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

    Research output: Contribution to journalArticle

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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

    Fingerprint

    Cyanogen Bromide
    Sequence Analysis
    Haptoglobins
    Serine
    Catalytic Domain
    Gels
    Chymotrypsinogen
    human HP protein
    Homoserine
    Carboxypeptidases
    Molecules
    Fibrinolysin
    Guanidine
    Chymotrypsin
    Serine Proteases
    Disulfides
    Alanine
    Sepharose
    Trypsin
    Gel Chromatography

    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.

    Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin. / Kurosky, Alexander; Hay, Regine E.; Kim, Han Hwa; Touchstone, Billy; Rasco, Marilyn A.; Bowman, Barbara H.

    In: Biochemistry, Vol. 15, No. 24, 1976, p. 5326-5336.

    Research output: Contribution to journalArticle

    Kurosky, A, Hay, RE, Kim, HH, Touchstone, B, Rasco, MA & Bowman, BH 1976, 'Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin', Biochemistry, vol. 15, no. 24, pp. 5326-5336.
    Kurosky A, Hay RE, Kim HH, Touchstone B, Rasco MA, Bowman BH. Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin. Biochemistry. 1976;15(24):5326-5336.
    Kurosky, Alexander ; Hay, Regine E. ; Kim, Han Hwa ; Touchstone, Billy ; Rasco, Marilyn A. ; Bowman, Barbara H. / Characterization of the cyanogen bromide fragments of the β chain of human haptoglobin. In: Biochemistry. 1976 ; Vol. 15, No. 24. pp. 5326-5336.
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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.",
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