Extent and topography of the acetylation of calcified chicken-bone collagen.
Acetic anhydride in anhydrous acetone-triethylamine reacts with decalcified bone collagen in two ways. It completely acetylates the e-amino groups of lysine and hydroxylysine and causes a progressive change in the protein matrix, such that increasing amounts of degraded peptides become soluble in alkaline phosphate solution. The peptides produced have molecular weights ranging from 100,000 to less than 2,000 daltons. The number of peptides obtained indicates that there are about 10 bonds cleaved per chain. Ball-mill grinding, particle size of the decalcified bone fragments, and age of the chicken do not affect the fraction of collagen that can be solubilized. The appearance of peptides begins at the point where 90% of the e-amino groups are modified. Both the phosphate-soluble and phosphate-insoluble fractions were essentially completely N-acetylated. Calcified bone under the same conditions is rapidly acetylated to only 45% of the total e-amino groups. With further additions of reagent, the degree of modification of the phosphate-insoluble matrix stabilizes at 50 +/- 4%. At the same time, progressively larger amounts of peptides, soluble in alkaline phosphate solution, are produced. The peptides, which approach 100% N-acetylation, have a distribution of molecular weight from 2000 to 75,000 daltons, indicating 10-15 disrupted peptide bonds per chain. The peptides are initially derived from the more accessible regions of the matrix and subsequently are produced from increasingly inaccessible regions. Chromatography of samples of calcified bone collagen, which were acetylated nearly to completion, indicate that the lysyl and hydroxylysyl residues which are most difficult to modify in the calcified bone are located along continuous regions of the collagen chains. Chromatography of partially acetylated samples also show that all regions of the chains are not uniformly accessible to modification, and further, no single region of every alpha chain is exposed to an identical environment.
D000085:Acetates / Q000494:pharmacology; D000107:Acetylation; D000812:Anhydrides; D000818:Animals; D001857:Bone Matrix / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism*; D002113:Calcification, Physiologic; D003094:Collagen / Q000032:analysis / Q000378:metabolism*; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism
D000085:Acetates; D000812:Anhydrides; D010455:Peptides; D003094:Collagen