National University of Singapore Tissue Engineering Program, Department of Orthopedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Acta Biomater. 2010 Aug;6(8):3146-51. doi: 10.1016/j.actbio.2010.02.004. Epub 2010 Feb 6.
Collagen contains the unique imino acid hydroxyproline (HyPro), which is involved in the stabilization of this triple helical molecule. The concentration of HyPro is customarily used to calculate the total collagen content in a cell culture environment and in acid hydrolysates of normal and pathophysiological tissues. Radiolabelling, chromatographic and calorimetric assays have been developed over the years for the accurate determination of collagen content through HyPro estimation. Recently, the Sircol Collagen Assay (SCA) has been almost exclusively adopted as the fastest and simplest colorimetric method for the determination of collagen concentration in complex protein solutions. We show here that the colorimetric SCA, which is based on the binding of Sirius red (SR) to collagen, is flawed by interference of non-collagenous proteins (e.g. serum). In fact, we demonstrate that SCA in cell culture systems and tissue hydrolysates results in a dramatic overestimation of collagen content ranging from 3- to 24-fold. In order to rescue this otherwise very practical assay, we introduce a simple purification procedure that allows the removal of interfering non-collagenous proteins from culture media and tissue samples so that accurate measurements with SCA are now possible.
胶原蛋白含有独特的亚氨基酸羟脯氨酸（HyPro），它参与了这种三螺旋分子的稳定。HyPro 的浓度通常用于计算细胞培养环境中和正常及病理组织酸水解物中的总胶原蛋白含量。多年来，已经开发了放射性标记、色谱和量热测定法，通过 HyPro 估计来准确测定胶原蛋白含量。最近，Sircol 胶原蛋白测定法（SCA）几乎被专门用作测定复杂蛋白质溶液中胶原蛋白浓度的最快和最简单的比色法。我们在这里表明，基于 Sirius 红（SR）与胶原蛋白结合的比色 SCA 受到非胶原蛋白（例如血清）的干扰。事实上，我们证明 SCA 在细胞培养系统和组织水解物中导致胶原蛋白含量的严重高估，范围从 3 倍到 24 倍。为了挽救这个非常实用的测定法，我们引入了一种简单的纯化程序，可以从培养基和组织样品中去除干扰的非胶原蛋白，从而可以使用 SCA 进行准确测量。
