as judged by the above different criteria. The amino acid composition and carbohydrate composition as well as the N-terminal amino acid were determined.
The β chain isolated in the -S-methyl-carboxyamide form was extensively aggregated. However, upon chemical modification using succinic anhydride, the modified β chain was completely disaggregated, and it was possible to determine the accurate molecular weight of the β chain using this succinyl β chain.
In order to elucidate the primary structure of the β chain, a cyanogen bromide reaction, which cleaved selectively at the methionine residues was performed. Though this cleavage was incomplete in β chain, three fragments were isolated and characterized.
The amino acid sequence of the N-terminal region, the α, β interchain disulfide region and the carbohydrate attachment
site region were determined. The alignment of some of the cyanogen bromide cleavage fragments were achieved using the methionine
peptides from a tryptic digest of the β chain to overlap these fragments. The methionine peptides were isolated by a selective diagonal paper electrophoretic technique.
The linkage between the protein and the carbohydrate in haptoglobin was established to be an aspartamido-glycosyl linkage This was demonstrated by the resistance of the linkage under mild alkali treatment and the actual isolation of an aspartic-carbo-hydrate complex.
A different approach was taken to determine the primary structure of the β chain. The β chain was maleylated at its e-amino lysyl groups with maleic anhydride and later digested with trypsin. The maleylated fragments were isolated and partially
characterized. Furthermore, five groups of arginine peptides were isolated. An overall structure of the β chain was deduced from the studies of the cyanogen bromide peptides, methionine peptides, tryptic maleylated peptides and the arginine peptides. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/34683 |
| Date | January 1970 |
| Creators | Hew, Choy-Leong |
| 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. |
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