The Limited Cleavage of Native Collagen with Chymotrypsin, Trypsin, and Cyanogen Bromide

Paul Bornstein, Andrew Kang, Karl A. Piez

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

Soluble rat skin collagen was cleaved with chymotrypsin, trypsin, and CNBr under conditions which did not disrupt the helical conformation of the protein. Chymotrypsin and CNBr reduced the content of double-chain β components and converted both α chains and β components to altered α chains. The amino acid compositions of the products of chymotryptic digestion (α1Chy and α2Chy) resembled that of the original α1 and α2 chains but were characterized by lower tyrosine and methionine contents. α1chy and α2Chy each possessed a single new N-terminal glycine and each lacked a sequence from the crosslink region of α1 and α2. Previous studies utilizing complete cleavage of α chains with CNBr have shown that these sequences give rise to peptides containing tyrosine and methionine-derived homoserine. These peptides exist in two forms in digests of both chains, a lysyl-containing form (α1-CB-1 and α2-CB-1) and a lysyl-derived aldehyde-containing form (α1-CB-1a and α2-CB-1a). The aldehydes have been implicated in the formation of intramolecular cross-links. Lysine is in position five in both α1-CB-1, which contains 15 amino acids, and α2-CB-1, which contains 14 amino acids. The altered a chains produced by limited cleavage with CNBr (α1CNBr and α2CNBr) resembled α1Chy and α2Chy in their amino acid composition and chromatographic and electrophoretic behavior. In addition the small peptides released by CNBr from a collagen in which lysyl-derived aldehydes were largely absent were shown to be α1-CB-1 and α2-CB-1. These data indicate that both chymotrypsin and CNBr cleave the chains of native collagen C terminal to the cross-linking site, near the N terminus of the molecule. This region is susceptible to enzymatic and chemical cleavage presumably because its amino acid composition prohibits the formation of the helical conformation typical of most of the collagen molecule. Because of its more limited specificity trypsin does not attack the N-terminal region of most native collagen preparations. However, in collagen in which the lysyl residues in α1-CB-1 and α2-CB-1 have not undergone transformation to aldehydes, cleavage does occur at these positions. Two peptides were isolated and their amino acid compositions were found to be identical with the first five amino acids in α1-CB-1 and α2-CB-1. These results confirm the findings of the chymotrypsin and CNBr experiments and demonstrate that the sequences represented by α1-CB-1 and α2-CB-1 are actually at the N-terminal ends of the α1 and α1 chains. The aldehyde-precursor lysyl residues are therefore located five amino acids from the N-terminal end of the collagen molecule.

Original languageEnglish (US)
Pages (from-to)3803-3812
Number of pages10
JournalBiochemistry
Volume5
Issue number12
DOIs
StatePublished - Dec 1 1966
Externally publishedYes

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Cyanogen Bromide
Collagen
Amino Acids
Aldehydes
Chymotrypsin
Peptides
Chemical analysis
Methionine
Molecules
Tyrosine
Conformations
Homoserine
Protein Conformation
trypsin drug combination chymotrypsin
Glycine
Trypsin
Lysine
Rats
Digestion
Skin

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

The Limited Cleavage of Native Collagen with Chymotrypsin, Trypsin, and Cyanogen Bromide. / Bornstein, Paul; Kang, Andrew; Piez, Karl A.

In: Biochemistry, Vol. 5, No. 12, 01.12.1966, p. 3803-3812.

