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

Abstract

Characterization of the cyanogen bromide (CNBr) fragments of the beta 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 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 alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. 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.

Model for haptoglobin heavy chain based upon structural homology.

“…This residue is an Asp in HpH, as pointed out by Kurosky et al (6), suggesting trypsin-like specificity. The restriction of the elastase substrate specificity to small side chains is believed to be due to blockage of the hydrophobic pocket that binds the side chain of the substrate by Val C216 and Thr C226, which replace glycines found in chymotrypsin and trypsin (12).…”

“…Kurosky et al (4)(5)(6) have suggested that the active-site residues of the serine proteases, Ser C195, His C57, and Asp C102, are Ala, Lys, and Asp in HpH, respectively. In addition, the Asp that precedes the reactive Ser is conserved.…”

“…A variety of experiments indicate that it is the Hp heavy chains (HpH) that bind a hemoglobin a# dimer during complex formation (2, 3). Kurosky et al (4)(5)(6) have reported that the sequence of HpH is clearly homologous to the mammalian serine protease family. The possibility exists, therefore, of determining the tertiary structure of HpH by fitting the sequence into the known structure for the serine proteases (7,8).…”

Covalent structure of human haptoglobin: a serine protease homolog.

“…Purification and characterization of the CNBr peptides were reported by Kurosky et al (24). Purified CNBr fragments II, III, IV, and V were subjected to hydrolysis* with chymotrypsin, trypsin, staphylococcal protease, and thermolysin.…”

Snake Venom Toxins

“…This could be due to an anomalous reaction of the expected aminoterminal tryptophan of CB3 during the digestion with cyanogen bromide. Anomalous cleavage of protein chains amino-terminal or carboxyl-terminal to tryptophan has been reported by, for example, Braunitzer and Aschauer [20] and Kurosky et al [21].…”