通过荧光法测定脯氨酸和羟脯氨酸对细胞培养中胶原代谢的研究。
A study of collagen metabolism in cell cultures by fluorometric determination of proline and hydroxyproline.
作者信息
Bellon G, Randoux A, Borel J P
出版信息
Coll Relat Res. 1985 Nov;5(5):423-35. doi: 10.1016/s0174-173x(85)80030-3.
A technique of derivatization of proline (Pro) and 4-hydroxyproline (Hyp) by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole permitted the measurement of Pro and Hyp radioactivities, concentrations, and specific activities in the main fractions separated from cultures of fibroblast cells (extracellular collagen and non-collagen proteins, intracellular free Pro and Hyp, Pro- and Hyp-containing peptides, procollagen, and non-collagen proteins). The evaluation of collagen in the medium was obtained from as few as 10(4) cells. The method might advantageously replace [14C] Pro or [3H] Pro incorporation studies. It permits measurement of the size of the Pro pool and the amount of peptides formed by intracellular catabolism of collagen. It demonstrates that the time necessary for a full equilibration of intracellular Pro and intracellular collagen is longer than is generally believed. It avoids the uncertainties of protein labelling, which may vary with uncontrolled variations of the intracellular Pro specific activity.
一种利用7-氯-4-硝基苯并-2-恶唑-1,3-二唑对脯氨酸（Pro）和4-羟脯氨酸（Hyp）进行衍生化的技术，能够测定从成纤维细胞培养物中分离出的主要组分（细胞外胶原蛋白和非胶原蛋白、细胞内游离Pro和Hyp、含Pro和Hyp的肽、前胶原和非胶原蛋白）中的Pro和Hyp放射性、浓度及比活性。仅从10⁴个细胞就能评估培养基中的胶原蛋白。该方法可能会有利地取代[¹⁴C]Pro或[³H]Pro掺入研究。它允许测量Pro池的大小以及胶原蛋白细胞内分解代谢形成的肽的量。结果表明，细胞内Pro与细胞内胶原蛋白完全平衡所需的时间比一般认为的要长。它避免了蛋白质标记的不确定性，这种不确定性可能会因细胞内Pro比活性的不受控制的变化而有所不同。
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