Studies on the Location of Intermolecular Cross-Links in Collagen. Isolation of a CNBr Peptide Containing S-Hydroxylysinonorleucinet

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Abstract

Purified soluble rat-tail tendon collagen, in vitro reconstituted native-type fibrils, and intact tail tendon were separately cleaved with cyanogen bromide at the methionyl residues after reduction with tritiated sodium borohydride, and the resulting peptides were fractionated by ion exchange and molecular sieve chromatography. The cross-link related compounds in the CNBr peptides were identified and located by amino acid analysis and a scintillation spectrometry after 6 n HC1 and 2 n NaOH hydrolyses. The soluble collagen yielded all 15 specific peptides (9 from al and 6 from a2) in the expected amounts. No 3-hydroxylysinonorleucine or posthistidine compound were found in any of the peptide fractions. In the digests of the reduced, reconstituted nativetype fibrils and in the intact tendons, however, the NH2-terminal peptides, al-CBlAlcl and a2-CBlAld, and fe-CBl and öiiCBl, were present in much decreased amounts. Most of the larger CNBr peptides contained partial residues of 5-hydroxylysinonorleucine, or the posthistidine compound, or both. These data indicate that during fibrillogenesis of rat- tail tendon in vitro and in vivo, the NH2-terminal, aldehyde-bearing peptides participate in the formation of intermolecular cross-links and further that the intermolecular cross-links form at several different sites along the neighboring molecules. Although all of the possible cyanogen bromide peptides bearing intermolecular cross-links were not isolated in pure form due to the complexity of the mixture, one such peptide of mol wt 18, 000 containing one residue of 5-hydroxylysinonorleucine was isolated and purified. The amino acid compositions of the peptide was consistent, within experimental error, with the sum of that for al-CBlAld and al-CB6, indicating its origin as an intermolecular amino-car-boxyl cross-link. The presence of βu-CBl and βirCBl, the intramolecular cross-link peptides, in the digests of soluble collagen and their disappearance in the digests of the nativetype fibrils and the intact tendons indicate that the intramolecular cross-links react further to form higher order aggregates.

Original languageEnglish (US)
Pages (from-to)1828-1835
Number of pages8
JournalBiochemistry
Volume11
Issue number10
DOIs
StatePublished - May 1 1972
Externally publishedYes

Fingerprint

Collagen
Peptides
Tendons
Bearings (structural)
Tail
Cyanogen Bromide
Rats
Amino Acids
Ion Exchange
Molecular sieves
Scintillation
Chromatography
Aldehydes
Spectrometry
Gel Chromatography
Hydrolysis
Spectrum Analysis
Ion exchange
Railroad cars
Molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Studies on the Location of Intermolecular Cross-Links in Collagen. Isolation of a CNBr Peptide Containing S-Hydroxylysinonorleucinet. / Kang, Andrew.

In: Biochemistry, Vol. 11, No. 10, 01.05.1972, p. 1828-1835.

Research output: Contribution to journalArticle

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abstract = "Purified soluble rat-tail tendon collagen, in vitro reconstituted native-type fibrils, and intact tail tendon were separately cleaved with cyanogen bromide at the methionyl residues after reduction with tritiated sodium borohydride, and the resulting peptides were fractionated by ion exchange and molecular sieve chromatography. The cross-link related compounds in the CNBr peptides were identified and located by amino acid analysis and a scintillation spectrometry after 6 n HC1 and 2 n NaOH hydrolyses. The soluble collagen yielded all 15 specific peptides (9 from al and 6 from a2) in the expected amounts. No 3-hydroxylysinonorleucine or posthistidine compound were found in any of the peptide fractions. In the digests of the reduced, reconstituted nativetype fibrils and in the intact tendons, however, the NH2-terminal peptides, al-CBlAlcl and a2-CBlAld, and fe-CBl and {\"o}iiCBl, were present in much decreased amounts. Most of the larger CNBr peptides contained partial residues of 5-hydroxylysinonorleucine, or the posthistidine compound, or both. These data indicate that during fibrillogenesis of rat- tail tendon in vitro and in vivo, the NH2-terminal, aldehyde-bearing peptides participate in the formation of intermolecular cross-links and further that the intermolecular cross-links form at several different sites along the neighboring molecules. Although all of the possible cyanogen bromide peptides bearing intermolecular cross-links were not isolated in pure form due to the complexity of the mixture, one such peptide of mol wt 18, 000 containing one residue of 5-hydroxylysinonorleucine was isolated and purified. The amino acid compositions of the peptide was consistent, within experimental error, with the sum of that for al-CBlAld and al-CB6, indicating its origin as an intermolecular amino-car-boxyl cross-link. The presence of βu-CBl and βirCBl, the intramolecular cross-link peptides, in the digests of soluble collagen and their disappearance in the digests of the nativetype fibrils and the intact tendons indicate that the intramolecular cross-links react further to form higher order aggregates.",
author = "Andrew Kang",
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N2 - Purified soluble rat-tail tendon collagen, in vitro reconstituted native-type fibrils, and intact tail tendon were separately cleaved with cyanogen bromide at the methionyl residues after reduction with tritiated sodium borohydride, and the resulting peptides were fractionated by ion exchange and molecular sieve chromatography. The cross-link related compounds in the CNBr peptides were identified and located by amino acid analysis and a scintillation spectrometry after 6 n HC1 and 2 n NaOH hydrolyses. The soluble collagen yielded all 15 specific peptides (9 from al and 6 from a2) in the expected amounts. No 3-hydroxylysinonorleucine or posthistidine compound were found in any of the peptide fractions. In the digests of the reduced, reconstituted nativetype fibrils and in the intact tendons, however, the NH2-terminal peptides, al-CBlAlcl and a2-CBlAld, and fe-CBl and öiiCBl, were present in much decreased amounts. Most of the larger CNBr peptides contained partial residues of 5-hydroxylysinonorleucine, or the posthistidine compound, or both. These data indicate that during fibrillogenesis of rat- tail tendon in vitro and in vivo, the NH2-terminal, aldehyde-bearing peptides participate in the formation of intermolecular cross-links and further that the intermolecular cross-links form at several different sites along the neighboring molecules. Although all of the possible cyanogen bromide peptides bearing intermolecular cross-links were not isolated in pure form due to the complexity of the mixture, one such peptide of mol wt 18, 000 containing one residue of 5-hydroxylysinonorleucine was isolated and purified. The amino acid compositions of the peptide was consistent, within experimental error, with the sum of that for al-CBlAld and al-CB6, indicating its origin as an intermolecular amino-car-boxyl cross-link. The presence of βu-CBl and βirCBl, the intramolecular cross-link peptides, in the digests of soluble collagen and their disappearance in the digests of the nativetype fibrils and the intact tendons indicate that the intramolecular cross-links react further to form higher order aggregates.

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