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

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Abstract
Characterization of the cyanogen bromide (CNBr) fragments of the .beta. chain of human haptoglobin revealed 5 major fragments resulting from cleavage of 4 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 5 cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact .beta. chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin .beta. chain and cyanogen bromide fragments identified 139 residues, or about 55% of the .beta.-chain molecule. The placement of the fragments within the .beta.-chain molecule was established by sequence analysis of whole .beta.-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 .beta.-chain fragment, CNBr III, covalently attached to the intact .alpha.1 chain by a single disulfide bond. The .beta. chain had 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; 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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