Hazra A K, Chock S P, Albers R W
Anal Biochem. 1984 Mar;137(2):437-43. doi: 10.1016/0003-2697(84)90110-6.
Conditions are described for precise quantitative measurement of microgram protein samples by spectrophotometric determination of the trinitrobenzene derivatives of amino acids in hydrolysates. The mean molar absorbances of individual amino acids were measured and the effective molar absorbance for use in protein measurements of 1.9 X 10(4) A M-1 cm-1 has been determined. From measurements using the trinitrobenzene sulfonate and fluorescamine reagents, and the published data on the o-phthaldialdehyde method, the molar absorption coefficients and the relative fluorescent yields are compared for the amino acids derivatives found in protein hydrolysates. The coefficients of variation for the trinitrobenzene derivatives are less than that for either the fluorescamine or the o-phthaldialdehyde derivatives. The color yields for five soluble proteins were also compared using the Lowry, Bradford, and trinitrobenzene sulfonate reagents. The results show that the described trinitrobenzene sulfonate method is more sensitive and produces a threefold smaller variation in absorbance per milligram protein than either the Lowry or the Bradford methods.
本文描述了通过分光光度法测定水解产物中氨基酸的三硝基苯衍生物来精确定量测量微克级蛋白质样品的条件。测量了各氨基酸的平均摩尔吸光度，并确定了用于蛋白质测量的有效摩尔吸光度为1.9×10⁴ A M⁻¹ cm⁻¹。通过使用三硝基苯磺酸盐和荧光胺试剂进行测量，并结合邻苯二甲醛法的已发表数据，比较了蛋白质水解产物中氨基酸衍生物的摩尔吸收系数和相对荧光产率。三硝基苯衍生物的变异系数小于荧光胺或邻苯二甲醛衍生物的变异系数。还使用洛瑞法、考马斯亮蓝法和三硝基苯磺酸盐试剂比较了五种可溶性蛋白质的显色产率。结果表明，所描述的三硝基苯磺酸盐方法更灵敏，每毫克蛋白质的吸光度变化比洛瑞法或考马斯亮蓝法小三倍。
