乙基紫分光光度法测定牛血清白蛋白

Ethyl Violet-based Spectrophotometric Determination of Bovine Serum Albumin

研究了乙基紫 (EV)与牛血清白蛋白 (BSA)的结合反应 .在pH =7.2的条件下 ,BSA的加入使乙基紫的最大吸收峰波长红移 5nm(5 95→ 6 0 0nm) ,并使染料吸光度增大 .6 0 0nm处吸光度与BSA在一定浓度范围内有线性关系 ,线性响应范围为 1.0 0× 10 -7～ 2 .0 0× 10 -6mol/L ,检测下限为 8.0 0× 10 -8mol/L .该法简便、快速、干扰少、灵敏度较高 ,用于牛血清白蛋白样品测定结果满意 .初步探讨了EV -BSA结合机理 ,Scatchard图表明有两类结合 ,第一类结合的最大结合数为 2 .3,结合常数为 6 .5× 10 5L/mol;第二类结合的最大结合数为 5 .6 ,结合常数为 2 .0× 10 5L/mol.讨论了乙醇、十六烷基三甲基溴化铵 (CTAB) ,十二烷基苯磺酸钠 (DBS)

The binding reaction of BSA with ethyl violet(EV) was investigated in a Tris-HCI buffer(pH= 7.2). Upon addition of BSA into an EV-containing solution, an increase with a red shift(595&rarr600 nm) in the peak absorbance of EV was observed. The peak absorbance at 600 nm was linear with the BSA concentration over a range of 1.00 × 10-7 ∼2.00 × 10-6 mol/L with a detection limit of 8.00 × 10-8 mol/L. A simple, rapid and sensitive method for BSA analysis was thus established, and it has been used to determine BSA in several samples with satisfactory results. In addition, the mechanism of the BSA-EV binding reaction was also explored, the Scatchard plot gave the first-class binding constant of 6.5 × 105 L/mol with a binding number of 2. 3, and the second-class binding constant of 2.0 × 105 L/mol with a binding number of 5.6. Effects of ethanol, CTAB, DBS on this binding reaction were also discussed.