Ryhänen L, Zaragoza E J, Uitto J
Arch Biochem Biophys. 1983 Jun;223(2):562-71. doi: 10.1016/0003-9861(83)90621-5.
Native collagen polypeptides exist in a unique triple helical conformation resistant to most proteinases. In this study, the stability of type I collagen triple helix, employing a mixture of trypsin and alpha-chymotrypsin as a proteolytic probe, was examined. The degradation of type I [3H]collagen was monitored as 3H-labeled peptides soluble in trichloroacetic acid (TCA) or by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis. In one set of experiments, collagen substrates were preincubated at various temperatures for up to 8 h, followed by a 15-min proteolytic treatment at the same temperature. At 43 degrees C, most of the collagen was degraded, while the fraction of the substrate degraded at 40, 38, and 35 degrees C was 53, 41 and 19%, respectively. This fraction was independent of the preincubation time which varied from 10 to 480 min. Thus, at any given temperature, a constant fraction of the collagen substrate was susceptible to proteolysis. Measurement of the midpoint temperature (Tm) of the helix to coil transformation for type I collagen, at neutral pH employing an increasing temperature gradient and brief proteolysis at the individual temperatures, indicated a value of 38.8 degrees C. However, determination of the Tm by employing proteolytic digestions at a constant temperature (30 degrees C) using conditions under which the nonhelical peptides are readily digested to TCA-soluble peptides while native collagen resists such proteolysis, indicated a value of 42.7 degrees C. In further studies, collagen was subjected to continuous proteolysis for up to 24 h. A large fraction of collagen was digested at 30 or 34 degrees C, temperatures well below the Tm of the helix to coil transformation. SDS-polyacrylamide gel electrophoresis of the degradation products obtained at these temperatures revealed multiple cleavage fragments. Finally, temperature double-jump experiments indicated that the destabilization of the triple helix is reversible provided that the Tm of the substrate is not exceeded. The results provide evidence for reversible and local relaxation of the collagen triple helix.
天然胶原蛋白多肽以独特的三螺旋构象存在，对大多数蛋白酶具有抗性。在本研究中，使用胰蛋白酶和α-胰凝乳蛋白酶的混合物作为蛋白水解探针，检测了I型胶原三螺旋的稳定性。通过监测可溶于三氯乙酸（TCA）的3H标记肽或通过十二烷基硫酸钠（SDS）-聚丙烯酰胺平板凝胶电泳来监测I型[3H]胶原的降解。在一组实验中，将胶原底物在不同温度下预孵育长达8小时，然后在相同温度下进行15分钟的蛋白水解处理。在43℃时，大部分胶原被降解，而在40℃、38℃和35℃下被降解的底物比例分别为53%、41%和19%。该比例与预孵育时间无关，预孵育时间从10分钟到480分钟不等。因此，在任何给定温度下，恒定比例的胶原底物易受蛋白水解作用。在中性pH条件下，采用升高的温度梯度并在各个温度下进行短暂蛋白水解，测量I型胶原螺旋-卷曲转变的中点温度（Tm），其值为38.8℃。然而，在恒定温度（30℃）下进行蛋白水解消化来测定Tm，在该条件下非螺旋肽很容易被消化成可溶于TCA的肽，而天然胶原则抵抗这种蛋白水解作用，其值为42.7℃。在进一步的研究中，将胶原进行长达24小时的连续蛋白水解。在30℃或34℃下，很大一部分胶原被消化，这两个温度远低于螺旋-卷曲转变的Tm。在这些温度下获得的降解产物的SDS-聚丙烯酰胺凝胶电泳显示出多个切割片段。最后，温度双跳实验表明，只要不超过底物的Tm，三螺旋的去稳定化是可逆的。这些结果为胶原三螺旋的可逆和局部松弛提供了证据。
