Chandrakasan G, Torchia D A, Piez K A
J Biol Chem. 1976 Oct 10;251(19):6062-7.
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.
对从大鼠皮肤和尾腱制备可溶性胶原蛋白的方法进行了回顾和修订，以制备具有完整非螺旋末端的天然单体胶原蛋白。完整性程度通过仅存在于非螺旋末端的酪氨酸含量以及α1链的COOH末端溴化氰肽在十二烷基硫酸钠凝胶上的迁移率来估计。通过对变性样品进行分子筛色谱法来估计存在的共价交联聚合物材料的量。在低温下快速纯化足以防止或大大减少蛋白水解改变。在酸性pH下通过盐沉淀进行分级分离有效地降低了聚合物材料的含量。大鼠尾腱产生了完全完整的天然胶原蛋白，但仍残留一些高分子量聚集体。来自患骨生成异常大鼠皮肤的胶原蛋白更容易获得无聚集体的状态，但即使在存在酶抑制剂的情况下分离，每条α1链也少约1个酪氨酸残基。这些制剂的变性酸性溶液的质子核磁共振光谱表明每条α链存在4至5个酪氨酸残基，证实了化学分析结果。天然分子的光谱表明每条链中大约相同数量的酪氨酸残基处于快速运动中，这与分子螺旋部分中的残基不同；这一结果表明天然分子的非螺旋末端在酸性溶液中是无结构的。
