Miller E J, Narkates A J, Niemann M A
Department of Biochemistry, University of Alabama, Birmingham 35294.
Anal Biochem. 1990 Oct;190(1):92-7. doi: 10.1016/0003-2697(90)90139-z.
A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).
最近描述的一种氨基酸分析方法已被修改并适用于定量胶原蛋白水解产物中常见的独特产物混合物。该方法包括用9-芴基甲基氯甲酸酯（FMOC-CL）对水解产物进行柱前衍生化，在反相柱上对衍生物和过量试剂进行色谱分离，并基于衍生物的荧光特性进行定量。该方法利用了FMOC试剂易于形成稳定衍生物的特点。使用三元梯度系统，可在35分钟内完成所有成分分辨率良好的完整氨基酸分析。该方法的灵敏度与其他衍生物所达到的水平相当，并且每种衍生物的荧光响应在1-800皮摩尔的整个范围内呈线性。鉴于这些参数，该方法允许对相当于一条胶原蛋白链（分子量100,000）一皮摩尔的100纳克胶原蛋白进行完整的氨基酸分析。
