An improved method for the determination of hydroxyproline in rat skin.
An improved method for the determination of hydroxyproline in rat skin.
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一种测定大鼠皮肤中羟脯氨酸的改进方法。
DOI：
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发表时间：
1969
影响因子：
6.5
通讯作者：
P. Cheng
中科院分区：
文献类型：
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作者：
P. Cheng
确定了大鼠皮肤水解羟脯氨酸的最佳工艺条件：4盐酸，温度110℃，在铁氟龙螺纹管中水解22小时。对Bergman和Loxley氯胺T氧化法测定羟脯氨酸的分光光度法进行了改进，结果重复性好，节省了大量时间。没有尝试对酸性水解产物进行蒸发，因为在本方案中，即使对水解产物进行中和也证明是不必要的。在测色前用异丙醇稀释最后一步省略。研究了NaCl对羟基脯氨酸溶液吸光度的“盐效应”；直到NaCl达到0.6 m时才检测到影响，这一信息对于使用高渗盐溶液从生物组织中提取中性可溶性胶原蛋白的研究人员来说是重要的。甘氨酸和脯氨酸，这是胶原蛋白的另外两个主要成分，被发现不干扰羟基脯氨酸的分析。在回收研究中，羟脯氨酸本身和添加到组织中的羟脯氨酸被定量回收，表明该亚胺酸在水解条件下没有被破坏。对未知溶液的显影光谱纯度进行了验证。
ABSTRACT The optimal conditions for hydrolysis of rat skin for maximal yield of hydroxyproline were determined:4 N HCl at a temperature of 110° C for 22 hours in Teflon-lined screw-capped tube. The spectrophotometry method of Bergman and Loxley for the determination of hydroxyproline based on oxidation by Chloramine T was modified to give reproducible results with considerable savings of time. Evaporation of the acid hydrolysate was not attempted, because even neutralization of the hydrolysate was proved unnecessary in the present scheme. The final dilution step using isopropanol before color measurement was omitted. However, the “salt effect” of NaCl on the absorbance of treated hydroxyproline solution was studied; no effect was detected until NaCl reached 0.6 M. This information is important for investigators employing hypertonic salt solution for the extraction of neutral soluble collagen from biological tissues. Glycine and proline, which are the other two major constituents of collagens were found not to interfere with the hydroxyproline analysis. Hydroxyproline by itself and hydroxyproline added to tissue were recovered quantitatively in recovery studies, indicating no destruction of this imino acid under the hydrolyzing conditions. The purity of spectra of the developed color for unknown solutions was demonstrated.