Simultaneous determination of the reducible and nonreducible cross-links of connective tissue. Analysis of mineralized and nonmineralized bone collagen.
Secondary amine cross-links occur in collagen and elastin from a number of tissue sources. Quantification of these cross-links by amino acid analysis is complicated by the problem of separating cross-links, which are often minor components, from the more common amino acids and also because relatively large amounts of a cross-link are required to determine a color factor. A specific radioactive labeling method has been developed and used to quantify cross-links in bone collagen. Primary amines such as lysine and hydroxylysine are first guanidinated with 3,5-dimethylpyrazole-1-carboxamidine nitrate (DMPC). Secondary amines, which are unreactive with DMPC, are then quantitatively cyanoethylated with [14C]acrylonitrile. This procedure can be used to detect any secondary amine cross-link, with higher sensitivity than ninhydrin analysis, in peptide form as well as in acid hydrolysates. It is applied here in conjunction with [3H]NaBH4 reduction to simultaneously quantify Schiff base cross-links and amounts of in vivo reduction of Schiff bases in mineralized versus nonmineralized bovine bone.
Graham
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Department of Biochemistry, University of North Carolina, Chapel Hill 27514.
Mechanic
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D000588:Amines; D000818:Animals; D015519:Bone Density; D001842:Bone and Bones / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002855:Chromatography, Thin Layer; D003094:Collagen / Q000032:analysis*; D003238:Connective Tissue / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000493:pharmacokinetics*; D006168:Guinea Pigs; D008239:Lysine; D014357:Trypsin
D000588:Amines; D003432:Cross-Linking Reagents; D003094:Collagen; D014357:Trypsin; D008239:Lysine