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Ehrlich chromogens, probable cross-links in elastin and collagen.

ABSTRACT: (1) Proteolytic digests of tissue elastin contain material which reacts with dimethylaminobenzaldehyde in acid solution (Ehrlich's reagent) to give a cherry-pink colour. This Ehrlich chromogen(s) [EC(s)] is similar to but not identical with EC(s) previously demonstrated in tissue collagens [Scott, Hughes & Shuttleworth (1979) Biosci. Rep. 1, 611-618]. Both ECs react with diazonium salts in acid to give coloured products. (2) Diazobenzene linked via a phenolic ester to polyacrylamide beads (Biogel P10) has been used to absorb ECs specifically and almost quantitatively from proteolytic digests. The coupled deeply coloured azo-EC-peptides were then recovered after mild alkaline cleavage from the support and purified by gel chromatography. (3) Using 15N-labelled NaNO2, the collagen azo-EC-peptides were prepared, and 15N abundance measured therein. The molar absorption coefficient of the azo-EC group was calculated (18,700) based on the assumption that each azo-EC group contained one 15N atom. (4) Collagen azo-EC-peptides contained glucose and galactose, whereas elastin azo-EC peptides did not. The amino acid patterns of the two peptides were quite different, the former being rich in polar amino acids, the latter containing much alanine. The patterns were compatible with an origin from the cross-linking regions of collagen and elastin respectively. (5) Quantitative (molar) comparisons of the azo-EC group content with amino acid, amino end-group and sugar contents, and azo-EC peptide molecular mass, suggest that a structure is present in the collagen azo-EC-peptides containing two EC groups shared between four peptide chains. Three peptide chains probably meet at each (cross-linking) EC group. (6) Based on this structure, about 15% of adult bovine skin collagen contains EC groups.

SUBMITTER: Kemp PD

PROVIDER: S-EPMC1149157 | biostudies-other | 1988 Jun

REPOSITORIES: biostudies-other

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Project description:The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

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Ehrlich chromogens, probable cross-links in elastin and collagen.

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