Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution.
Procedures for the preparation of soluble collagen from rat skin and tail tendon were reviewed and revised to permit the preparation of native monomeric collagen with intact nonhelical ends. The degree of intactness was estimated from the tyrosine content, which is present only in the nonhelical ends, and by mobility of the COOH-terminal cyanogen bromide peptide of the alpha1 chain on sodium dodecyl sulfate gels. The amount of covalently cross-linked polymeric material present was estimated by molecular sieve chromatography of denatured samples. Rapid purification in the cold was sufficient to prevent or greatly reduce proteolytic alteration. Fractionation by salt precipitation at acid pH was effective in reducing the content of polymeric material. Rat tail tendon yielded completely intact native collagen, but some high molecular weight aggregates remained. Collagen from the skin of lathyritic rats was easier to obtain free of aggregates, but contained about 1 less tyrosine residue per alpha1 chain even when isolated in the presence of enzyme inhibitors. Proton NMR spectra of denatured acidic solutions of these preparations showed that 4 to 5 tyrosine residues per alpha chain were present, confirming the chemical analysis. Spectra of the native molecule showed that about the same number of tyrosine residues per chain are in rapid motion, unlike residues in the helical portion of the molecule, a result which shows that the nonhelical ends of the native molecule are unstructured in acidic solution.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism; D007842:Lathyrism / Q000378:metabolism; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D009928:Organ Specificity; D011487:Protein Conformation; D051381:Rats; D012867:Skin / Q000032:analysis*; D013623:Tail; D013710:Tendons / Q000032:analysis* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D003094:Collagen