QUANTITATION OF EPSILON-AMINO GROUP USING AMINO-ACIDS AS REFERENCE-STANDARDS BY TRINITROBENZENE SULFONIC-ACID - A SIMPLE SPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF HAPTEN TO CARRIER PROTEIN RATIO
QUANTITATION OF EPSILON-AMINO GROUP USING AMINO-ACIDS AS REFERENCE-STANDARDS BY TRINITROBENZENE SULFONIC-ACID - A SIMPLE SPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF HAPTEN TO CARRIER PROTEIN RATIO
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DOI：
10.1016/0022-1759(94)90081-7
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发表时间：
1994-01-03
影响因子：
2.2
通讯作者：
RAMANA, D
中科院分区：
文献类型：
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作者：
SASHIDHAR, RB;CAPOOR, AK;RAMANA, D
以游离氨基酸L-赖氨酸和L-谷氨酸为参比标准品，以2，4，6-三硝基苯1-磺酸（TNBS）为显色剂，建立了一种简便、灵敏、直接测定载体蛋白中L-赖氨酸ε-氨基的分光光度法。使用标准曲线直接定量与半抗原偶联后载体蛋白中存在的ε-氨基的量，所述标准曲线由L-赖氨酸和L-谷氨酸与TNBS试剂反应后观察到的吸光度差异产生。TNBS的L-赖氨酸和L-谷氨酸（27.36 nmol）衍生物在335 nm处的光谱分析表明，TNP-L-赖氨酸的吸光度是TNP-L-谷氨酸的两倍，因为TNBS试剂分别与L-赖氨酸中存在的α-氨基和ε-氨基以及L-谷氨酸的α-氨基平等地相互作用。发现吸光度与ε-氨基浓度（高达16 μ g/ml）之间呈线性关系。载体蛋白如BSA，HSA，甲状腺球蛋白和酶，辣根过氧化物酶中存在的赖氨酸的ε-氨基的数量进行了分析，通过本方法，并被发现是类似的报道值。制备各种载体蛋白-半抗原缀合物（蛋白-真菌毒素/维生素/类固醇激素缀合物），并通过开发的方法进行分析，以确定它们的摩尔比。
A simple, sensitive and direct spectrophotometric method is presented for the determination of epsilon-amino groups of L-lysine present in carrier proteins, using the free amino acids L-lysine and L-glutamic acid as reference standards, and 2,4,6-trinitrobenzene 1-sulfonic acid (TNBS) reagent. The amount of epsilon-amino group present in the carrier protein after coupling with hapten is directly quantitated using the standard curve generated by the difference in absorbance observed with L-lysine and L-glutamic acid after their reaction with TNBS reagent. Spectral analysis of L-lysine and L-glutamic acid (27.36 nmol) derivatives of TNBS at 335 nm showed that TNP-L-lysine had twice the absorbance of TNP-L-glutamic acid, since TNBS reagent interacts equally with the alpha-amino and epsilon-amino groups present in L-lysine and the alpha-amino group of L-glutamic acid, respectively. The relationship between absorbance and concentration of epsilon-amino groups (up to 16 mu g/ml) was found to be linear. The number of epsilon-amino groups of lysine present in carrier proteins such as BSA, HSA, thyroglobulin and the enzyme, horseradish peroxidase, were analyzed by the present method and were found to be similar to the reported values. Various carrier protein-hapten conjugates (protein-mycotoxin/vitamin/steroid hormone conjugates) were made and analyzed by the method developed in order to determine their mole to mole ratio.