Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin

Alexander Kurosky, Regine E. Hay, Han Hwa Kim, Billy Touchstone, Marilyn A. Rasco, Barbara H. Bowman

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    Abstract

    Characterization of the cyanogen bromide (CNBr) fragments of the β chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact β chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin β chain and cyanogen bromide fragments identified 139 residues, or about 55% of the β-chain molecule. The placement of the fragments within the β-chain molecule was established by sequence analysis of whole β chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a β-chain fragment, CNBr III, covalently attached to the intact α1 chain by a single disulfide bond. The β chain was shown to have primary structural similarities to the chymotrypsin family of serine proteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.

    Original languageEnglish (US)
    Pages (from-to)5326-5336
    Number of pages11
    JournalBiochemistry
    Volume15
    Issue number24
    StatePublished - 1976

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    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Kurosky, A., Hay, R. E., Kim, H. H., Touchstone, B., Rasco, M. A., & Bowman, B. H. (1976). Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin. Biochemistry, 15(24), 5326-5336.