乙基曙红分光光度法测定蛋白质

Speotrophtometric Determination of Protein with Ethyl Eosin

在pH3 5～4 0的NaAc HCl介质中,乙基曙红与蛋白质结合形成复合物,溶液颜色发生变化,最大吸收波长为548nm,比乙基曙红红移了28nm,并在520nm处产生最大褪色,其吸收或褪色作用均可用于蛋白质的分光光度测定.在最大吸收波长548nm处,蛋白质的浓度在0 23～20 0μg·mL-1(HSA)和0 25～17 5μg·mL-1(BSA)范围内与吸光度成正比,其表观摩尔吸光系数分别为1 33×106L·mol-1·cm-1(BSA)和1 26×106L·mol-1·cm-1(HSA);而在最大褪色波长520nm处测量,上述范围的蛋白质浓度也与褪色程度成直线关系,其表观摩尔吸光系数分别为2 02×106L·mol-1·cm-1(BSA)和2 06×106L·mol-1·cm-1(HSA),褪色光度法灵敏度更高.并且方法选择性好,用于尿液及人血清样品中总蛋白的测定,所得结果与考马斯亮蓝法基本一致.

In pH 35-40 NaAcHCl buffer medium, ethyl eosin reacts with protein to form a complex, The colors of solutions change obviously, which has a maximum absorption wavelength at 548 nm, which exhibits a bathochromic shift of 28 nm compared with the maximum absorption wavelength of ethyl eosin, and there exists a maximum fading wavelength at 520 nm, absorption or fading can used to the determination of protein with spectrophotometric method. The absorbance of the binding system is proportional to the concentration of protein in the range of 023-200 μg·mL-1 (HSA) and 025-175 μg·mL-1 (BSA) at the maximum absorption wavelength 548 nm, the apparent molar absorptivities are 133×106 L·mol-1·cm-1 (BSA) and 126×106 L·mol-1·cm-1 (HSA), respectively. At maximum fading wavelength of 520 nm, the fading intensity is also proportional to the concentration of protein in abovementioned linear range. The apparent molar absorptivities are 202×106 L·mol-1·cm-1 (BSA) and 206×106 L·mol-1·cm-1 (HSA), the sensitivity of the fading spectrophotometric method is higher. The method has good selectivity. It is used for the determination of total protein in urine and human serum samples, the rusult obtained by this method agreeds well with those obtained by Coomassie brillant blue G250 method.