Research output: Contribution to journalArticle

Bornstein, Paul ; Kang, Andrew ; Piez, Karl A. / The Limited Cleavage of Native Collagen with Chymotrypsin, Trypsin, and Cyanogen Bromide. In: Biochemistry. 1966 ; Vol. 5, No. 12. pp. 3803-3812.
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N2 - Soluble rat skin collagen was cleaved with chymotrypsin, trypsin, and CNBr under conditions which did not disrupt the helical conformation of the protein. Chymotrypsin and CNBr reduced the content of double-chain β components and converted both α chains and β components to altered α chains. The amino acid compositions of the products of chymotryptic digestion (α1Chy and α2Chy) resembled that of the original α1 and α2 chains but were characterized by lower tyrosine and methionine contents. α1chy and α2Chy each possessed a single new N-terminal glycine and each lacked a sequence from the crosslink region of α1 and α2. Previous studies utilizing complete cleavage of α chains with CNBr have shown that these sequences give rise to peptides containing tyrosine and methionine-derived homoserine. These peptides exist in two forms in digests of both chains, a lysyl-containing form (α1-CB-1 and α2-CB-1) and a lysyl-derived aldehyde-containing form (α1-CB-1a and α2-CB-1a). The aldehydes have been implicated in the formation of intramolecular cross-links. Lysine is in position five in both α1-CB-1, which contains 15 amino acids, and α2-CB-1, which contains 14 amino acids. The altered a chains produced by limited cleavage with CNBr (α1CNBr and α2CNBr) resembled α1Chy and α2Chy in their amino acid composition and chromatographic and electrophoretic behavior. In addition the small peptides released by CNBr from a collagen in which lysyl-derived aldehydes were largely absent were shown to be α1-CB-1 and α2-CB-1. These data indicate that both chymotrypsin and CNBr cleave the chains of native collagen C terminal to the cross-linking site, near the N terminus of the molecule. This region is susceptible to enzymatic and chemical cleavage presumably because its amino acid composition prohibits the formation of the helical conformation typical of most of the collagen molecule. Because of its more limited specificity trypsin does not attack the N-terminal region of most native collagen preparations. However, in collagen in which the lysyl residues in α1-CB-1 and α2-CB-1 have not undergone transformation to aldehydes, cleavage does occur at these positions. Two peptides were isolated and their amino acid compositions were found to be identical with the first five amino acids in α1-CB-1 and α2-CB-1. These results confirm the findings of the chymotrypsin and CNBr experiments and demonstrate that the sequences represented by α1-CB-1 and α2-CB-1 are actually at the N-terminal ends of the α1 and α1 chains. The aldehyde-precursor lysyl residues are therefore located five amino acids from the N-terminal end of the collagen molecule.

AB - Soluble rat skin collagen was cleaved with chymotrypsin, trypsin, and CNBr under conditions which did not disrupt the helical conformation of the protein. Chymotrypsin and CNBr reduced the content of double-chain β components and converted both α chains and β components to altered α chains. The amino acid compositions of the products of chymotryptic digestion (α1Chy and α2Chy) resembled that of the original α1 and α2 chains but were characterized by lower tyrosine and methionine contents. α1chy and α2Chy each possessed a single new N-terminal glycine and each lacked a sequence from the crosslink region of α1 and α2. Previous studies utilizing complete cleavage of α chains with CNBr have shown that these sequences give rise to peptides containing tyrosine and methionine-derived homoserine. These peptides exist in two forms in digests of both chains, a lysyl-containing form (α1-CB-1 and α2-CB-1) and a lysyl-derived aldehyde-containing form (α1-CB-1a and α2-CB-1a). The aldehydes have been implicated in the formation of intramolecular cross-links. Lysine is in position five in both α1-CB-1, which contains 15 amino acids, and α2-CB-1, which contains 14 amino acids. The altered a chains produced by limited cleavage with CNBr (α1CNBr and α2CNBr) resembled α1Chy and α2Chy in their amino acid composition and chromatographic and electrophoretic behavior. In addition the small peptides released by CNBr from a collagen in which lysyl-derived aldehydes were largely absent were shown to be α1-CB-1 and α2-CB-1. These data indicate that both chymotrypsin and CNBr cleave the chains of native collagen C terminal to the cross-linking site, near the N terminus of the molecule. This region is susceptible to enzymatic and chemical cleavage presumably because its amino acid composition prohibits the formation of the helical conformation typical of most of the collagen molecule. Because of its more limited specificity trypsin does not attack the N-terminal region of most native collagen preparations. However, in collagen in which the lysyl residues in α1-CB-1 and α2-CB-1 have not undergone transformation to aldehydes, cleavage does occur at these positions. Two peptides were isolated and their amino acid compositions were found to be identical with the first five amino acids in α1-CB-1 and α2-CB-1. These results confirm the findings of the chymotrypsin and CNBr experiments and demonstrate that the sequences represented by α1-CB-1 and α2-CB-1 are actually at the N-terminal ends of the α1 and α1 chains. The aldehyde-precursor lysyl residues are therefore located five amino acids from the N-terminal end of the collagen molecule.

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